Benzimidazol-2-ylidene gold(I) complexes are thioredoxin reductase inhibitors with multiple antitumor properties

Article abstract of DOI:10.1021/jm100801e

Gold(I) complexes such as auranofin have been used for decades to treat symptoms of rheumatoid arthritis and have also demonstrated a considerable potential as new anticancer drugs. The enzyme thioredoxin reductase (TrxR) is considered as the most relevan

Full text of DOI:10.1021/jm100801e

8608 J. Med. Chem. 2010, 53, 8608–8618
DOI: 10.1021/jm100801e
Benzimidazol-2-ylidene Gold(I) Complexes Are Thioredoxin Reductase Inhibitors with
Multiple Antitumor Properties
Riccardo Rubbiani,† Igor Kitanovic,‡ Hamed Alborzinia,‡ Suzan Can,‡ Ana Kitanovic,‡ Liliane A. Onambele,§
Maria Stefanopoulou, Yvonne Geldmacher, William S. Sheldrick, Gerhard Wolber,^ Aram Prokop,§
‡
Stefan Wolfl, and Ingo Ott*
,†
€
†
€
Institute of Pharmaceutical Chemistry, Technische Universitat Braunschweig, Beethovenstrasse 55, 38106 Braunschweig, Germany,
€
‡
Institut fu€r Pharmazie und Molekulare Biotechnologie, Ruprecht-Karls-Universitat Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg,
Germany, §Department of Paedriatric Oncology, Childrens Hospital Cologne, Amsterdamer S^trasse 59, 50735 Cologne, Germany,
€
€
Lehrstuhl fu€r Analytische Chemie, Ruhr-Universitat Bochum, 44780 Bochum, Germany, and Institute of Pharmacy, Freie Universitat Berlin,
€
Konigin-Luise-Strasse 2 þ 4, 14195 Berlin, Germany
Received June 29, 2010
Gold(I) complexes such as auranofin have been used for decades to treat symptoms of rheumatoid
arthritis and have also demonstrated a considerable potential as new anticancer drugs. The enzyme
thioredoxin reductase (TrxR) is considered as the most relevant molecular target for these species. The
here investigated gold(I) complexes with benzimidazole derived N-heterocyclic carbene (NHC) ligands
1a-4a represent a promising class of gold coordination compounds with a good stability against the
thiol glutathione. TrxR was selectively inhibited by 1a-4a in comparison to the closely related enzyme
glutathione reductase, and all complexes triggered significant antiproliferative effects in cultured tumor
cells. More detailed studies on a selected complex (2a) revealed a distinct pharmacodynamic profile
including the high increase of reactive oxygen species formation, apoptosis induction, strong effects on
cellular metabolism (related to cell surface properties, respiration, and glycolysis), inhibition of
mitochondrial respiration and activity against resistant cell lines.
Introduction
Cisplatin and other platinum species belong to the block-
busters of anticancer drugs sold worldwide. However, remaining
problems such as severe side effects and resistance phenomena
trigger an increasing demand for novel innovative drugs with
a mode of action differing from that of the platinum generation
of cancer chemotherapeutics.1-3 In recent years especially,
gold complexes have attracted major attention due to their
enzyme inhibiting acitivities related to cancer development
and progression (e.g., interaction with cathepsins, tyrosine
phosphatases, or cyclooxygenases). The antirheumatic gold(I)
phosphine drug auranofin emergedas the lead compound also
for the class of antiproliferatively active gold species, and in
the recent years, an increasing number of bioactive complexes
(e.g., phosphine derivatives, gold carbene complexes, or sev-
eral gold(III) agents) have shown promising biological activity
(see Figure 1 for some relevant examples).4-12
Figure 1. Bioactive gold(I) species.
On the basis of the structural diversity of the reported
complexes and the huge amount of so far available biological
data, a common mode of action for gold complexes is unlikely
to exist. However, the enzyme thioredoxin reductase (TrxRa)
is nowadays considered as the most relevant target for bioactive
gold coordination compounds.5-7 Human TrxR is an enzyme
belonging to the antioxidant cellular network and involved in
many aspects of tumor pathophysiology (e.g., proliferation,
apoptosis, metastasis). It is an ubiquitaryNADPH-dependent
flavoprotein with a cysteine-cysteine bridge at the N-terminal
end and a selenocysteine-cysteine bridge at the C-terminal end
(also termed the interface domain of the protein), from which
it differs from the otherwise closely related enzyme glu-
tathione reductase (GR).13,14 Gold complexes derived from
the lead compound auranofin have demonstrated a considerable
selectivity for the inhibition of TrxR over GR or other struc-
turally similar enzymes.8,15,16 This selectivity is commonly
attributed to the higher affinity of gold for selenium (as present
*To whom correspondence should be addressed. Phone: þ49 531
3912743. Fax: +495313918456. E-mail: ingo.ott@tu-bs.de.
a Abbreviations: GR, glutathione reductase; LDH, lactate dehydro-
genase; NHC, N-heterocyclic carbene; TNB, 2-nitro-5-thiobenzoic acid;
ROS, reactive oxygen species; DTNB, 5,50-dithiobis-(2-nitrobenzoic
acid); TrxR, thioredoxin reductase.
r
pubs.acs.org/jmc
Published on Web 11/17/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8609
Figure 2. Left: overlay of TrxR (blue) and GR (green). Middle: organic residue of GOPI in GR. Hydrophobic contacts are indicated as yellow
spheres, the binding pocket surface is color-coded by hydrophobicity (yellow = hydrophobic, blue = hydrophilic). Right: structural features
of the novel target compounds.
in TrxR but not in GR) compared to sulfur and has also been
experimentally confirmed by using mutant forms of TrxR as
well as by advanced mass spectrometry studies on the C-term-
inal motif of TrxR.17,18
However, auranofin is strongly metabolized and much evidence
exists that it represents a prodrug for several bioactive meta-
bolites.7,19-21 For example, in serum, a conjugate with albumin
is readily formed under replacement of the thiolate ligand.19
So far this instability has prevented a more rational develop-
ment of novel gold(I) drugs, and it is thus not surprising that
the number of available structure-activity relationship stud-
ies on auranofin derivatives is rather limited.4,5,22,23
Interestingly, in recent years, many highly promising results
with novel gold(I) species have been reported, shedding also
more light on the issue of drug design.11,16,17,23-25 For example,
for the gold phosphol complex [1-phenyl-2,5-di(2-pyridyl)-
phosphole]AuCl (GoPI), which showed EC50 values in the
low nanomolar range against both TrxR and GR, a crystal
structure with GR was published. This provided an experimental
proof for the covalent binding of gold to relevant cysteine resi-
dues in GR and thereby opened also some options for a more
rational design of bioactive gold species.17,26 Additionally,
gold(I) NHC complexes developed by the group of Berners-
Price have shown exciting biological properties including the
inhibition of TrxR, selectivity for tumorous tissues over normal
tissue, apoptosis induction, or preferential binding to selenols
over thiols.25,27-29 NHCs are strong σ-donors stabilized by π-
backbonding, which can coordinate with metals to complexes
of comparable stability with gold(I) phosphines.30,31 Thus,
NHC gold compounds may also represent a type of bioorga-
nometallics exhibiting an enhanced stability under biologi-
cally relevant conditions.32 The general potential of NHC
ligands for the use in drug development is also reflected by an
increasing number of studies reporting formidable pharmaco-
logical properties for complexes with various metals (including,
e.g., silver, platinum) going back to first reports on antimicrobal
derivatives.32-38
Structural Considerations and Design Strategy of the Target
Compounds
For the rational design of new bioactive gold(I) NHC
coordination compounds, we used the recently published
crystal structure of human GR containing the gold phosphole
inhibitor GoPI as a starting point.17 An initial overlay of
human TrxR with human GR (see Figure 2, left) had con-
firmed the close structural analogy of both protein structures,
indicating thatthe observed binding sites of GoPIin GR could
also be used todesign potentinhibitorsofTrxR. Selectivityfor
TrxR over GR should finally be achieved due to the higher
affinity of the gold central atom to the selenocysteine residue
in TrxR compared to the cysteine residue in GR.
GR incubated with GoPI showed two mechanisms taking
place: a cross-linking of two cysteines in the active site by a
gold atom, which was set free upon complete ligand dissocia-
tion of GoPI and the covalent binding of a gold phosphole
moiety to the surface exposed Cys-284 under loss of a chloride
ligand (see Figure 2 middle).17 The existence of such structural
data opens some options for the structure based design of
related potential inhibitors and the development of novel
compounds might in turn also provide information on the
relevance of the Cys-284 binding site.
Evaluation of the structural features of the gold phosphole
attached to Cys-284 indicated that the two pyridine rings and
the cyclohexane ring annelated to the phosphol ring were
involved in hydrophobic interactions (mainly with Leu-183,
Leu-261, and Ileu-175, see Figure 2 middle), whereas the
phenyl ring was located outside the binding site. Relevant
polar interactions or hydrogen bonding to nearby residues of
the GR protein backbone could not be identified. Taking this
information into account, we designed a short series of gold(I)
NHC species which exhibit the following features: a central
benzimidazol-2-ylidene carbene core, substituents with varying
lipophilicity and surface volume for an improved interaction
with the binding site, and a chloride leaving group enabling
the necessary covalent binding (see Figure 2 right).
Motivated bythe greatpotentialofNHC complexes indrug
design and by our recent results with gold(I) species11,23,24 we
developed and studied a series of novel gold(I) NHC com-
plexes. The target compounds are based on a benzimidazol-2-
ylidene core and were designed based on available crystal
structure data of the gold phosphole GoPI in the active site of
GR (see below). The synthesis, stability (against inactivation
by glutathione), and the intensive biological investigation are
reported here and demonstrate a huge potential of this type of
bioorganometallics in medicinal chemistry.
Synthesis
The target compounds were prepared according to a con-
venient established procedure starting from benzimidazole
(see Scheme 1).27,39-41 Alkylation of the aromatic nitrogens
was achieved by refluxing the educt with an excess of the
respective alkyl halogenide in the presence of a mild base in
acetonitrile. The benzimidazol-2-ylidene gold complexes were
obtained by treating the benzimidazolium salts with Ag2O

8610 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Rubbiani et al.
(leading to the formation of an intermediate silver complex)
followed by a ligand exchange reaction with chlorodimethyl-
sulfide gold(I). The target compounds were finally purified by
37 ꢀC was confirmed exemplarily for 2a (see Supporting Infor-
mation, Figure S1).
For the subsequent experiments on possible inactivation by
glutathione, we used the 5,50-dithiobis-(2-nitrobenzoic acid)
(DTNB, also known as the Ellmans reagent), which can be
used to quantify the thiol content of biological samples based
on a rapid and stochiometric reaction but which also allows to
monitor the activity of NADPH metabolizing enzymes such
as TrxR.15,42-44 In contrast to enzymatic reduction assays
where one equivalent of DTNB is reduced under formation of
two equivalents of of 2-nitro-5-thiobenzoic acid (TNB, Figure 3a)
DTNB reacts with a thiol to a mixed disulfide and one
equivalent of TNB (see Figure 3b), which can finally be
measured photometrically. The formation of gold complexes
at the cysteine thiol of reduced glutathione (or a metal
mediated oxidation of the tripeptide) would lower the avail-
able amount of free thiol able to react with DTNB. This assay
thus provides an efficient tool to screen the stability of gold
complexes against inactivation by glutathione.
1
filtration over Celite and characterized by H NMR, mass
spectrometry, and elemental analysis. A characteristic feature
confirming the complex formation was the disappearing ofthe
carbene proton signal in 1H NMR spectra upon coordination.
13C NMR spectra were exemplarily taken for 2 and 2a and
again confirmed complex formation as the signal of the carbene
carbon was shifted from 141 ppm in 2 to 177 ppm in 2a.
Reaction with Glutathione
For inactivation of the anticancer drug cisplatin and many
other metal based drugs, thiols such as the tripeptide glu-
tathione play an important role. Similarly, the gold(I) phos-
phine lead compound auranofin is biologically processed and
metabolized in thiol ligand exchange processes.7,19,20 As metal
NHC complexes supposedly represent biologically stable co-
ordination compounds, it was of interest to study their inter-
action with glutathione under physiological conditions in
comparison to relevant gold(I) phosphine complexes. Initially,
the stability of gold(I) NHC complexes in buffer solution at
For the experiments, we incubated an excess of the respec-
tivegold complex withreduced glutathione at37ꢀC for 20and
60 min. Chloro(dimethylsulfide)gold(I) (Me2SAuCl) was
used as a positive control and as expected led to an almost
complete reduction of TNB product formation under the
chosen conditions (see Figure 4).
Scheme 1. Synthesis Procedure for 1a-4aa
Quite astonishingly, the gold(I) NHC species 1a-4a did not
influence the reaction significantly and can thus be considered
as sufficiently stable against thiols under biologically relevant
conditions, a property highly desirable in the drug development
of novel metal coordination compounds. Only 1a showed a
minor inhibition of approximately 10% after 60 min. This
might be attributed to the lower steric hindrance of an attack
at the gold center by the less bulky methyl side chains. In fact,
ligand replacement processes upon reaction with cysteine over
a (a) Alkylhalide, K2CO3, CH3CN, reflux, 6 h; (b) Ag2O in CH2Cl2,
6 h; (c) chloro(dimethylsulfide)gold(I), 4 h.
Figure 3. Possible reactions taking place with the Ellmans reagent: (a) reduction by NADPH metabolizing enzymes; (b) disulfide bond
exchange with thiols (here reduced glutathione).
Figure 4. Interaction of gold(I) complexes with glutathione and DTNB; left, 20 min exposure; right, 60 min exposure.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8611
an extended period of time have been reported for gold(I)
NHC derivatives.25
that the gold(I) center is necessary for the inhibition of the
enzymes and support the assumption of the preferential bind-
ing to the selenocysteine residue present in the active site of
TrxR. One striking feature observed in these enzymatic
studies is that the strong selectivity for TrxR inhibition over
GR inhibition, which had been previously described for
various gold phosphine derivatives,15,16 was also found for
the here reported series of gold(I) NHC complexes (at least
20-fold lower values for TrxR inhibition compared to GR
inhibition).
With the exception of the diphenylmethylene derivate 4a,
which exhibited a comparably low solubility in the used assay
buffer that might explain its significantly lower activity, the
gold(I) NHC derivatives displayed strong EC50 values against
TrxR well below 0.5 μM. Higher activity, however, was noted
for the investigated gold(I) phosphine derivatives, which also
represent a kinetically more reactive class ofgold(I) complexes
(as studied in more detail above).
Unexpectedly, gold(I) phosphine complexes led to a strong
increase in TNB release during the reaction, which was most
marked after the shorter exposure period (20 min). Due to
stochiometric reasons, an exceeding of 100% is not possible
(see Figure 3b) and therefore the occurrence of an additional
breaking/reduction of the DTNB disulfide bond had to be
taken into account. For auranofin, it had been reported that
after an initial formation of an albumin-auranofin adduct, in
which the thiocarbohydrate was replaced by a cysteine of
albumin, the phosphine neutral ligand dissociated off the gold
atom and was oxidized nonenzymatically to the respective
phosphine oxide.7,19,20 Thus, an analogous “activation” by
glutathione and subsequent oxidation of the phosphine moi-
ety might in turn lead to the reduction of DTNB, resulting in
the observed “overshot” of TNB. In fact the formation of
several intermediates related to a breakage of the DTNB di-
sulfide bond (as well as coordinative bonds) could be observed
in experiments on triphenylphosphine gold(I) chloride
(TPPAuCl) using 31P NMR and MS/MS spectroscopy (see
Supporting Information, Figures S2 and S3, Scheme S1).
Effects on Cell Proliferation, Apoptosis, ROS Formation, Cell
Metabolism and Mitochondrial Respiration
The triggering of antiproliferative effects by the target
coordination compounds was investigated in various tumor
cell lines (namely, MCF-7 human breast adenocarcinoma,
HT-29 and HCT-116 colon carcinoma, and HEP-G2 hepato-
cellular carcinoma). Relevant activities could be noted for all
the complexes in these cell lines. The observed IC50 values (in
the range of 4.6-14.9 μM with the exception of lower activity
of 4a in HCT-116 and HEP-G2 cells) were generally within the
same order of magnitude as noted for gold(I) phosphine
species (IC50 values in the range of 1-5 μM in the used
assay11,23,24). These results are in good agreement with other
reports on bioactive gold carbene species, which reached
activities in the low micromolar range.25,45 The inactivity
(IC50 values >100 μM) of the free benzimidazolium salt 2
confirmed that the gold center was necessary to obtain bioac-
tive species. Interestingly, the tumor selective behavior of other
gold(I) NHC complexes25 could not be confirmed for 1a-4a as
comparative experiments in nontumorigenic cell lines (HEK-
293 human embryonic kidney cells and HFF human foreskin
fibroblasts) afforded similar activities (IC50 values in the range
of 7.6-32 μM, see Supporting Information, Table S1).
Inhibition of the Disulfide Reductases TrxR and GR
The inhibition of the activity of the target enzyme TrxR and
the structurally closely related GR by the NHC gold complexes
1a-4a, the benzimidazolium salt 2 (as negative control) as well
as the gold(I) phosphine species auranofin, triethylphosphine
gold(I) chloride (TEPAuCl), and TPPAuCl (as positive controls)
was performed using the isolated enzymes.11 Whereas the non-
gold-containing compound 2 was devoid of any activity against
both enzymes strong inhibitory effects against TrxR (EC50
values between 0.009 and 4.0 μM) and more moderate effects
against GR (4.2 to 94 μM) could be noted for all gold(I)
complexes (see Table 1). Overall, these results clearly demonstrate
Table 1. Inhibition of TrxR and GR; Results Are Expressed As Means
(( Error) of at Least Two Independent Experiments
EC50 TrxR (μM)
EC50 GR (μM)
selectivity (x-fold)
auranofin
0.009 (( 0.000)
0.037 (( 0.005)
0.256 (( 0.002)
>50 μM
15 (( 0.1)
7.9 (( 0.4)
4.2 (( 0.7)
>50 μM
1666
213
16
TEPG
TPPG
2
To evaluate the biological potential of benzimidazol-2-
ylidene gold(I) complexes in more detail, we investigated 2a
exemplarily for more specific chemotherapeutic properties in
various cancer and leukemia cell lines. Video phase contrast
microscopic imaging of HT-29 cells exposed to 7.0 μM of 2a
1a
2a
0.399 (( 0.040)
0.361 (( 0.040)
0.465 (( 0.006)
4.0 (( 1.0)
30 (( 2.5)
8.7 (( 0.0)
9.3 (( 1.7)
94 (( 15)
75
24
20
23
3a
4a
Figure 5. Left: induction of ROS formation by 2 and 2a in Jurkat cells after 48 h exposure. Similar results were observed after 24 h exposure
(see Supporting Information, Figure S5); right: annexin/PI assay for Jurkat cells exposed to 2a for 48 h. In the Annexin/PI assay, vital cells
(double negative) can be distinguished from early apoptotic (AnnVþ), late apoptotic (AnnV/PIþ), and necrotic (PIþ) cells.48 CMPT served as
a positive control.

8612 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Rubbiani et al.
Figure 6. Influence of 2a on DNA fragmentation (72 h exposure, left) and viability (2 h exposure, LDH release assay, right) in BJAB cells. 2
showed no relevant effects in both assays (see Supporting Information, Figure S7 and Figure S8). Values are given in % of control ( SD (n = 3).
Table 2. IC50 Values for Antiproliferative Effects in MCF-7, HT-29, HCT-116, and HEP-G2 Cells, Results Are Expressed As Means (( Error) of at
Least Two Independent Experiments
IC50 MCF-7 (μM)
IC50 HT-29 (μM)
IC50 HCT-116 (μM)
IC50 HEP-G2 (μM)
2
>100
>100
>100
>100
1a
2a
3a
4a
7.5 (( 0.6)
4.6 (( 0.0)
10.2 (( 0.1)
10.3 (( 0.7)
13.3 (( 4.4)
6.4 (( 2.0)
11.8 (( 1.9)
12.3 (( 3.3)
6.7 (( 0.6)
8.4 (( 2.2)
10.0 (( 0.4)
24.6 (( 2.8)
4.9 (( 0.8)
11.2 (( 1.6)
14.9 (( 3.9)
60.7 (( 10.6)
Figure 7. Influence of 2 and 2a on cell impedance (a), cell respiration (b) and acidification rate (c) in MCF-7 cells; RM, “running medium”
(without compound).
for 14 h (Figure S4 and the video files in the Supporting
Information) showed the rounding, detachment, and “dying”
of single cells, but the cell biomass was overall not strongly
reduced during this period. Comparable effects were also
observed in an untreated control culture. Therefore, it can
be concluded that the above-described effects on cultured
tumor cells are rather related to a proliferation inhibition than
to a direct cytotoxic effect on the adherent cell monolayer.
Reactive oxygene species (ROS) are products of the phy-
siological mitochondrial cell metabolism and are involved in
cellular redox homeostasis. Their induction indicates a per-
turbation of the cellular antioxidant defense system. In good
agreement with the results of the proliferation assay, concen-
trations higher than 2.5 μM of 2a strongly induced cellular
ROS levels (see Figure 5 left). In contrast, 2 did not trigger
ROS production. This pattern correlates also well with the
above notion that 2a selectively affected the activity of the
redox relevant enzyme TrxR, whereas 2 had no effect on
enzymatic activities. The ability to inhibit the activities of
disulfide reductases also under cell culture conditions was
confirmed for 2a and afforded an EC50 value of 19.1 μM (see
Supporting Information, Figure S6).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8613
Figure 8. Respiration of freshly isolated mouse liver mitochondria. Mitochondrial activity leads to a decrease in oxygen saturation, which
decreases over time (control). Inhibition of mitochondrial activity blocks oxygen consumption, resulting in continuous high oxygen
concentration (rotenone, an inhibitor of respiratory chain complex I); decoupling of respiration by carbonyl cyanide 3-chlorophenylhydrazone
(CCCP) leads to increased oxygen consumption. The gold complex 2a leads to a concentration-dependent inhibition of mitochondrial
respiration while treatment with 2 shows no effect compared to the untreated control; controls: “blank”, buffer without test compound;
“DMF”, buffer containing the organic solvent but no compound; “respiration buffer”, experiment without mitochondria.
Figure 9. Wildtype and daunorubicine resistant (Nalm/DR) Nalm-6 cells were treated with different concentrations of 2a and daunorubicine
(DR) for 72 h and DNA fragmentation was measured. Relevant doses of DR did not cause a significant DNA fragmentation in Nalm/DR cells.
For the treatment with 2a, both Nalm-6 and Nalm/DR were affected (IC50 values: 2.9 μM in Nalm-6 and 3.1 μM in Nalm/DR). Cells treated
with blank or DMSO containing cell culture media served as controls. Values are given as percentages of cells with hypodiploid DNA ( sd (n = 3).
Experiments on the apoptosis/necrosis inducing activity of
2a by the Annexin/propidium iodide (PI) assay showed a
strong reduction of vital cells accompanied by a significant
relative increase of apoptotic cells in concentrations of 2.5 μM
and higher (see Figure 5 right). Additionally, DNA fragmen-
tation, another marker for apoptosis, was strongly increased
after exposure to 2a but not after incubation with 2 (see Figure 6
left).
Short exposure (2 h) lactate dehydrogenase (LDH) release
experiments (see Figure 6, right) at higher concentrations
(>10 μM) of 2a showed a relevant loss of cell vitality, which
indicated that first effects took place within a rather short time
frame. In overall correlation with its inactivity in the prolif-
eration experiments (see Table 2), 2 did not influence cell
viability in this assay up to the highest concentration investi-
gated (100 μM).
cells started to decrease in a concentration dependent manner
after an exposure of approximately 7 h to 2a. This indicated
morphological changes of the cells, changes in cell membranes,
cell-cell contacts, and cellular adhesion, reflecting the induc-
tion and onset of cell death (see Figure 7a). Immediate effects
of the gold species were reduced oxygen consumption and an
increased glycolysis starting at the same time. The latter only
increased initially, suggesting a compensatory enhanced glycol-
ysis to compensate for the reduced respiration (see Figure 7b,c).
After longer exposure also the acidification rate decreased in a
concentration dependent manner, which can be ascribed to
the “dying” of the cells. Interestingly, there was no recovery of
cellular metabolism when 2a was withdrawn after 24 h, which
indicates that the effects were irreversible. Compound 2
triggered only minor effects at higher concentration on cell
impedance but was inactive otherwise. This again demon-
strates the importance of the gold(I) central atom.
Next the influence of 2 and 2a on cellular metabolism was
monitored online by use of a metabolic sensor chip analysis
system, which allows the evaluation of the impedance of the
cell layer and the respiration rate (oxygen consumption) as
well as the acidification rate (glycolysis) of living cells over an
extended time span (see Figure 7).46,47 The impedance of the
As the inhibition of TrxR should lead to antimitochondrial
effects, which have also been reported for an increasing
number of gold metallodrugs,25,29 we evaluated the effects
of 2 and 2a on mitochondrial respiration of isolated mice liver
mitochondria (see Figure 8). In this assay, the oxygen saturation

8614 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Rubbiani et al.
of a buffer medium is lowered by respirating (functionally
active) mitochondria. Impairment of mitochondrial vital fun-
ctions will antagonize this effect. As expected, exposure to 2a
led to a concentration dependent decrease of mitochondrial
respiration while 2 remained again without effect.
(as evidenced by a decrease of mitochondrial respiration).
Cellular metabolism studies on the influence on cell impe-
dance, respiration, and acidification rate indicated that the
described effects on cell physiology were induced within a
rather short time frame of a few hours and were generally
irreversible. In analogy, LDH release studies showed that cell
viability could be affected after short exposure to 2a at higher
concentrations. It should also be noted that the free ligand 2
was ineffective in all comparative experiments, which con-
firmed that the presence of the gold(I) center was necessary to
obtain compounds with the described bioactivities. Experi-
ments in P-glycoprotein overexpressing daunorubicine, vin-
cristine, and doxorubicine resistant cell lines indicated that
the here investigated type of bioorganometallics might find
also application in the treatment of certain drug resistant
malignancies.
Effects on Drug Resistant Cell Lines
Drugresistancephenomena have a majorimpact oncurrent
therapy regimens and cause frequent treatment failures. The
major reasons are found with alterations in drug transport
processes or the up/down regulation of relevant metabolizing
enzymes or molecular targets, thus causing a complex multi-
factorial event. Novel compounds with different chemical
structures and biological properties in some cases can address
these highly relevant issues.
Here we studied the effects of 2a on the apoptosis induction
in wild type and doxorubicin, daunorubicin, and vincristin
resistant BJAB and Nalm-6 leukemia cells. Western blot studies
confirmed the overexpression of P-glycoprotein in the resistant
cells (see Supporting Information, Figure S9). The most marked
effects of 2a were observed in daunorubicin resistant Nalm-6
cells, where 2a caused in the whole investigated concentration
range (1.0-15.0 μM) the same extent of DNA fragmentation
as in the respective wild type cells (see Figure 9). In vincristine
resistant Nalm-6 cells, similar effects were observed at con-
centrations above 7.5 μM of 2a (see Supporting Information,
Figure S10). In the resistant types of BJAB cells, however, the
resistance effects could only be partially reversed (see Sup-
porting Information, Figures S11 and S12).
Overall, the here observed overall pharmacodynamic pat-
tern of gold(I) NHC species exhibits several important key
features of state-of-the-art novel anticancer drugs, suggesting
further development of the here studied class of gold(I) NHC
complexes.
Experimental Section
General. All reagents and the solvents were used as received
1
from Sigma, Aldrich, or Fluka. H NMR and 13C NMR were
recorded on a Bruker DRX-400 AS NMR System, MS spectra
were recorded on a Finnigan MAT 4515. The purity of the target
compounds (>95%) was confirmed by elemental analysis
(Flash EA112, Thermo Quest Italia). For all compounds under-
going biological evaluation, the experimental values differed less
than 0.5% from the calculated ones.
Molecular Modeling. As a starting point for rational design,
we used the X-ray structure of GR complexed with the GOPI
residue 2-(2-phenyl-3-pyridin-2-yl-4,5,6,7-tetrahydro-2H-iso-
phosphoindol-1-yl)pyridine (PDB entry 1AAQ) and derived a
3D pharmacophore using the software LigandScout 3.0 as shown
in Figure 2.17,50,51 LigandScout identified lipophilic contacts to
Leu261, Leu183, and Ile175, which served as a basis for assump-
tions leading to the new compounds presented in this work. The
3D overlay of TrxR (2CFY) and GR (2AAQ) was performed
using the molecular modeling package MOE (Molecular Oper-
ating Environment, version 2009.10, Chemical Computing
Group, Montreal, Canada) and is based on a sequence align-
ment of the two proteins with a subsequent 3D overlay. The
alignment confirmed the close relationship between TrxR and
GR described by Sandalova et al. previously.52
Synthesis. General Procedure for Synthesis of the Benzimida-
zolium Halide Salts. Benzimidazole (0.118 g, 1.0 mmol), the
respective alkyl halogenide (3.0 mmol) and K2CO3 (0.138 g, 1.0
mmol) were heated under reflux conditions in acetonitrile for
8 h. The solvent of the resulting suspension was removed under
reduced pressure, and the residue was resuspended in dichloro-
methane and filtered to remove the formed potassium halo-
genide. The filtrate was evaporated under reduced pressure, the
residue was resuspended in tetrahydrofuran and filtered to give
the pure product.
Conclusions
A series of benzimidazol-2-ylidene gold(I) complexes was
prepared, structurally characterized, and biologically investi-
gated. Stability studies against inactivation by the tripeptide
glutathione showed that the here presented gold(I) NHC
complexes 1a-4a remained rather unaffected. The comparable
strong “thiol-based” metabolism of gold(I) phosphines such
as auranofin has so far hampered the development of novel
drug candidates out of this class.19,20,49 Accordingly, the choice
of NHC ligands probably provides an useful strategy for
gold(I) drugdesignin terms of an enhancedbiologicalstability
and also more predictable drug-target interactions.
Studies on the inhibition of disulfide reductases (see Table 2)
pointed overall to a strong and selective inhibition of the
selenocysteine containing TrxR and confirmed this enzyme as
one of the major targets for the here studied gold(I) com-
plexes. The interaction with GR (and the putative Cys284
binding site) supposedly play a role only at higher exposure
concentrations. This pattern is also in good agreement with
the low reactivity against the thiol of reduced glutathione.
Concerning the enzymatic inhibition studies, this is to the best
ofour knowledge the first reportprovidingEC50 values for the
direct and selective TrxR inhibition by gold(I) NHC com-
plexes while the inhibition of TrxR in cellular lysates has
already been reported previously.25 However, the expected
increase in activity with increasing the lipophilicity/surface
volume of the residues at the benzimidazol-2-ylidene nitro-
gens could not be observed (compare results for 1a-4a in
Table 2). Antiproliferative effects for 1a-4a were noted in the
low micromolar range, and more detailed experiments on 2a
showed that these effects could be related to the induction of
ROS formation, finally resulting in apoptosis and necrosis of
the cells as well as to direct effects on mitochondrial integrity
1,3-Dimethylbenzimidazoliumiodide (1).53 Yield: 0.238 g (0.8
mmol, 87%) white powder; mp 217 ꢀC. 1H NMR (DMSO-d6):
(ppm) 4.08 (s, 6H, CH3), 7.71 (dd, 2H, J = 3.2 Hz, J = 6.3 Hz,
ArH4/7), 8.02 (ddd, 2H, J = 3.2 Hz, J = 6.3 Hz, ArH5/6), 9.64 (s,
1H, NHC). Elemental analysis for C9H11N2I (% calcd/found):
C (39.44/39.04), H (4.05/4.54), N (10.22/10.00).
1,3-Diethylbenzimidazoliumiodide (2).53 Yield: 0.272 g (0.9
mmol, 90%) white powder; mp 240 ꢀC. 1H NMR (DMSO-d6):
(ppm) 1.55 (t, 6H, J = 9.7 Hz, CH3), 4.52 (q, 4H, J = 9.7 Hz,
CH2), 7.68 (dd, 2H, J = 4.2 Hz, J = 8.4 Hz, ArH4), 8.10 (dd, 2H,
J = 4.2 Hz, J = 8.4 Hz, ArH5), 9.8 (s, 1H, NHC). 13C NMR
(DMSO-d6): (ppm) 14.1 (CH3) 42.0 (CH2) 113 (ArC8) 126

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8615
(ArC4) 130 (ArC5) 141 (NHC). Elemental analysis for C11H15-
N2I (% calcd/found): C (43.73/43.80), H (5.00/5.02), N (9.27/
9.13).
(both from Sigma-Aldrich) were used and diluted with distilled
water to achieve a concentration of 2.0 U/mL. The compounds
were freshly dissolved as stock solutions in DMF. To each,
25 μL aliquots of the enzyme solution each 25 μL of potassium
phosphate buffer pH 7.0 containing the compounds in graded
concentrations or vehicle (DMF) without compounds (control
probe) were added and the resulting solutions (final concentra-
tion of DMF: 0.5% V/V) were incubated with moderate shaking
for 75 min at 37 ꢀC in a 96-well plate. To each well, 225 μL of
reaction mixture (1000 μL reaction mixture consisted of 500 μL
potassium phosphate buffer pH 7.0, 80 μL 100 mM EDTA
solution pH 7.5, 20 μL BSA solution 0.05%, 100 μL of 20 mM
NADPH solution, and 300 μL of distilled water) were added and
the reaction started by addition of 25 μL of an 20 mM ethanolic
DTNB solution. After proper mixing, the formation of 5-TNB
was monitored with a microplate reader (Perkin-Elmer Victor X4)
at 405 nm in 10 s intervals for 6 min. The increase in 5-TNB
concentration over time followed a linear trend (r2 g 0.99), and
the enzymatic activities were calculated as the slopes (increase in
absorbance per second) thereof. For each tested compound, the
noninterference with the assay components was confirmed by a
negative control experiment using an enzyme-free solution. The
EC50 values were calculated as the concentration of compound
decreasing the enzymatic activity of the untreated control by
50% and are given as the means and error of repeated experiments.
Cell Culture. MCF-7 breast adenocarcinoma, HT-29 and
HCT-116 colon carcinoma, and HEP-G2 hepatocellular carcinoma
cells were maintained in DMEM high glucose (PAA) supplemented
with 50 mg/L gentamycin and 10% (V/V) fetal calf serum (FCS)
prior to use. Leukemia B-cell precursor (Nalm-6) and its Vincristin
and Daunorubicin resistant sublines, Burkitt-like lymphoma
cells (BJAB) and its Doxorubicin resistant (7CCA) and Vincristin
resistant (BIBO) sublines, were cultured in RPMI 1640 supple-
mented with 10% FCS.
Antiproliferative Effects in MCF-7, HT-29, HCT-116, and
HEP-G2 Cells. The antiproliferative effects in MCF-7, HT-29,
HCT-116, and HEP-G2 cells after 72 h (HT-29, HCT-116) or
96 h (MCF-7, HEP-G2) exposure to the gold complexes were
evaluated according to an established procedure.23 For the
experiments, the compounds were prepared freshly as stock
solutions in DMF and diluted with the cell culture medium to
the final assay concentrations (0.1% V/V DMF). The IC50 value
was described as that concentration reducing proliferation of
untreated control cells by 50%.
ROS Formation. Jurkat cells were cultivated under standard
conditions and cells were incubated with the compounds for 24 h
as indicated. After incubation, cells were collected, centrifuged
at 0.2g (1500 rpm), and resuspended in FACS buffer (D-PBS,
Gibco, þ 1% BSA, PAA). Cell suspensions were treated with
DHE (dihydroethidium, SIGMA, 5 μL of 5 mM stock solution
per 1.0 mL of cell suspension containing 106 cells) at room
temperature in the dark for 15 min, washed one more time with
FACS buffer, and immediately analyzed using a FACSCalibur
(Becton Dickinson) and CellQuest Pro (BD) analysis software.
Excitation and emission settings were 488 nm and 564-606 nm
(FL2 filter), respectively. Important note: although DHE is
known to interact only with superoxide anion, the intensity of
fluorescence is commonly considered as a reflection of total
intracellular ROS.
1,3-Dibenzylbenzimidazoliumbromide (3).54 Yield: 0.323 g (0.8
mmol, 76%) white powder; mp 150 ꢀC. 1H NMR (DMSO-d6):
(ppm) 5.81 (s, 4H, CH2), 7.46 (m, 10H, ArH), 7.64 (dd, 2H, J =
3.1 Hz, J = 6.3 Hz, ArH4/7), 7.98 (ddd, 2H, J = 3.2 Hz, J =
6.3 Hz, ArH5/6), 10.12 (s, 1H, NHC). Elemental analysis for
C21H19N2Br (% calcd/found): C (66.30/65.41), H (5.26/5.20), N
(7.36/6.79).
1,3-Bis-(diphenylmethyl)benzimidazoliumchloride (4).55 Yield:
1
0.462 g (0.8 mmol, 80%) white powder; mp 254 ꢀC. H NMR
(CDCl3): (ppm) 7.45 (m, 24H, ArH), 7.82 (s, 2H, CH), 10.68 (s,
1H, NHC). Elemental analysis for C33H27N2Cl (% calcd/found):
C (81.48/80.99), H (5.55/5.54), N (5.76/5.60).
General Procedure for Synthesis of the Gold NHC Complexes.
The respective benzimidazolium salt (1.0 mmol) was treated
with Ag2O (0.116 g, 0.5 mmol) under vigorous stirring in dichloro-
methane for 5 h. After the color change, dimethylsulfidegold(I)
(0.296 g, 1.0 mmol) was added and the reaction was stirred for
another 10 h. The obtained suspension was filtered over Celite
(281 nm), and the solvent was evaporated under reduced pressure
to give the pure product.
Chloro-(1,3-dimethylbenzimidazol-2-ylidene)gold(I) (1a).53 Yield:
0.095 g (0.3 mmol, 25%) yellow powder. 1H NMR (CDCl3): (ppm)
3.99 (s, 6H, CH3), 7.46 (m, 4H, ArH). MS(EI): 378 (M þ Hþ).
Elemental analysis for C9H10AuClN2 (% calcd/found): C (28.55/
28.56), H (2.60/2.69), N (7.40/6.98).
Chloro-(1,3-diethylbenzimidazol-2-ylidene)gold(I) (2a).53 Yield:
0.122 g (0.3 mmol, 30%) pale-yellow powder. 1H NMR (CDCl3):
(ppm) 1.55 (t, 6H, J = 5.9 Hz, CH3), 4.54 (q, 4H, J = 9.8 Hz,
CH2), 7.46 (m, 4H, ArH). 13C NMR (CDCl3): (ppm) 15.4 (CH3),
43.9 (CH2), 111 (ArC8), 124 (ArC4), 132 (ArC5), 177 (NHC).
MS(EI) 406 (M þ Hþ). Elemental analysis for C11H14AuClN2 (%
calcd/found): C (32.49/32.03), H (3.47/3.38), N (6.89/6.91).
Chloro-(1,3-dibenzylbenzimidazol-2-ylidene)gold(I) (3a). Yield:
0.265 g (0.5 mmol, 50%) white powder. 1H NMR (CDCl3):
(ppm) 5.76 (s, 4H, CH2), 7.35 (m, 14H, ArH). MS(EI): 530
(M - Hþ). Elemental analysis for C21H18AuClN2 (% calcd/found):
C (47.52/47.72), H (3.42/3.58), N (5.28/5.25).
Chloro-[1,3-bis-(diphenylmethyl)benzimidazol-2-ylidene]gold-
(I) (4a). Yield: 0.375 g (0.6 mmol, 55%) white powder. 1H NMR
(CDCl3): (ppm) 7.05 (dd, 2H, J = 3.5 Hz, J = 6.5 Hz, ArH4/7),
7.36 (m, 22H, ArH), 7.89 (s, 2H, CH). MS (EI) 682 (M þ Hþ).
Elemental analysis for C33H26AuClN2 (% calcd/found): C
(58.03/57.75), H (3.84/3.87), N (4.10/3.87).
Reaction with Glutathione. The gold(I) complexes were pre-
pared as stock solutions in dimethylformamide (DMF) and
diluted with potassium phosphate buffer pH 7.0 to achieve a
final concentration of 500 μM (DMF: 0.2% V/V). To 25 μL of
250 μM aqueous solutions of reduced glutathione, each 25 μL of
the respective potassium phosphate buffer solution (containing
the compounds or only the DMF vehicle as control) and 25 μL
100 mM aqueous EDTA solution pH 7.5 were added and the
resulting solutions were incubated with moderate shaking in a
96-well plate at 37 ꢀC for 20 or 60 min. To each well, 200 μL of
reaction mixture (1000 μL reaction mixture consisted of 620 μL
potassium phosphate buffer pH 7.0, 80 μL 100 mM EDTA
solution pH 7.5, and 300 μL distilled water) were added, and the
reaction was started with the addition of 25 μL of a 20 mM
ethanolic solution of DTNB. After proper mixing, the formation
of 5-TNB was monitored in a microplate reader (Perkin-Elmer
Victor X4) at 405 nm. For each tested compound, the noninter-
ference with the assay components was confirmed by a negative
control experiment using a glutathione free solution. Results are
presented as means of two independent experiments.
Annexin V/PI Staining. Jurkat cells were treated with the
indicated concentration of the substance for 48 h, collected, and
stained with Annexin V-FITC (eBioscience) according to the
manufacturers recommendation. Briefly, approximately 5.0 ꢀ
105 cells were resuspended in 50 μL of Annexin V staining buffer
(10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4),
2.5 μL of Annexin V conjugate, and 1.25 μL of PI solution (1 mg/mL)
were added and the probes were incubated in the dark at room
temperature for 15 min. Signal intensity was analyzed using a
FACSCalibur (Becton Dickinson) and CellQuest Pro (BD)
analysis software. Excitation and emission settings were 488 nm,
TrxR and GR Inhibition Assay. To determine the inhibition of
TrxR and GR an established microplate reader based assay was
performed with minor modifications.11,56 For this purpose,
commercially available rat liver TrxR and baker’s yeast GR

8616 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Rubbiani et al.
515-545 nm (FL1 channel) for Annexin V-FITC and 564-
606 nm (FL2 channel) for PI.
repeated. After resuspending the final mitochondria pellet in
isolation buffer, the protein content was estimated by the Bradford
Assay.
LDH Release Assay. After incubation with different concen-
trations of 2 and 2a for 2 h, LDH released by BJAB cells was
measured in cell culture supernatants using a cytotoxicity detec-
tion kit from Boehringer Mannheim (Mannheim, Germany).
The supernatants were centrifuged at 300g for 5 min. Cell-free
supernatants (20 μL) were diluted with 80 μL of PBS and 100 μL
of reaction mixture were added. Then, the time-dependent
formation of the reaction product was quantified photometri-
cally at 490 nm. The maximum amount of LDH activity released
by the cells was determined by lysis of the cells using 0.1% Triton
X-100 in culture medium and was set as 100% cell death.
Measurement of DNA Fragmentation. Apoptotic cell death
was determined by a modified cell cycle analysis, which detects
DNA fragmentation at the single cell level. For measurement of
DNA fragmentation cells were seeded at a density of 1 ꢀ 105
cells/mL and treated with different concentrations of 2 and 2a.
After 72 h of incubation, cells were collected by centrifugation at
30g for 5 min, washed with PBS at 4 ꢀC, and fixed in PBS/2%
(v/v) formaldehyde on ice for 30 min. After fixation, cells were
incubated with ethanol/PBS (2:1, v/v) for 15 min, pelleted, and
resuspended in PBS containing 40 μg/mL RNase A. After
incubation for 30 min at 37 ꢀC, cells were pelleted again and
finally resuspended in PBS containing 50 μg/mL PI. Nuclear
DNA fragmentation was then quantified by flow cytometric
determination of hypodiploid DNA. Data were collected and
analyzed using a FACScan (Becton Dickinson, Heidelberg,
Germany) equipped with the CELLQuest software. Data are
given in % hypoploidy (subG1), which reflects the number of
apoptotic cells.
Measurement of Mitochondrial Oxygen Consumption. The
measurement was performed using OxoPlate (PreSens, Germany)
96-well plates which contain an immobilized oxygen sensor at
the bottom of each well. Fluorescence is measured in dual mode
(excitation 540 nm and emission 650 nm) with reference emission
590 nm. The signal ratio 650/590 nm corresponds to the oxygen
partial pressure. The calibration of the fluorescence reader is
performed using a two-point calibration with oxygen-free water
(1% Na2SO3) and air-saturated water with oxygen partial
pressure corresponding to 0% and 100% respectively. Then
18 μg of freshly isolated mitochondria were suspended in 100 μL
of respiration buffer (25 mM sucrose, 100 mM KCl, 75 mM
mannitol, 5 mM MgCl2, 10 mM KH2PO4, 0.5 mM EDTA,
10 mM TRIS, 0.1% fatty acid-free BSA, pH 7.4) containing
10 mM pyruvate, 2 mM malate, 2 mM ADP, and 0.5 mM ATP
to activate oxidative phosphorylation. The mitochondrial sus-
pensions also contained the test compounds in the indicated
concentrations. Fluorescence was measured continuously for
400 min with kinetic intervals of 5 min by a Tecan Safire2 (Tecan,
Maennedorf, Switzerland) microplate reader at 37 ꢀC. During
the measurements, the plates were sealed with a breathable
membrane (Diversified Biotech, Boston, MA). Additional controls
were 5 μM rotenone (Sigma-Aldrich) as inhibitor of respiratory
chain complex I and 1 μM CCCP (carbonyl cyanide 3-chloro-
phenylhydrazone, Sigma-Aldrich) as uncoupling agent, capable
of increasing electron flow through the respiratory chain there-
by increasing the oxygen consumption.
Effects on Cell Metabolism. Online measurement of cell meta-
bolism and morphological changes was done using a Bionas
2500 sensor chip system (Bionas, Rostock, Germany). The
metabolic sensor chips (SC 1000) include ion-sensitive field-
effect transistors to record pH changes, an oxygen electrode to
monitor oxygen consumption, and interdigitated electrode
structures to measure impedance under the cell layer. Approxi-
mately 1.5 ꢀ 105 cells were seeded directly onto each sensor chip
in 450 μL of DMEM (PAA, E15-883) with penicillin/strepto-
mycin and 10% (v/v) FCS (PAA) and incubated at 37 ꢀC, 5%
CO2, and 95% humidity for 24 h. The cell number used resulted
in approximately 80-90% confluence of the cells on the chip
surface after 24 h. This was the starting condition for online
monitoring. Sensor chips with cells were then transferred to the
Bionas2500 analyzer in which medium is continuously ex-
changed in 8 min cycles (4 min exchange of medium and 4 min
without flow), during which the parameters were measured. The
running medium used during analysis was DMEM without
carbonate buffer and only weakly buffered with 1 mM Hepes
and reduced FCS (0.1%). For drug activity testing, the follow-
ing steps were included: (1) 5 h equilibration with running
medium (RM), (2) drug incubation with substances freshly
dissolved in medium at indicated concentrations and indicated
incubation time, (3) a regeneration step in which cells are again
fed with running medium without substances, and (4) at the end
of each experiment, the cell membrane was damaged by addition
of 0.2% Triton X-100 to obtain a basic signal without living cells
on the sensor surface as a negative control.
Acknowledgment. Financial support by Deutsche Forsch-
ungsgemeinschaft (DFG, grant FOR-630) is gratefully
acknowledged.
Supporting Information Available: More details on the reac-
tion of a gold(I) phosphine complex with glutathione (31P NMR
and ESI-MS experiments), additional data on the effects on cell
proliferation of non tumorigenic cells, video microscopic ima-
ging, ROS formation, inhibition of disulfide reductases in cells,
DNA fragmentation, cell viability, and effects in resistant cell
lines are presented as a PDF file and as two video files. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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