Impedance technology reveals correlations between cytotoxicity and lipophilicity of mono and bimetallic phosphine complexes

Article abstract of DOI:10.1007/s10534-015-9851-y

Label free impedance technology enables the monitoring of cell response patterns post treatment with drugs or other chemicals. Using this technology, a correlation between the lipophilicity of metal complexes and the degree of cytotoxicity was observed. A

Full text of DOI:10.1007/s10534-015-9851-y

Biometals
DOI 10.1007/s10534-015-9851-y
Impedance technology reveals correlations
between cytotoxicity and lipophilicity of mono
and bimetallic phosphine complexes
•
P. Fonteh A. Elkhadir B. Omondi
•
•
•
I. Guzei J. Darkwa D. Meyer
•
Received: 18 February 2015 / Accepted: 25 March 2015
Ó Springer Science+Business Media New York 2015
Abstract Label free impedance technology enables
the monitoring of cell response patterns post treatment
with drugs or other chemicals. Using this technology,
a correlation between the lipophilicity of metal
complexes and the degree of cytotoxicity was ob-
served. Au(L1)Cl (1), AuPd(L1)(SC4H8)Cl3 (1a) and
Au(L2)Cl (2) [L1 = diphenylphosphino-2-pyridine;
L2 = 2-(2-(diphenylphosphino)ethyl)-pyridine] were
synthesised, in silico drug-likeness and structure–
activity relationship monitored using impedance tech-
nology. Dose dependent changes in cytotoxicity were
observed for the metal complexes resulting in IC50s of
12.5 2.5, 18.3 8.3 and 16.9 0.5 lM for 1, 1a
and 2 respectively in an endpoint assay. When a real
time impedance assay was used, dose-dependent
responses depicting patterns that suggested slower
uptake (at a toxic 20 lM) and faster recovery of the
cells (at the less toxic 10 lM) of the bimetallic
complex (1a) compared to the monometallic com-
plexes (1 and 2) was observed. These data agreed with
the ADMET findings of lower aqueous solubility of 1a
and non-ideal lipophilicity (AlogP98 of 6.55) over
more water soluble 1 and 2 with ideal lipophilicity
(4.91 and 5.03 respectively) values. The additional
coordination of a Pd atom to the nitrogen atom of a
pyridine ring, the sulfur atom of a tetrahydrothiophene
moiety and two chlorine atoms in 1a could be
contributing to the observed differences when com-
pared to the monometallic complexes. This report
presents impedance technology as a means of corre-
lating drug-likeness of lipophilic phosphine complex-
es containing similar backbone structures and could
prove valuable in filtering drug-like compounds in a
drug discovery project.
Electronic supplementary material The online version of
this article (doi:10.1007/s10534-015-9851-y) contains supple-
mentary material, which is available to authorized users.
P. Fonteh ꢀ D. Meyer
Department of Biochemistry, University of Pretoria,
Pretoria 0002, South Africa
A. Elkhadir ꢀ B. Omondi ꢀ J. Darkwa
Department of Chemistry, University of Johannesburg,
Auckland Park, Johannesburg 2006, South Africa
I. Guzei
Department of Chemistry, University of Wisconsin-
Madison, 1101 University Ave, Madison, WI 53706, USA
Keywords Impedance ꢀ Cytotoxicity ꢀ Uptake ꢀ
Recovery ꢀ Lipophilicity ꢀ Phosphine gold complexes
Present Address:
D. Meyer (&)
Abbreviations
ADMET Absorption, distribution, metabolism,
excretion, toxicity
Department of Biochemistry, University of Johannesburg,
P.O. Box 524, Auckland Park, Johannesburg 2006, South
Africa
e-mail: dmeyer@uj.ac.za
123

Biometals
identifying drug-like compounds. One such method-
ology is electrical detection based on cell substrate
impedance measurement (Giaever and Keese 1984).
The principle of the technique is based on the fact that
most non hematopoietic cells attach firmly to the ideal
substrate while injured cells will round up and finally
detach, which is associated with cell death (Kepp et al.
2011). By measuring the impedance of the surface
occupied by the cells, an indirect indicator of viability
(Solly et al. 2004; Atienza et al. 2005; Xing et al. 2005)
or cytotoxicity (Boyd et al. 2008; Xing et al. 2005;
Xing et al. 2006) is obtained. The advantages of
impedance technology makes the data relevant in
complementing these end point assays by providing
additional information on cellular parameters such as
cytotoxicity, cell proliferation, cell recovery and cell
response patterns (Xing et al. 2005) in real time. This
technique is therefore ideal for determining drug-like
properties of potential drugs and was further used here
to correlate structure, uptake, toxicity and lipophilicity
profiles.
AlogP98 Atom based log P
BBB
IC50
CI
Blood–brain barrier
50 % inhibitory concentration
Cell index
CIs
Cell indices
CYP
DS
Cytochrome P450
Discovery studio
HIA
HIV
MTT
Human intestinal absorption
Human immunodeficiency virus
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
Normalised cell index
Plasma protein binding
Post treatment
NCI
PPB
PT
RT-CES Real time cell electronic sensing
SAR Structure–activity relationship
Introduction
The study of drug-like or absorption, distribution,
metabolism, excretion and toxicity (ADMET) prop-
erties of new chemicals early in drug discovery has
gained significant importance over the last decade due
to the high rate of late drug failures during clinical
trials (Hamid et al. 2004), in addition to other adverse
effects. In silico prediction models provides insight
into potential toxicity and can significantly shorten the
drug discovery time line (Lobell and Sivarajah 2003).
It is however important that these data be comple-
mented with in vitro experimental studies because of
the physiological relevance of the latter. This is one of
the main reasons why cell-based assays continue to
grow in importance in drug discovery especially since
the data can be extrapolated to pharmacological
endpoints (McGuinness 2007).
Gold-based compounds have been widely exploited
for anti-cancer activity (Nobili et al. 2010; Sun and
Che 2009; Monim-ul-Mehboob et al. 2013) as well as
for inhibition of various pathogens such as the human
immunodeficiency virus (HIV) (Shapiro and Masci
1996; Fonteh et al. 2009; Fonteh et al. 2011). One
limitation of the earliest anti-HIV gold-based drugs
such as the injectable gold compound, aurothioglucose
and its metabolites was the fact that they could not be
taken up by cells due to poor lipophilicity (Okada et al.
1993; Zhang et al. 1995). Aurothioglucose unlike
auranofin lacks a phosphine moiety; which is known to
confer the lipophilic property and hence oral avail-
ability of the latter group (Shaw et al. 1994). The
addition of a second similar (homo-bimetal) or
different metal (hetero-bimetal) has shown improved
activity due to synergism between the metals or dual
biological activity since each metal maintains its
chemical identity with the resultant inhibition of
multiple sites (Rupesh et al. 2006). Palladium is one
such metal which has been considered in bimetallic
synthesis since compounds of this metal have
biological properties such as anti-cancer activity
(Ray et al. 2007; Wang et al. 2011). The addition of
a second metal however affects lipophilicity and hence
drug-likeness of the complexes.
Until recently, toxicity has largely been tested using
end point assays which unfortunately are not dynamic
and focus on measuring just one parameter (Xing et al.
2006). These end point assays usually include the use
of a label for monitoring various cellular functions that
are then detected using methods such as optical
absorbance, fluorescence, luminescence or radioiso-
topic techniques (Solly et al. 2004). The labels
sometimes interfere with test compounds, involve
time consuming labelling and wash steps and could
potentially affect physiological conditions in cell
culture (Solly et al. 2004). The use of label-free
technology has therefore been emerging as a way of
Lipophilicity is used to assess biological pa-
rameters relevant to drug action such as lipid
123

Biometals
Instrumentation
solubility, tissue distribution, receptor binding, cellu-
lar uptake, metabolism and bioavailability (Ghose
et al. 1998). In this study, one novel bimetallic
complex (1a) and two monometallic gold-based
phosphine complexes (1 and 2) were synthesised and
characterised. Cytotoxicity effects of the compounds
was determined on TZM-bl cells using the 3-(4,5-
dimethylythiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) tetrazolium dye. The impedance tech-
nology of the real time cell electronic sensing (RT-
CESTM, ACEA Biosciences Inc., San Diego, USA)
device was used to further complement MTT findings
while lipophilicity predictions were done using the
ADMET prediction protocol in DSÒ. The cell respon-
se patterns with regards to compound toxicity (both
MTT and RT-CESTM) as well as uptake and recovery
after uptake in the RT-CESTM assay were shown to
correlate with the liphophilicity levels obtained from
in silico ADMET predictions. These findings demon-
strate the ability of impedance technology in correlat-
ing uptake of chemicals or recovery of cells after
treatment with lipophilic phosphine gold monometal-
lic and bimetallic gold–palladium complexes. These
findings are useful in complementing ADMET studies
and could aid in reducing the attrition rate for these
types of complexes or others in a drug discovery study.
1H NMR and 13C{1H} NMR spectra were recorded in
chloroform-d (CDCl3) on a Varian Gemini 2000
1
instrument (at 300 MHz for H NMR, at 75.46 MHz
for 13C{1H} NMR and at 121.49 MHz for 31P{1H}
NMR) and on a Bruker Ultrashield 400 instrument {at
1
400 MHz for H NMR, at 100.61 MHz for 13C[1H]
NMR and at 161.97 MHz for 31P[1H] NMR} at room
1
temperature. H and 13C{1H} NMR chemical shifts
were referenced to the residual signals of the protons
or carbons of the NMR solvents and are quoted in d
(ppm): CDCl3 at 7.24 and 77.00 ppm for 1H and
13C{1H} NMR spectra respectively. Coupling con-
stants are measured in Hertz (Hz). Elemental analyses
were performed on a Vario Elementar III microcube
CHNS analyzer at Rhodes University, South Africa.
Synthesis of palladium(tetrahydrothiophene)
[(diphenylphosphino-2-pyridyl)]AuCl chloride (1a)
To a chloroform solution (10 ml) of diphenylphos-
phino-2-pyridine (0.05 g, 0.10 mmol) was added
[Au(THT)Cl] (0.03 g, 0.10 mmol). The solution was
stirred for 20 min before [Pd(NCMe)2Cl2] (0.01 g,
0.05 mmol) was added and stirring continued for a
further 30 min. Hexane (10 ml) was added to pre-
cipitate the crude product, which was filtered and dried
in vacuo. Analytically pure product was obtained from
re-crystallization of the crude product in a chloroform-
hexane mixture to afford bright yellowish-orange
Materials and Methods
For the synthetic processes, all manipulations were
carried out under argon by standard Schlenk techniques.
All Solvents were of analytical grade and were dried
using a Braun MB SPS-800 drying solvent system.
Diphenylphosphino-2-pyridine (L1) and 2-(2-
(diphenylphosphino)ethyl)pyridine (L2) were pur-
chased from Sigma-Aldrich and were used as received.
The gold and palladium starting materials,
chloroauric acid H[AuCl4] and palladium dichloride
[PdCl2]n, were purchased from South Africa Precious
Metals. The starting materials gold(tetrahydrothio-
phene)chloride [Au(THT)Cl] (Uson et al. 1989) and
bis(acetonitrile)-dichloropalladium(II) [Pd(NCMe)2-
Cl2] (Ru¨lke et al. 1990) were synthesized following
literature procedures. Diphenylphosphino-2-pyridyl-
gold(I) chloride (1) and 2-(2-(diphenylphosphi-
no)ethyl)pyridyl)gold(I) chloride (2) were prepared
using the method by Calhorda et al. (2010) and hence
there is no need for discussion of the spectral data.
1
crystals. A yield of 80 % (0.06 g) was obtained. H
NMR (CDCl3): d 1.94 (s, 2H) 2.31 (s, 2H), 2.77 (s,
2H), 3.61 (s, 2H), 7.35 (t, 1H), 7.46 (m, 4H), 7.56 (m,
6H), 7.69 (q, 1H, J = 5.6, 7.6 and 8.0 Hz), 7.79 (t,
1H), 9.39 (d, 1H, J = 5.2 Hz). 31P{1H} (CDCl3) 37.54
(s, 1P). 13C{1H} NMR (CDCl3) 29.5; 30.1; 37.7; 38.5;
126.9; 127.1; 127.8; 129.3; 132.3, 132.4, 132.7, 132.8;
134.2, 134.4; 138.1; 155.2, 155.3; 156.3. Anal. Calcd.
for C21H22PAuCl3NPdS.CHCl3, C, 30.01; H, 2.63; N,
1.59; S, 3.64 %. Found: C, 30.07; H, 2.52; N, 1.47; S,
3.64 %.
X-ray structure determination
Data collection
A crystal of 2 (as a representative compound) of
dimensions 0.32 9 0.06 9 0.02 mm3 was selected and
123

Biometals
Culturing of TZM-bl cells
glued on to the tip of a glass fibre. The crystal was then
mounted in a stream of cold nitrogen at 100(1) K and
centred in the X-ray beam using a video camera. The
crystal evaluation and data collection were performed
on a Bruker APEXII diffractometer with Mo Ka
The TZM-bl cell line from Dr. John C. Kappes, Dr.
Xiaoyun Wu and Tranzyme Inc, (Takeuchi et al. 2008;
Wei et al. 2002; Derdeyn et al. 2000; Platt et al. 1998)
was used in determining toxicity and proliferation.
This is an adherent HeLa cell line engineered to stably
express CD4, CXCR4 and CCR5 and containing
reporter genes controlled by the HIV-1 promoter
enabling HIV-1 infectivity determination. These cells
are a model cell line for testing HIV infectivity and
was used because of our previous work with HIV and
also because we aimed in a separate study to determine
the effect of the compounds on HIV infectivity.
˚
(k = 0.71073 A) radiation and the diffractometer to
crystal distance of 4.00 cm. The initial cell matrix was
obtained from three series of scans at different starting
angles. Each series consisted of 12 frames collected at
intervals of 0.5° in a 6° range about with the exposure
time of 10 s per frame. The reflections were success-
fully indexed by an automated indexing routine built in
the APEXII program suite (Bruker 2009). The final cell
constants were calculated from a set of 6460 strong
reflections from the actual data collection.
The cells were sub-cultured in T-75 tissue culture
flasks (NuncTM
, Roskilde, Denmark) with ap-
The data were collected by using the full sphere
data collection routine to survey the reciprocal space
˚
to the extent of a full sphere to a resolution of 0.75 A.
proximately 106 cells in 15 ml of complete Dulbec-
co’s Modified Eagle Medium (DMEM) with L-
glutamine (Sigma Aldrich, Missouri, USA) containing
sodium pyruvate (100 mM, Thermo Scientific,
HyCloneÒ, UT, USA), hepes buffer (1 M, Gibco
BRL Life Technologies, Grand Island, USA), sodium
bicarbonate (3.70 g/l, Merck, Wadeville, RSA), an-
tibiotics (50 lg/ml gentamycin sulphate, Sigma
Aldrich, Missouri, USA) and 10 % (v/v) heat inacti-
vated fetal calf serum (Thermo Scientific, HyCloneÒ,
UT, USA). Sub-culturing was done every 2 or 3 days
when confluency (surface area of the culture flask
occupied by cells) was about 90 %. Cell culture
conditions for the bioassays (MTT and impedance),
which included cell number, media volume and
incubation time, were established through titration
experiments on the RT-CESTM analyser. The pre-
determined conditions were maintained for both
bioassays since a similarity in the efficiency for viable
cell count has been reported for the two (Xing et al.
2006).
A total of 4426 data were harvested by collecting 1109
frames at intervals of 0.5° scans in 6° and u with
exposure times of 15 s per frame. These highly
redundant datasets were corrected for Lorentz and
polarization effects. The absorption correction was
based on fitting a function to the empirical transmis-
sion surface as sampled by multiple equivalent
measurements (Bruker 2009).
Structure solution and refinement
The systematic absences in the diffraction data were
uniquely consistent for the space group P21/c that
yielded chemically reasonable and computationally
stable results of refinement. A successful solution by
the direct methods of SIR92 (Altomare et al. 1993)
provided all non-hydrogen atoms from the E-map. All
non-hydrogen atoms were refined with anisotropic
displacement coefficients. All hydrogen atoms except
those on the solvent water molecules were included in
the structure factor calculation at idealized positions
and were allowed to ride on the neighbouring atoms
with relative isotropic displacement coefficients. The
final least-squares refinement of 205 parameters
against 4286 data resulted in residuals R (based on
F2 for I C 2r) and wR (based on F2 for all data) of
0.0198 and 0.0515, respectively. The final difference
Fourier map was featureless. The molecular diagrams
are drawn with 50 % probability ellipsoids (Palatinus
and Chapuis 2007; Farrugia 1999; Farrugia 1997;
Sheldrick 2008).
Cytotoxicity determination with MTT
To obtain cells in the exponential phase of growth,
100 ll of TZM-bl cells at 2 9 105 cells/ml in com-
plete DMEM medium were plated in 96 well tissue
culture plates to a final volume of 200 ll. The cells
were allowed to adhere and reach confluency (after
22–24 h) in a 5 % CO2 incubator (37 °C, 95 %
humidity). Compound stocks were dissolved in
DMSO and further diluted with culture medium to a
final DMSO concentration B0.5 % (v/v). That DMSO
123

Biometals
was a suitable solvent was evident from the consis-
tently reproducible responses of gold(I) phosphine
complexes during biological analysis (Fonteh and
Meyer 2009). NMR spectral profiles show
gold(I) phosphine complexes to be stable in DMSO
in the time required before further dilution with culture
medium (Gandin et al. 2010; Fonteh 2011). Recent
work by Altaf et al. (2015) again highlighted the
stability of gold(I) phosphine complexes in DMSO-d6
72 h post dissolution.
response patterns. Impedance measurements were
performed on the TZM-bl cells using the RT-CESTM
xCELLigence system (ACEA Biosciences Inc., San
Diego, USA) and following the manufacturer’s in-
structions. This technique enables the measurement of
cell response patterns in real time (providing results
not possible in end point assays) and resulting in
multivariate data from a single experiment. In addi-
tion, because it is label free, further downstream
analysis of the same cells can be performed. The RT-
CESTM system has previously been described (Solly
et al. 2004; Atienza et al. 2005). The system that was
used in this study is known as the dual plate (DP) and
employs 3 9 16 well electronic plates (E-plates). The
E-plate contains integral sensor electrode arrays that
allows impedance measurement read out as cell index
(CI) resulting from the interaction of attaching cells
with the electrodes (Xing et al. 2006). This provides
information on the viability or cytotoxicity as CI
increases or decreases respectively. Pre-determined
assay parameters such as cell culture conditions, cell
number (2 9 105 cells/ml) and media volume were
used. After plating the cells, the E-plates were left at
room temperature for 30 min so as to reduce vari-
ability resulting from edge effects (Lundholt et al.
2003) before being transferred to the RT-CESTM
analyser. After approximately 22–24 h of allowing the
cells to attach and attain confluency of between 60 and
70 %, the test compounds were added to the wells to
final concentrations of 5, 10 and 20 lM (250 ll final
volume). The dynamic monitoring of cell response
patterns, depicting death and recovery, was auto-
matically recorded every minute (short term) for 1 h
and then every 30 min (long term) for a further
48–72 h. Short term monitoring allows for the iden-
tification of immediate and transient compound effects
while long term monitoring enables sufficient time for
the compounds to interact with the cells and modulate
their targets, enabling distinctions in cell response
patterns (Abassi et al. 2012). Certain compounds can
immediately result in cell death (drop in CI) with the
possibility of recovery (increase CI) while others are
taken up at varying degrees depicted by an increase in
CI over a given time period prior to a decrease. These
differences in uptake, recovery and or toxicity levels
could also be related to the lipophilicity of the
particular compound. A normalised CI (NCI) was
used to compare the effect of the compounds on the
cells where impedance is set at 1 and at the time point
The concentrations of the compounds attained in
wells ranged from 0.8 to 100 lM (250 ll final
volume). An untreated control sample contained cells
and culture medium only while a blank control sample
contained medium only. The cells were further
incubated for 48 h (37 °C, 90 % humidity, 5 %
CO2) after which 190 ll of spent medium was
discarded and replaced with 140 ll of complete
medium and 20 ll of 5 mg/ml MTT. Colour devel-
opment was analysed after 2 h following solubilisa-
tion of the MTT formazan product using acidified
isopropanol in a 1:9 ratio (one part of 1 M HCl and
nine parts of isopropanol). Absorbance was measured
at 550 nm and a reference wavelength of 690 nm on a
Multiskan AscentÒ spectrophotometer (Labsystems,
Helsinki, Finland). Cell viability was calculated using
the formula: (absorbance of sample-absorbance of
medium/absorbance of control-absorbance of medi-
um) 9 100 for n = 4 experiments. The IC50s were
then graphically obtained after generating a dose
response curve using Graphpad PrismÒ software
(California, USA).
Impedance measurement using the xCELLigence
system
The MTT assay developed by Mosmann in 1983 is an
endpoint assay widely used for the quantitative
assessment of cellular viability and proliferation.
Unfortunately because of limitations such as poor
linearity with changing cell number, sensitivity to
environmental conditions and the fact that it depends
on only one parameter, which is the cells’ metabolism
of formazan (Boyd 1989; Haselsberger et al. 1996;
Denizot and Lang 1986), there is the need to use
additional confirmatory methods to conclude on cell
status. Measuring several parameters in the same assay
using dynamic conditions like the impedance tech-
nology has been shown to provide better insight on cell
123

Biometals
where the cells were treated with compounds (22–24 h
post seeding). When normalisation is set, it corrects
for any small differences that might have occurred
between cells before the addition of compounds such
that only compound effects are visible.
like properties such as lipid solubility, tissue distribu-
tion, receptor binding, cellular uptake, metabolism and
bioavailability (Ghose et al. 1998), our main focus
here was to compare the lipophilicity outcome from
the ADMET predictions to the uptake profile from the
real time studies. The anticipation was that the
predicted lipophilicity values will correlate with the
uptake, cell response and recovery patterns from the
impedance study.
ADMET predictions to determine lipophilicity
ADMET predictions were performed using DSÒ, as
previously described for some gold(III) thiosemicar-
bazonate complexes (Fonteh et al. 2011). The
molecular structure of the compounds in the structural
data file (sdf) format were geometrically optimised
and subjected to an ADMET run. The protocol
consists of various models that enable prediction of
human intestinal absorption (HIA), aqueous solubility,
blood brain barrier penetration (BBB), cytochrome
P450 (CYP) inhibition, hepatotoxicity and plasma
protein binding (PPB). It also predicts lipophilicity
levels in the form of atom-based LogP (AlogP98). We
have previously shown a similarity between these in
silico lipophilicity predictions and those obtained
from the wet lab shake flask method for two gold-
based compounds (Fonteh et al. 2011). Considering
that lipophilicity provides information on other drug-
Results and Discussion
Synthesis and characterization of the complexes
Compounds 1 (Alcock et al. 1982) and 2 (Calhorda
et al. 2010) have previously been synthesised but in
this study both compounds were done by a slightly
modified method that led to better yields; whilst
compound 1a which was prepared by adopting a one-
pot approach is novel. All complexes were synthesized
in good to excellent yields. The procedure outlined in
Scheme 1 represents a general route for synthesis of
gold(I) complexes 1, 2 and bimetallic complex 1a. The
complexes were characterized by NMR spectroscopy
N
P
N
P
N
(L1)
(L2)
N
P
P
Au
Cl
Au
Cl
L2
L1
(1)
2
( )
S Au Cl
[Pd(NCMe)2Cl2]
[Pd(NCMe)2Cl2]
L1
Cl
Au
N
P
Cl Pd Cl
N
N
Cl Pd
S
P
P
Au
Cl
Cl
Au
Cl
(2b)
(1a)
Scheme 1 Preparation of complexes
123

Biometals
and elemental analysis as well as X-ray diffraction
studies for complexes 1a and 2.
A novel bimetallic complex, 1a, was synthesized by
the reaction of L1 with [Au(THT)Cl] and [PdCl2(-
NCMe)2] in a one-pot reaction with equimolar quan-
tities of all three reagents (Scheme 1). Complex 1a
was isolated in a very good yield. The 1H NMR
spectrum of 1a similarly showed the characteristic
phenyl resonances found in 1. In addition to pyridine
ring resonances that were observed as a doublet at
9.39 ppm corresponding to the proton ortho to the
pyridine ring that had been shifted downfield due to
coordination, and a triplet at 7.79 ppm, a quartet at
7.67 ppm and another triplet at 7.35 ppm. The
tetrahydrothiophene (THT) protons appeared as broad
singlets at 3.61 ppm, 2.77 ppm, 2.31 ppm and
1.94 ppm. The 31P{1H} NMR spectrum of the
monometallic gold(I) complex showed one resonance
at 32.21 ppm, however, the 31P{1H} NMR spectrum
of complex 1a showed single resonance at 37.56 ppm
which confirmed further coordination to palladium.
The 13C{1H} NMR spectrum of dichlorobis-(tetrahy-
The molecular structure of 2 (Fig. SI in ESM)
which was confirmed by single crystal X-ray diffrac-
tion is presented in the online resource section. The
complex crystallized with one molecule in the asym-
metric unit. Crystal data collection and refinement
parameters are given in Table SI in ESM (online
resource section) and a selection of bond lengths and
angles are listed in Table SII in ESM. The coordina-
tion around the gold center deviates by about 2.7° from
a perfect linear geometry. The bond angle P-Au–Cl
deviates from linearity like (triphenylphosphine)-
gold(I) chloride complex which is 179.63(8)°. P-Au
distance is within expected values while the Au–Cl
distance is slightly longer than the distance for
˚
(triphenylphosphine)gold(I) choride 2.235(3) A
(Baenziger et al. 1976).
drothiophene)-palladium(II) has
a
doublet at
29.9 ppm and a singlet at 42.1 ppm whilst for 1a
these carbons resonated as two doublets at 29.54 and
37.7 ppm. This was further evidence that compound
1a had a phosphine ligand, as the two doublets in the
13C spectrum are the result of 31P coupling with 13C.
The molecular structure of 1a was confirmed using
single crystal X-ray diffraction as shown in Fig. 1.
Crystal data collection and refinement parameters are
given in Table SI in ESM, and selected bond lengths
and angles for 1a are listed in Table 1. The geometry of
1a has a nitrogen atom of the pyridine ring, two chlorine
atoms as well as the sulfur atom of THT bound to the
palladium in a four coordinate geometry. The reaction
of [Au(THT)Cl] with L1 leads directly to 1. The
bounded THT is indicative of a re-arrangement that led
to the final product. In fact, the released THT moiety
that is present in the reaction mixture filled the fourth
coordination site in 1a by coordinating to palladium
through the sulphur atom. The geometry around the
gold center in 1a deviates from linearity, with a bond
angle P1-Au1-Cl3 [170.09(2)°] which deviates from
linearity much more than the corresponding angle in
(triphenylphosphine)gold(I) chloride [179.63(8)°] re-
ported in the literature (Baenziger et al. 1976).
Fig. 1 Molecular structure of 1a with 50 % probability
ellipsoids. The ORTEP diagram is presented for the purpose
of showing the connectivity of the atoms. Hydrogen atoms are
omitted for clarity
˚
Table 1 Selected bond distances (A) and angles (°) for 1a
˚
Bond length (A)
Bond angles (°)
Au1–P1
Au1–Cl3
Pd–N1
2.2199 (0.0001)
P1–Au1–Cl3
N1–Pd1–Cl1
N1–Pd–Cl2
S1–Pd1–Cl2
170.09 (2)
88.13 (6)
90.71 (6)
87.37 (4)
2.2907 (0.0001)
2.058 (2)
The
gold–phosphorous
˚
distance
P1–Au1
[2.199(10) A] is however slightly shorter than the
distance for (triphenylphosphine)gold(I) choride
˚
[2.235(3) A]. The geometry around the Pd atom in
The numbers in parenthesis are estimated standard deviations
123

Biometals
the complex is a distorted square planar geometry with
complexes were much lower suggesting increased
toxicity resulting from complexation of the ligands
N1–Pd1–Cl1 [88.13(6)°], N1–Pd1–Cl2 [90.71(6)°],
S1–Pd1–Cl1
[87.37(4)°],
and
S1–Pd1–Cl2
with
metals.
These
were
12.5 2.5 lM,
[92.87(4)°]. For trans-dichlorobis(tetrahydrothio-
phene)palladium(II) the similar angles for S1–Pd1–
Cl1 and S1–Pd1–Cl2 are 84.33(5)° and 95.67(5)°
18.3 8.3 lM, and 16.9 0.5 lM for complexes
1, 1a and 2 respectively. A notable observation was the
fact that 1 was more toxic than 2. The presence of the
ethyl group in L2 and thus 2 which is absent in 1 (L1)
might play a role in the reduced toxicity of the former.
Although the monometallic complex appeared slightly
more toxic than the bimetallic complex e.g. complex 1
with an IC50 of 12.5 2.5 lM compared to 1a with
IC50 of 18.3 8.3 lM, the higher deviation from the
mean as seen in the standard error of the means for the
bimetallic complex over the monometallic ones
appeared to cancel this out. This high deviation for
the bimetal could be attributable to the structural
differences such as the presence of the dichloro(te-
trahydrothiophene)palladium(II) moiety and hence
differences in drug-like properties such as lipophili-
city, uptake and hence metabolism. The lipophilicity
outcome and cytotoxicity correlation are further
discussed in the real time and the in silico ADMET
prediction studies to further clarify this difference.
´
respectively (Noren et al. 1997). The N1–Pd1
˚
˚
[2.058(2) A), Pd1–Cl1 [2.2976(90) A] and Pd1–Cl2
˚
[2.3054(9) A] bond distances are shorter than the
corresponding distances for [Pd(CH3)Cl(C26H24NP)]
(Agostinho et al. 2006). The solvent chloroform has
the expected structure, with bond lengths Cl4–
˚
C22 = 1.747(3) A, Cl5–C22 = 1.767(3) A, and
˚
˚
Cl6–C22 = 1.762(3) A.
An attempt to synthesise a second bimetallic
complex (2a) with formula AuPd(L2)(SC4H8)Cl3,
from L2 in order to investigate the structure–activity
relationship (SAR) with 1, 1a and 2 was initiated.
Unfortunately 2a was found to be unstable and was
only considered for the in silico ADMET predictions
and not for the bioassays. The reason for this is
because the in silico analysis depend on computer
aided structure predictions only and can potentially
provide some predictive information on 2a’s effect on
cells thereby enabling comparison with those of 1, 1a
and 2.
RT-CESTM findings show distinguishing uptake
and recovery patterns between mono- and bi-
metallic compounds
Further investigation by NMR and X-ray spec-
troscopy of the reaction mixture of the supposed
instable second bimetal (2a) confirmed the presence of
traces of 2b, Au2Pd(L2)2Cl4, in solution (Scheme 1),
also previously synthesised by Calhorda et al. (2010),
hence no spectral data is presented. This complex
unlike 1a and the proposed 2a structure is structurally
bulkier containing two Au atoms, each independently
linked to L2 and bridged by a Pd atom coordinated to
the nitrogen atom of the pyridine ring of both L2
moieties. Interestingly, the impedance data and
ADMET predictions for 2b were not comparable to
that of 1a probably because of these significant
structural differences and as such are only briefly
discussed.
Results for the impedance profiles of the complemen-
tary metallic complexes (1 and 1a) of L1 are shown in
Fig. 3. Three concentrations of 5, 10 and 20 lM were
tested and only one concentration of 10 lM for L1
(insert Fig. 3). A dose dependent decrease in CI was
observed from 5 to 20 lM, representative of increas-
ing cell death or decreasing cell proliferation when the
TZM-bl cells were treated with the metal complexes.
At 10 lM, the effect of L1 did not result in CI
decreases supporting the MTT findings where the IC50
was reported to be [100 lM (insert Fig. 3). This
finding also supports the fact that the activity/toxicity
of the metal complex is as a result of the presence of
the metal entity (Navarro 2009; Beraldo and Gambino
2004) which in addition to structurally stabilising the
organic moiety or ligand (Navarro 2009), also im-
proves activity.
MTT assay for toxicity determination
In the MTT study, dose dependent increases in toxicity
were observed for the complexes and not the ligands
(Fig. 2). At the tested concentrations, ligands L1 and
L2 had no adverse effects on the viability of the cells
(IC50s [ 100 lM). IC50 values for the metal
Following complex addition, there was an observed
steady increase in CI which peaked by 1 h for 1 and
second hour for 1a compared to the untreated cells.
This peaking in CI has been associated with compound
123

Biometals
Fig. 2 Effect of compounds on the viability of TZM-bl cells
analysed using the MTT tetrazolium dye. Two fold serially
diluted concentrations from 100 to 0.8 lM were tested and a
dose dependent decrease in viability was observed for the mono-
and bi-metallic compounds. IC50 values obtained were
[100 lM for L1 and L2 and 12.5 2.5 lM, 18.3 8.3 lM
and 16.9 0.5 lM for complexes 1, 1a, and 2 respectively. The
monometallic compounds, 1 and 2 were more toxic than
bimetallic compound, 1a. A 10 lM the IC50 value of auranofin
which was used as control was 4.9 1.4 lM. The IC50 was
calculated from four independent experiments performed in
triplicate
Fig. 3 Effect of the Au monometallic complex 1 and the Au–
Pd bimetallic complex 1a on the impedance profile of TZM-bl
cells. TZM-bl cells were plated and allowed to adhere for
23.30 h before treatment with 5, 10 and 20 lM of 1 and 1a.
Normalisation was done 23.30 h. Dose dependent changes in CI
were observed for 1 and 1a at the tested concentrations with 1
being more toxic than 1a
uptake due to swelling (Thakur et al. 2012) or to result
from cell fusion forming multinuclear bodies (Xing
et al. 2005). Four independent Sybergreen staining
assays, detected using flow cytometry to determine
multinuclear body formation indicated that only 1N
nuclei and not 2N were present (data not shown). The
resultant increase in CI shortly after the addition of 1
and 1a was thus more likely as a result of uptake and
swelling of the cells and not multinuclear fusion. This
uptake was expected since lipophilic metal complexes
are known to enter cells by passive diffusion across the
membrane potential (Puckett et al. 2010). The
variation in uptake time (peak area) prior to the time
point when a sustained decrease in CI (below cell
control) was observed was used in comparing the
lipophilicity and hence uptake profiles of the phos-
phine metal complexes in this study. At the more toxic
20 lM concentration (also [IC50), the uptake time
(and subsequent cell death) for cells treated with 1 was
shorter than for cells treated with 1a suggesting that
123

Biometals
(a)
(b)
Fig. 4 Comparative uptake and recovery profiles between the
mono and bimetallic complexes. A comparison of uptake of 1
with that of 1a at 20 lM showed that 1 was taken up more
rapidly than 1a (a). As a result, recovery of cells treated with 1 at
10 lM was slower than for 1a (a). A quantitative representation
of complex mediated effect post treatment is shown in b. CI
increases were observed for L1, L2 and the untreated control at
24 h post treatment depicting cell viability but at 48 h PT,
decreases were observed probably as a result of overcrowding
and hence cell death. For the complexes, the reverse was
observed with CI decreases 24 h PT and increases at 48 h PT
which is associated with the observed recovery. PT stands for
post treatment. Recovery was better for the bimetallic complex
over the monometallic ones. At 10 lM, auranofin which was
used as control for cell death resulted in CI values similar to
those obtained for the media control confirming *100 % cell
death
more of 1a accumulated in the cells and not 1
(Fig. 4a). The area under this peak for three indepen-
dent experiments was found to be 0.2 0.2 and
1.3 0.4 for 1 and 1a respectively, further supporting
this observation.
compound resulted in quicker recovery over the other
since the staining process does not allow for real time
monitoring (Xing et al. 2006).
The real time studies were performed at least three
times for 1 and 1a at 5, 10 and 20 lM and at these
concentrations, similar uptake and recovery patterns
were obtained. The data shown in Fig. 3 is a repre-
sentative of the three while two additional profiles are
shown in Fig. SII of the online resource data. This data
further confirms the relatedness between uptake,
recovery, lipophilicity and structure. The main differ-
ences in proliferation patterns and toxicity of 1 and 1a
could be attributed to the presence of the dichloro(te-
trahydrothiophene)palladium(II) moiety present in 1a
which is absent in 1. The uptake patterns seen for the
bimetal 1a, where there was a significant increase in
CI upon compound addition has previously been
shown for a bimetallic gold(I) phosphine complex
designated EK207 (Fonteh 2011).
At the non toxic 10 lM (\IC50), no significant
differences in the uptake pattern of the complexes
were observed compared to untreated cells (Fig. 4a).
Instead, recovery patterns, where near constant cell
indices (CIs) started increasing with time, were
observed. These observations were more noticeable
and occurred earlier for 1a than for 1 (Fig. 4a) further
supporting the MTT end point findings where 1a was
shown to be less toxic than 1. CI decreases caused by 1
took much longer to reach a near constant point
(10 h 30 min post treatment or PT) and longer
(40 h 30 min PT) to reach recovery point compared
to 1a for which there was a brief or absent near
constant point due to a quicker recovery time starting
10 h 30 min PT (Fig. 4a). In an end point assay such
as MTT, it would be difficult to determine cell
recovery rate and time and thus determine which
In the impedance studies, a dose dependent decrease
in CI was also observed for 2 at 5, 10 and 20 lM
(depicted in Fig SIII in ESM). At 10 lM recovery was
123

Biometals
slower, findings that were similar to those of 1 (Fig. SIV
in ESM). When the proliferation patterns of 1 and 2
were compared (Fig. SIV in ESM), it was evident that
recovery for 2 was better than for 1.This again may be
linked to the presence of the ethyl group and supports
the MTT findings where 1 was more toxic than 2 (IC50
12.5 2.5 and 16.9 0.5 lM respectively)..
get into the more costly, late drug developmental
phases (Donato et al. 2008). The use of the rule of five
which states that poor absorption or permeation is
more likely when there are [5 H-bond donors, [10
H-bond acceptors, the molecular weight (Mr) is[500
and when the calculated log P is [5 (Lipinski et al.
1997) is an example of one such prediction method.
Here we have employed the ADMET protocol in DSÒ
which aids in the prediction of aqueous solubility,
BBB penetration, CYP inhibition, hepatotoxicity, HIA
and PPB. AlogP98 and polar surface area (PSA,
related to the H-bonding ability) are also some of the
predicted parameters. To enable comparison of the
lipophilicity profile of the monometallic and bimetal-
lic complexes, predictions were also done for 2a. The
instability reported for 2a in the wet lab synthesis was
not a concern here since this is an in silico computer
aided study and does not require the synthetic product.
For the purposes of this study, the predicted
lipophilicity values (AlogP98) will be the main focus
of the discussion since lipophilicity provides infor-
mation on other drug-like properties such as lipid
solubility, tissue distribution, receptor binding, cellu-
lar uptake, metabolism and bioavailability (Ghose
et al. 1998). The lipophilicity levels were compared to
the xCELLigence profiles to determine if there was
correlation in uptake and recovery patterns. These will
be further discussed with respect to aqueous solubility
and HIA. The predicted HIA, aqueous solubility and
lipophilicity values are shown in Table 2. Ligands L1
and L2 and the monometallic complexes 1 and 2 were
predicted as having ideal lipophilicity (AlogP98 B 5),
HIA and aqueous solubility based on the rankings that
were returned unlike the bimetallic ones, 1a and 2a
respectively (Table 2).
To further compare uptake and recovery patterns
between the monometallic and bimetallic complexes,
a cumulative time point plot of the CIs at 24 h and
48 h PT and at 10 uM only were compared (Fig. 4b).
The average of at least three different repeats were
analysed. At these two time points, the CI for the
monometallic compounds (1 and 2) were lower than
those of the bimetallic complex (1a) especially at
48 h, further supporting the toxicity and recovery
trends observed in the real time and MTT study. Over
time (from 24 to 48 h PT), CI was seen to decrease for
L1 and L2 and the untreated control (probably due to
overcrowding and hence cell death) while an increase
was observed for the complexes as a result of recovery.
Based on this time study, the gold complexes appeared
slightly more toxic after 24 h but not after 48 h (when
recovery was occurring), findings which will other-
wise not be noticeable in the end point MTT assay. For
the complexes, the CIs were always higher for the
bimetallic than for the monometallic complexes
further correlating the fact that the former were less
toxic compared to the latter.
ADMET predictions to determine lipophilicity
The importance of determining drug-likeness in the
early stages of drug discovery has resulted in the
development of numerous prediction tools. These
tools enable the sorting of new drug candidates early
on in the discovery process such that more safe drugs
Four prediction levels (0–4) are provided to aid in
the classification of HIA. A classification of zero
Table 2 ADMET prediction scores for the compounds
L1
1
1a
L2
2
2a
Auranofin
HIA
0
0
2
0
0
2
0
Aqueous solubility level
Alogp98
2
2
1
2
2
1
4
4.56
4.91
6.55
5.03
4.84
6.48
1.41
These scores were predicted using DSTM (Accelrys, California, USA). Various rankings come with the protocol. For L1, L2, 1 and 2,
a ranking of 0 represents good HIA while 2 for bimetallic compounds, 1a and 2a suggested a low HIA. Aqueous solubility level of 2
is classified as low for L1, L2, 1 and 2 while 1a and 2a were classified as possible (1). An AlogP98 or lipophilicity[5 is poor and was
shown for 1a and 2a and B5 is ideal lipophilicity as was the case for L1, L2, 1 and 2
123

Biometals
indicates good HIA while 4 is very poor absorption.
The ligands and monometallic compounds were all
predicted as having an HIA of 0 (good) while the
bimetallic compounds resulted in an HIA level of 2
(low). With regards to aqueous solubility, six levels
are described were 0 is extremely low, 1 very low but
possible, 2 is low, 3 good, 4 optimal, 5 too soluble and
6 a warning suggesting that molecules with one or
more unknown AlogP98 types are present. Ligands
L1, L2, and monometallic complexes, 1 and 2 were
again predicted to have a ranking of 2 (low) unlike
bimetallic ones 1a and 2a with a prediction of 1(very
low but possible). With regards to lipophilicity, the
Lipinski’s rule of 5 (Lipinski et al. 1997) was used as a
guide to classify predictions where compounds with
lipophilicity of [5 were considered to have poor
absorption and permeation. Based on this classifica-
tion, the bimetallic complexes again were the least
drug-like with AlogP98 values of 6.55 and 6.48 for 1a
and 2a respectively while the ligands and monometal-
lic complexes were all predicted as having AlogP98
values of 5 and below (Table 2).
assay for 1a. Such a limitation will be obviated in the
xCELLigence assay considering the label-free nature.
It was therefore exciting to note that despite the
possible MTT limitation, there was correlation be-
tween the two studies. The lipophilicity prediction
value of 1.41 for auranofin (Table 2), which was used
as a control in this study, supports literature reports
where the latter compound has been orally used as an
anti-arthritic gold-based compound because of its ideal
lipophilicity (Shapiro and Masci 1996).
Toxicity, impedance and ADMET findings for
complex 2b (product obtained when 2a synthesis was
attempted) are shown in Table SIII in ESM. The
complex was slightly more toxic as seen from both the
IC50 and impedance profile. The predicted AlogP98
was 10.66 indicative of poor HIA and aqueous
solubility (Table SIII in ESM). This compound which
has two gold atoms and lacks the THT moiety present
in 1a presented a profile which was not supportive of
the SAR. This finding was not surprising but suggests
that using impedance for drug-likeness correlation
should only serve to complement other techniques
since different ligands or moieties which coordinate to
metals differently could alter ADMET properties
significantly.
The bimetallic complexes which were less toxic in
the MTT study (Fig. 2) were predicted to have non
ideal lipophilicity values ([5) according to Lipinski
et al. (1997), limited aqueous solubility and HIA
levels and thus poor absorption. Generally, com-
pounds with ideal lipophilicity values, like the case
of L1, L2, 1 and 2, demonstrate good aqueous
solubility and are usually taken up better compared to
poorly soluble compounds. The presence of the ethyl
moiety in 2 and not 1 appeared to also influence
lipophilicity (4.91 and 4.84 respectively) and to
minimally affect uptake and recovery compared to 1a
(Fig. SIV in ESM). This was also observed in the
slight differences seen in toxicity patterns (IC50 of
12.5 2.5 for 1 and 16.9 0.5 lM for 2).
Considering that lipophilicity predictions and thus
HIA and aqueous solubility were limiting for 1a and
2a, it is not surprising that uptake of 1a (a bimetal) at
the toxic concentration of 20 lM occurred at a slower
pace (peak area of 1.3 0.4 compared to 0.2 0.17
for 1) such that recovery was quicker at 10 lM
(Fig. 4a). The poor aqueous solubility as a result of
high lipophilicity also meant more of the free com-
pound was available in solution. If this was the case, the
likelihood of the free compound interfering with MTT
metabolism was higher and might be the reason for the
high standard error of means observed in the MTT
Conclusion
Testing new chemical entities for cytotoxicity and
drug-likeness are common in the very early stages of a
drug discovery project so as to limit failure later in the
more costly phases. Rational drug design has been
instrumental in this process but the focus has also been
on designing libraries based on biological principles
(McGuinness 2007). Metal compounds such as gold
and palladium have been studied for anticancer
activity and for their effects on a wide range of
microorganisms making them candidate compounds
in drug discovery as either in the mono or bimetallic
forms. While it has been reported that varying the
lipophilicity and hydrophilicity of some gold-based
compounds results in better selectivity and hence
reduced toxicity (Berners-Price et al. 1999) due to the
correlation in uptake (McKeage et al. 2000), to the best
of our knowledge, using impedance technology to
monitor these correlations and how it is altered in the
presence of a second metal or group to show SAR has
not been reported.
123

Biometals
Xu X (2012) Dynamic monitoring of beating periodicity of
stem cell-derived cardiomyocytes as a predictive tool for
preclinical safety assessment. Br J Pharmacol 165:1424–
1441. doi:10.1111/j.1476-5381.2011.01623.x
In this study, the drug-likeness of two monometallic
gold-based compounds (1 and 2) and a bimetallic Au–
Pd complex (1a) were investigated using an end point
cytotoxicity assay, a real time impedance assay (which
produces multivariate data) and lipophilicity predic-
tions were done using an in silico technique. The
monometallic complexes were slightly more toxic
than the bimetal as depicted by IC50 values but
presented ideal lipophilicity, aqueous solubility and
hence better cellular uptake. Overall, these qualities
classify the bimetallic complex as a poor drug
candidate since compounds with limited uptake will
generally not reach cellular targets (thus not orally
available) but will cause irreversible cell membrane
damage especially at high concentrations. Although
the findings appear to be applicable to compounds
with minimal diversity in structure, the multivariate
information obtained from the RT-CES study could be
useful in the filtering out these types of compounds to
identify the most drug-like for further biological
testing.
Agostinho M, Banu A, Braunstein P, Welter R, Morise X (2006)
New palladium complexes with phosphino- and phos-
phinitopyridine ligands. Acta Crystallogr C 62:m81–m86.
doi:10.1107/S0108270106001296
Alcock NW, Moore P, Lampe PA, Mok K (1982) Crystal and
molecular structures of two complexes of diphenyl (2-
pyridyl) phosphine (L):[AuClL] and [Ag2Cl2L3]. J Chem
Soc Dalton Trans. doi:10.1039/dt9820000207
Altaf M, Monim-ul-Mehboob M, Seliman AAA, Sohail M,
Wazeer MIM, Isab AA, Li L, Dhuna V, Bhatia G, Dhuna K
(2015) Synthesis, characterization and anticancer activity
of gold(I) complexes that contain tri-tert-butylphosphine
and dialkyl dithiocarbamate ligands. Eur J Med Chem.
doi:10.1016/j.ejmech.2015.03.019
Altomare A, Cascarano G, Giacovazzo C, Guagliardi A (1993)
Completion and refinement of crystal structures with
SIR92. J Appl Crystallogr 26:343–350
Atienza JM, Zhu J, Wang X, Xu X, Abassi Y (2005) Dynamic
monitoring of cell adhesion and spreading on microelec-
tronic sensor arrays. J Biomol Screen 10:795–805
Baenziger N, Bennett W, Soborofe D (1976) Chloro (triph-
enylphosphine) gold (I). Acta Crystallographica B Struct
Crystallogr Cryst Chem 32:962–963
Beraldo H, Gambino D (2004) The wide pharmacological ver-
satility of semicarbazones, thiosemicarba-zones and their
metal complexes. Mini Rev Med Chem 4:31–39
Acknowledgments The authors would like to thank the
Technology Innovation Agency (TIA) and the University of
Pretoria for financial support. A. Elkhadir is grateful to the
Organization for Women in Science for the Developing World
(OWSD) formerly Third World Organization for Women in
Science (TWOWS) and University of Johannesburg for
financial support.
Berners-Price SJ, Bowen RJ, Galettis P, Healy PC, McKeage MJ
(1999) Structural and solution chemistry of gold (I) and
silver (I) complexes of bidentate pyridyl phosphines: se-
lective antitumour agents. Coord Chem Rev 185:823–836
Boyd MR (1989) Status of the NCI preclinical antitumor drug
discovery screen. In: DeVita VT, Hellman S, Rosenberg
SA (eds) Cancer: principles and practice of oncology up-
dates, vol 3. J. B. Lippincott, Philadelphia, pp 1–12
Boyd JM, Huang L, Xie L, Moe B, Gabos S, Li XF (2008) A
cell-microelectronic sensing technique for profiling cyto-
toxicity of chemicals. Anal Chim Acta 615:80–87. doi:10.
1016/j.aca.2008.03.047
Conflict of interest The authors declare that they have no
conflict of interest.
Appendix: online resource data
Bruker A (2009) APEX2—software suite for crystallographic
programs. Bruker AXS Inc., Madison
CCDC numbers \884806[ and \960995[ contains
the online crystallographic data for 2 and 1a, respec-
tively. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or
from the Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 1EZ, UK; fax: (?44)
1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
Calhorda MJ, Ceamanos C, Crespo O, Gimeno MC, Laguna A,
Larraz C, Vaz PD, Villacampa MD (2010) Heteropolynu-
clear gold complexes with metallophilic interactions:
modulation of the luminescent properties. Inorg Chem
49:8255–8269
Denizot F, Lang R (1986) Rapid colorimetric assay for cell
growth and survival. Modifications to the tetrazolium dye
procedure giving improved sensitivity and reliability.
J Immunol Method 89:271–277
Derdeyn CA, Decker JM, Sfakianos JN, Wu X, O’Brien WA,
Ratner L, Kappes JC, Shaw GM, Hunter E (2000) Sensi-
tivity of human immunodeficiency virus type 1 to the fu-
sion inhibitor T-20 is modulated by coreceptor specificity
defined by the V3 loop of gp120. J Virol 74:8358–8367
Donato MT, Lahoz A, Castell JV, Gomez-Lechon MJ (2008)
Cell lines: a tool for in vitro drug metabolism studies. Curr
Drug Metab 9:1–11
References
Abassi YA, Xi B, Li N, Ouyang W, Seiler A, Watzele M,
Kettenhofen R, Bohlen H, Ehlich A, Kolossov E, Wang X,
123

Biometals
Farrugia LJ (1997) ORTEP-3 for windows-a version of ORTEP-
III with a graphical user interface (GUI). J Appl Crystallogr
30:565
Farrugia LJ (1999) WinGX suite for small-molecule single-
crystal crystallography. J Appl Crystallogr 32:837–838
Fonteh PN (2011) Gold Compounds with Anti-HIV and Im-
munomodulatory Activity. PhD thesis. University of Pre-
toria, Pretoria, South Africa
Fonteh P, Meyer D (2009) Novel gold (I) phosphine compounds
inhibit HIV-1 enzymes. Metallomics 1:427–433
Fonteh PN, Keter FK, Meyer D, Guzei IA, Darkwa J (2009)
Tetra-chloro-(bis-(3,5-dimethylpyrazolyl)methane)-
gold(III) chloride: an HIV-1 reverse transcriptase and
protease inhibitor. J Inorg Biochem 103:190–194. doi:10.
1016/j.jinorgbio.2008.10.001
McKeage MJ, Berners-Price SJ, Galettis P, Bowen RJ, Brouwer
W, Ding L, Zhuang L, Baguley BC (2000) Role of lipo-
philicity in determining cellular uptake and antitumour
activity of gold phosphine complexes. Cancer Chemother
Pharmacol 46:343–350
Monim-ul-Mehboob M, Altaf M, Fettouhi M, Isab AA, Wazeer
MI, Shaikh MN, Altuwaijri S (2013) Synthesis, spectro-
scopic characterization and anti-cancer properties of new
gold (III)–alkanediamine complexes against gastric, prostate
and ovarian cancer cells; crystal structure of [au\ sub[
2(\i[ pn)\ sub[ 2(cl)\ sub[ 2] cl\ sub[ 2ꢀH\ sub[ 2
O. Polyhedron 61:225–234
Mosmann T (1983) Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity
assays. J Immunol Methods 65:55–63
Fonteh PN, Keter FK, Meyer D (2011) New bis(thiosemicar-
bazonate) gold(III) complexes inhibit HIV replication at
cytostatic concentrations: potential for incorporation into
virostatic cocktails. J Inorg Biochem 105:1173–1180.
doi:10.1016/j.jinorgbio.2011.05.011
Navarro M (2009) Gold complexes as potential anti-parasitic
agents. Coord Chem Rev 253:1619–1626. doi:10.1016/j.
ccr.2008.12.003
Nobili S, Mini E, Landini I, Gabbiani C, Casini A, Messori L
(2010) Gold compounds as anticancer agents: chemistry,
cellular pharmacology, and preclinical studies. Med Res
Rev 30:550–580
Gandin V, Fernandes AP, Rigobello MP, Dani B, Sorrentino F,
¨
Tisato F, Bjornstedt M, Bindoli A, Sturaro A, Rella R
´
Noren B, Oskarsson A, Svensson C (1997) Cis/trans influences
(2010) Cancer cell death induced by phosphine gold
(I) compounds targeting thioredoxin reductase. Biochem
Pharmacol 79:90–101
on bond distances in square-planar complexes. Crystal
structure of trans-dichlorobis (tetrahydrothiophene) palla-
dium (II) and trans-dichlorobis (tetrahydrothiophene)
platinum (II). Acta Chem Scand 51:289–294
Ghose AK, Viswanadhan VN, Wendoloski JJ (1998) Prediction
of hydrophobic (lipophilic) properties of small organic
molecules using fragmental methods: an analysis of
ALOGP and CLOGP methods. J Phys Chem A 102:3762–
3772
Giaever I, Keese CR (1984) Monitoring fibroblast behavior in
tissue culture with an applied electric field. Proc Natl Acad
Sci 81:3761–3764
Hamid R, Rotshteyn Y, Rabadi L, Parikh R, Bullock P (2004)
Comparison of alamar blue and MTT assays for high
through-put screening. Toxicol In Vitro. doi:10.1016/j.tiv.
2004.03.012
Haselsberger K, Peterson DC, Thomas DG, Darling JL (1996)
Assay of anticancer drugs in tissue culture: comparison of a
tetrazolium-based assay and a protein binding dye assay in
short-term cultures derived from human malignant glioma.
Anticancer Drugs 7:331–338
Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G (2011) Cell
death assays for drug discovery. Nat Rev Drug Discov
10:221–237. doi:10.1038/nrd3373
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997)
Experimental and computational approaches to estimate
solubility and permeability in drug discovery and devel-
opment settings. Adv Drug Deliv Rev 23:3–25. doi:10.
1016/S0169-409X(96)00423-1
Lobell M, Sivarajah V (2003) In silico prediction of aqueous
solubility, human plasma protein binding and volume of
distribution of compounds from calculated pKa and
AlogP98 values. Mol Divers 7:69–87
Lundholt BK, Scudder KM, Pagliaro L (2003) A simple tech-
nique for reducing edge effect in cell-based assays.
J Biomol Screen 8:566–570
Okada T, Patterson BK, Ye SQ, Gurney ME (1993) Aurothio-
lates inhibit HIV-1 infectivity by gold(I) ligand exchange
with a component of the virion surface. Virology
192:631–642. doi:10.1006/viro.1993.1079
Palatinus L, Chapuis G (2007) Superflip—a computer program
for the solution of crystal structures by charge flipping in
arbitrary dimensions. J Appl Crystallogr 40:786–790
Platt E, Wehrly K, Kuhmann S, Chesebro B, Kabat D (1998) 819
effects of CCR5 and CD4 cell surface concentrations on
infections by 820 macrophage tropic isolates of human
immunodeficiency virus type 1. J Virol 821:2855–2864
Puckett CA, Ernst RJ, Barton JK (2010) Exploring the cellular
accumulation of metal complexes. Dalton Trans 39:1159–
1170
Ray S, Mohan R, Singh JK, Samantaray MK, Shaikh MM,
Panda D, Ghosh P (2007) Anticancer and antimicrobial
metallopharmaceutical agents based on palladium, gold,
and silver N-heterocyclic carbene complexes. J Am Chem
Soc 129:15042–15053
Ru¨lke R, Han I, Elsevier C, Vrieze K, Van Leeuwen P, Roobeek
C, Zoutberg M, Wang Y, Stam C (1990) New neutral and
cationic methylpalladium (II) complexes containing triden-
tate nitrogen ligands. synthesis, reactivity and x-ray crystal
structure of r-\ i[ N-2-(\ i[ N-isopropylcarbaldimino)-
6-(\i[N-isopropylcarbaldimino)-r-\i[N-pyridyl}(chloro)
methylpalladium (II) and [{r\ sup[ 3-\ i[ N,\ i[ N0,
\ i[ N00-2, 20: 60, 200-terpyridyl methylpalladium (II)]
chloride dihydrate. Inorg Chim Acta 169:5–8
Rupesh K, Deepalatha S, Krishnaveni M, Venkatesan R, Jay-
achandran S (2006) Synthesis, characterization and in vitro
biological activity studies of Cu–M (M = cu\ sup[ 2,
co\ sup[ 2, ni\ sup[ 2, mn\ sup[ 2, zn\ sup[ 2)
bimetallic complexes. Eur J Med Chem 41:1494–1503
McGuinness R (2007) Impedance-based cellular assay tech-
nologies: recent advances, future promise. Curr Opin
Pharmacol. doi:10.1016/j.coph.2007.08.004
123

Biometals
Shapiro DL, Masci JR (1996) Treatment of HIV associated
psoriatic arthritis with oral gold. J Rheumatol 23:1818–
1820
Shaw CF III, Isab AA, Hoeschele JD, Starich M, Locke J,
Schulteis P, Xiao J (1994) Oxidation of the phosphine from
the auranofin analog, triisopropylphosphine (2,3,4,6-tetra-
O-acetyl-1-thio-beta-D-glucopyranosato-S) gold (I), via a
protein-bound phosphonium intermediate. J Am Chem Soc
116:2254–2260
Sheldrick GM (2008) A short history of SHELX. Acta Crys-
tallogr A Found Crystallogr 64:112–122
Solly K, Wang X, Xu X, Strulovici B, Zheng W (2004) Appli-
cation of real-time cell electronic sensing (RT-CES)
technology to cell-based assays. Assay Drug Dev Technol
2:363–372
tumor-targeted toxins and saponins using impedance
measurement. Biosens Bioelectron 35:503–506
Uson R, Laguna A, Laguna M, Briggs D, Murray H, Fackler J
(1989) (Tetrahydrothiophene) gold (I) or gold (III) com-
plexes. Inorg Synth 26:85–91
Wang C, Shih W, Chang HC, Kuo Y, Hung W, Ong T, Li W
(2011) Preparation and characterization of amino-linked
heterocyclic carbene palladium, gold, and silver complexes
and their use as anticancer agents that act by triggering
apoptotic cell death. J Med Chem 54:5245–5249
Wei X, Decker JM, Liu H, Zhang Z, Arani RB, Kilby JM, Saag
MS, Wu X, Shaw GM, Kappes JC (2002) Emergence of
resistant human immunodeficiency virus type 1 in patients
receiving fusion inhibitor (T-20) monotherapy. Antimicrob
Agents Chemother 46:1896–1905
Sun RW, Che C (2009) The anti-cancer properties of gold (III)
compounds with dianionic porphyrin and tetradentate li-
gands. Coord Chem Rev 253:1682–1691
Takeuchi Y, McClure MO, Pizzato M (2008) Identification of
gammaretroviruses constitutively released from cell lines
used for human immunodeficiency virus research. J Virol
82:12585–12588. doi:10.1128/JVI.01726-08
Thakur M, Mergel K, Weng A, Frech S, Gilabert-Oriol R,
Bachran D, Melzig MF, Fuchs H (2012) Real time
monitoring of the cell viability during treatment with
Xing JZ, Zhu L, Jackson JA, Gabos S, Sun XJ, Wang XB, Xu X
(2005) Dynamic monitoring of cytotoxicity on microelec-
tronic sensors. Chem Res Toxicol 18:154–161. doi:10.
1021/tx049721s
Xing JZ, Zhu L, Gabos S, Xie L (2006) Microelectronic cell sensor
assay for detection of cytotoxicity and prediction of acute
toxicity. Toxicol In Vitro. doi:10.1016/j.tiv.2005.12.008
Zhang Y, Hess EV, Pryhuber KG, Dorsey JG, Tepperman K, Elder
RC (1995) Gold binding sites in red blood cells. Inorg Chim
Acta 229:271–280. doi:10.1016/0020-1693(94)04254-S
123