Selenium- and tellurium-containing redox modulators with distinct activity against macrophages: Possible implications for the treatment of inflammatory diseases

Article abstract of DOI:10.1016/j.tet.2012.09.021

Various selenium- and tellurium-containing molecules are able to modulate the intracellular redox state of cells, an effect which may be used for the (selective) targeting of cancer cells, which are naturally under oxidative stress (OS). As macrophages also generate an environment rich in Reactive Oxygen Species (ROS) and nitric oxide (NO), they may represent an additional, prime target for such redox-modulating agents. A range of selenium and tellurium-containing quinones have therefore been synthesized and subsequently tested in macrophage culture. The tellurium agents were generally cytotoxic at very low concentrations, and their mode of action seemed to involve the upregulation of intracellular ROS levels. This redox-modulating effect was confirmed by simple yeast-based chemogenetic analysis in conjunction with in vitro redox assays and electrochemistry. Together, these studies point towards an intracellular build-up of superoxide radicals as the most likely cause of toxicity. In contrast, some of the selenium derivatives were less toxic and exerted a pronounced inhibitory effect on the formation of lipopolysaccharide- induced NO production. Whilst the Te-analogues therefore may enable the resolute, effective and fairly selective targeting of macrophages, the selenium agents could act less severely, but equally effectively by interfering with inflammatory signalling molecules.

Full text of DOI:10.1016/j.tet.2012.09.021

Tetrahedron 68 (2012) 10577e10585
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Selenium- and tellurium-containing redox modulators with distinct activity
against macrophages: possible implications for the treatment
of inflammatory diseases
a
b
c
a
ꢀ
ꢀ d
Mandy Doering , Britta Diesel , Martin C.H. Gruhlke , Uma M. Viswanathan , Dominika Manikova ,
Miroslav Chovanec d, Torsten Burkholz a, Alan J. Slusarenko c, Alexandra K. Kiemer b, Claus Jacob a
,
*
a Division of Bioorganic Chemistry, School of Pharmacy, Saarland State University, D-66123 Saarbruecken, Germany
b Department of Pharmaceutical Biology, School of Pharmacy, Saarland State University, D-66123 Saarbruecken, Germany
c Department of Plant Physiology (BioIII), RWTH Aachen University, D-52056 Aachen, Germany
d Laboratory of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2012
Received in revised form 29 August 2012
Accepted 4 September 2012
Available online 11 September 2012
Various selenium- and tellurium-containing molecules are able to modulate the intracellular redox state
of cells, an effect which may be used for the (selective) targeting of cancer cells, which are naturally
under oxidative stress (OS). As macrophages also generate an environment rich in Reactive Oxygen
Species (ROS) and nitric oxide (ꢀNO), they may represent an additional, prime target for such redox-
modulating agents. A range of selenium and tellurium-containing quinones have therefore been syn-
thesized and subsequently tested in macrophage culture. The tellurium agents were generally cytotoxic
at very low concentrations, and their mode of action seemed to involve the upregulation of intracellular
ROS levels. This redox-modulating effect was confirmed by simple yeast-based chemogenetic analysis in
conjunction with in vitro redox assays and electrochemistry. Together, these studies point towards an
intracellular build-up of superoxide radicals as the most likely cause of toxicity. In contrast, some of the
selenium derivatives were less toxic and exerted a pronounced inhibitory effect on the formation of
Keywords:
Redox modulation
Macrophages
Chemogenetic analysis
Nitric oxide
Reactive oxygen species
iNOS
ꢀ
lipopolysaccharide-induced NO production. Whilst the Te-analogues therefore may enable the resolute,
Lipopolysaccharide
effective and fairly selective targeting of macrophages, the selenium agents could act less severely, but
equally effectively by interfering with inflammatory signalling molecules.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
the homeless, which links OS not only to various diseases but also
to age and lifestyle.10,11
The last two decades have witnessed a growing interest in se-
lenium- and tellurium-containing agents with potential ‘redox-
modulating’ properties.1e6 This interest is based on the fact that
many human diseases are associated withdif not caused
bydoxidative stress (OS).7e9 The latter represents an imbalance in
the cellular redox homeostasis, which is characterized by an in-
tracellular build-up of Reactive Oxygen Species (ROS), and fre-
quently also diminished levels of reduced glutathione (GSH). OS is
found, for instance, in many auto-inflammatory, immune and in-
fectious diseases, in cancer, diabetes, glaucoma and neurodegen-
erative disorders to name just a few.7,9 An increase in intracellular
levels of ROS is also frequently observed in older people and even in
Not surprisingly, pharmaceutical research has long focused on
antioxidants, which may counteract the damaging effects of ROS
and related species. Within this context, selenium-based agents
have obtained a certain prominence as they can mimic the activity
of the selenocysteine-containing antioxidant enzyme glutathione
peroxidase (GPx); selenocysteine is also found in the human anti-
oxidant enzyme thioredoxin reductase (TrxR).12 With the notable
exception of the Se-based agent ebselen,13 however, most of these
Se-compounds have not (yet) progressed to clinical trials. In con-
trast, tellurium-containing compounds have hardly been consid-
ered as potential drugs, as tellurium is not an essential trace
element, seems to be ‘alien’ to the human body and is often asso-
ciated with outright toxicity.6
Only recently, have first attempts been undertaken to employ
tellurium agents as redox modulators in the context of cancer
therapy.3,5,6,14,15 Here, the ability of such tellurium (and selenium)
agents to significantly change the pre-existing redox balance in the
* Corresponding author. Tel.: þ49 681 302 3129; fax: þ49 681 302 3464; e-mail
address: c.jacob@mx.uni-saarland.de (C. Jacob).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.021

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M. Doering et al. / Tetrahedron 68 (2012) 10577e10585
cell towards more oxidizing conditions is used to push oxidatively
stressed cancer cells over the critical ‘redox threshold’ and induce
cancer cell death by apoptosis.3,16 As normal, healthy cells often
contain less ROS compared to cancer cells, these redox-modulating
agents may act fairly selectively against cancer cells, which bode
well for possible therapeutic applications.17 During the last couple
of years, research into such redox-modulating agents has seen
considerable progress: The first animal studies have been com-
pleted successfully, there is some evidence of selective action
against patient-derived chronic lymphocytic leukaemia cells (CLL
cells) compared to healthy peripheral blood mononuclear cells
(PBMCs), and we now have a clearer insight into the possible bio-
chemical mode(s) of action which often involve the cellular thiol-
Fig. 1. Previous studies have indicated that multifunctional agents,
which combine a ‘ROS generating’ quinone moiety with a catalytic,
‘ROS using’ chalcogen group are particularly effective (and selec-
tive) in cancer cell-based test systems.5,23 Hence compounds con-
taining selenium as well as tellurium, benzoquinone as well as
naphthoquinone, were chosen for this study.
stat (e.g.,
b-tubulin) and various regulatory pathways associated
with it.5,18
Whilst a number of important questions still need to be an-
swered as far as the anti-cancer activity of such agents is concerned,
a new and equally important target for redox-modulating agents
has been emerging in the context of inflammatory diseases (e.g.,
rheumatoid arthritis (RA)).19e21 Macrophages, like cancer cells
under OS, generate a highly oxidizing environment, which can
cause severe damage not only to invading microorganisms (such as
bacteria), but also to the surrounding tissue. In the case of RA and
related inflammatory diseases, such macrophages are ‘out of con-
trol’ in two aspects. Firstly they generate ROS, nitric oxide (ꢀNO) and
cytokines for no apparent reason (and hence damage the human
joint, causing severe inflammation), and secondly, they are re-
sistant towards the rather large amounts of ROS and ꢀNO that they
generate.
One may therefore speculate that redox-modulating agents
brought into contact with such macrophages could use the high
local ROS levels to inflict severe damage and hence kill these cells
fairly effectivelydyet also selectively. Indeed, selectivity may be
expected to result from the gradient in ROS concentration, which is
highest in, at or near the ROS generating macrophages themselves
and lower in the surrounding tissue. If this were the case, most
macrophages could be destroyed rather selectively by redox-
modulating agents. Subsequently, ROS and cytokine levels would
be expected to decrease and the inflammation may ultimately
subside. This rather unconventional strategy of ‘fighting fire with
fire’ may ultimately be superior to conventional antioxidant strat-
egies, which only counteract existing ROS but cannot interfere with
ROS formation itself.22
Fig. 1. Chemical structures of organochalcogens and reference compounds used in this
study. These compounds are multifunctional and were selected on the basis of pre-
vious results obtained in cancer cells. Structures containing two (redox active) chal-
cogen centres in addition to
a quinone redox centre are of particular interest.
Compounds 1e4 have not been reported in the literature before and their synthesis is
described in the experimental section.
Some of these compounds were already known in the literature
and could be synthesized according to established literature
methods, while others have been produced for the first time fol-
lowing specifically adapted synthetic procedures. Where appro-
priate, these synthetic avenues are discussed in the experimental
section. Ultimately, 14 compounds (four of them novel) have been
obtained in sufficient amounts and purity and were used for this
study. The tellurium analogues of compounds 1e3 have also been
synthesized and characterized chemically, but they were not of
sufficient purity at the time to be employed in biological cytotox-
icity assays.
Besides simply destroying macrophages, redox modulators may
ꢀ
also target either the production or stability of NO, a redox sensi-
tive inflammatory signalling molecule, which is produced by an
ꢀ
inducible enzyme, inducible NO synthase (iNOS), upon activation
of the innate immune system. Redox modulators may therefore
ꢀ
also act as anti-inflammatory agents via disruption of the NO sig-
nalling network.
Here, we provide initial evidence that certain compounds con-
taining tellurium and quinone moieties are able to prevent the
proliferation, and/or induce cell death of macrophages in cell cul-
ture when applied at (sub-)micromolar concentrations, most likely
via a redox-mediated mechanism. Whilst the sulfur and selenium
analogues generally are less cytotoxic, some of them are able to
2.2. Cytotoxicity in macrophage culture
Whilst compounds such as 8 and 11 have been studied in cancer
cell models before,5 there is no data available on possible cytotoxic
effects of these compounds on macrophages. As part of our studies,
we have therefore measured the impact of compounds
1e14dtogether with ‘chalcogen-free’ benzoquinone and naph-
thoquinone controlsdon RAW 264.7 macrophage viability using
the MTT assay. Among the various compounds tested, the tellurium
compounds, in particular, exhibit a significant cytotoxic effect, re-
ducing cell viability in a concentration dependent manner and with
IC50 values in the sub-micromolar range (Fig. 2). Table 1 summa-
rizes the IC50 values obtained in the macrophage viability assay
ꢀ
prevent the synthesis of NO by macrophages and hence may be
employed to disrupt pro-inflammatory cellular signalling.
2. Results/discussion
2.1. Selection and synthesis of compounds
In order to investigate the potential use of redox-modulating
selenium and tellurium agents, several suitable agents were se-
lected. The chemical structures of these compounds are shown in

M. Doering et al. / Tetrahedron 68 (2012) 10577e10585
10579
as it seems to identify macrophages as (the) prime target of such
tellurium-based redox agents. If confirmed in more complex sys-
tems, such as animals, such a selective activity against macrophages
at compound concentrations below 1 mM would be rather exciting,
as it may provide a new avenue to target macrophages with con-
siderable precision.
A
120
100
80
60
40
20
0
**
***
***
***
***
***
2.3. Tellurides: redox modulation as possible mode of action
***
***
The cytotoxicity assays support the general hypothesis that
macrophages, like cancer cells under OS, may represent targets of
redox-active compounds; they do not, however, explain why and
how such compounds may act on these cells. In order to obtain
some basic and certainly preliminary insights into the possible
mode(s) of action, we have therefore used cell-based assays to
control 0.025 0.05 0.10 0.20 0.39 0.78 1.56
100
concentration [µM]
MTT assay of compound 14
B
120
100
80
60
40
20
0
ꢀ
measure ROS and NO production in these cells.
n.s.
In the case of the tellurium compounds, the 20,70-dichlor-
odihydrofluorescein diacetate (DCF-DA)-based assay for in-
tracellular ROS has been employed and a small increase of
intracellular ROS levels (around 1 to 1.3-fold) could be observed for
some of the tellurium-containing compounds, which reached
a plateau after 40 min of incubation and then diminished again
(data not shown).
***
***
***
***
***
***
***
Since intracellular ROS levels in macrophages were difficult to
follow and quantify, a yeast-based chemogenetic screen has been
employed as a more reliable and also more informative analytical
alternative. The latter relies on a range of yeast mutants, which are
deleted in various, mostly redox-related enzymes, such as super-
oxide dismutase (SOD), catalase (CAT) or glutathione disulfide re-
ductase (GR). Each of these mutants is exposed to the test
compounds, and mutants particularly sensitive to the compounds
point towards a possible link between the (missing) enzyme and its
cellular role(s) on the one hand, and the biochemical action of the
compounds tested on the other. Importantly, for both, the enzymes
of the glutathione metabolism and glutathione reductase, the yeast
genome contains only one gene for the particular enzyme; and for
the superoxide dismutases and catalase, the yeast genome has only
one gene for a cytosolic (sod1) and a mitochondrial (sod2) enzyme
and only one for cytosolic catalase, respectively.26 This easy genetic
accessibility of particular biochemical key players, as well as an
easy experimental handling of yeast, turns this organism into
a reasonable and advantageous system for chemogenetic screen-
ing.27 As yeast features a cellular redox biochemistry, which is
comparable to human cells (but less complex), certain comparisons
are possible. Nonetheless, one should always be careful not to
overstrain the analogy between this fungus and human cells.
Interestingly, the yeast-based chemogenetic analysis confirms
a significant cytotoxicity for the most active tellurium compounds 8
and 14 (compound 11 was not tested in this assay). Some of the
selenium compounds show a similar cytotoxicity, highlighting
differences in susceptibility towards selenium- and tellurium
compounds, which seem to exist between yeast cells on the one
hand and macrophages on the other. As far as the different cyto-
toxicity of selenium and tellurium compounds in yeast is con-
cerned, it is possible that yeast cells are either more sensitive
towards selenium compounds or can cope better with tellurium
compounds as may be expected from previous data obtained for
human cells. Such considerations are interesting in the context of
antifungal activity and should be investigated further in the future.
Compounds 8 and 14 are particularly toxic against strains lack-
ing mitochondrial manganese-containing superoxide dismutase
control 2.5
5
7.5
10
15
20
25
100
concentration [µM]
MTT assay of compound 12
Fig. 2. Impact of compounds on macrophage viability measured in the MTT assay. Cells
were incubated with the relevant compound for 24 h. Panel A shows the concentration
dependence of cytotoxicity induced by the tellurium compound 14, which is among
the most active compounds studied in this assay. This compound is active in the sub-
micromolar range (IC50¼160 nM). In contrast, the selenium analogue, compound 12, is
considerably less cytotoxic, with an IC50 value of 9.2 mM. Shown are the significances
calculated with the Bonferroni test compared to control.
Table 1
Summary of the various selenium- and tellurium-containing compounds used as
part of this study and IC50 values obtained in the macrophage survival assay
Compound
Chalcogen
IC50
1
2
3
4
Se
Se
Se
Se
Se
d
d
S
Se
Te
S
Se
Te
Se
Te
Te
20.46
24.95
26.11
5.27
43.77
27.56
8.88
5.97
7.71
0.30
15.15
20.32
0.30
9.20
1.20
5
bq
nq
6
7
8
9
10
11
12
13
14
0.16
after 24 h. Compounds 8, 11 and 14 are particularly active, with
IC50 values of 0.30, 0.30 and 0.16 mM, respectively. In contrast, the
sulfur and selenium analogues were considerably less toxic, with
IC50 values in the micromolar range (e.g., compounds 9 and 10 with
an IC50 of 6.0 and 7.7 mM, respectively).
These findings agree with previous reports, which generally
point towards a considerably higher toxicity for the tellurium
compounds in mammalian cells when compared to the corre-
sponding selenium and sulfur analogues.24,25 Interestingly, the
cytotoxicity of 8 and 11 against macrophages is also at least 10
times higher when compared to values obtained by us for a range of
cancer cells or healthy cells.5 This finding is particularly significant,
(sod2
a cytosolic zinc, copper SOD (sod1
the screen also shows that mutants lacking cytosolic catalase
(ctt1 ) or glutathione reductase (glr1 ) are less affected. Further-
more, mutants that are affected by/impaired in glutathione
D) and, in some instances, are also toxic against strains lacking
D) (Fig. 3). Perhaps surprisingly,
D
D

10580
M. Doering et al. / Tetrahedron 68 (2012) 10577e10585
involvement of Oꢀ2ꢁ and H2O2 in the cytotoxic effects of certain
compounds on the other is still valid.31,32 Nonetheless, such studies
need to be taken with some caution.
Secondly, mitochondria are not the only source of Oꢀ2ꢁ. It is
therefore difficult to assign the effects observed here solely to an
interaction with mitochondria or SOD2. Alternative, maybe more
complex interactions are also possible (for instance involving
NADPH oxidases) and cannot be ruled out at this point.33
The same considerations also apply to strains whose GSH syn-
thesis is impaired or which are deficient in GR. Here, the results
obtained so far count against the direct involvement of GSH or
glutathione disulfide (GSSG) in the mechanism of the compounds’
cytotoxic action. One should note, however, that other redox-
modulating agents targeting the cellular thiolstat, such as allicin,
do have a pronounced effect on strains lacking GR. This difference
in toxicity, which apparently exists between most of the quinones
tested on the one hand, and compounds such as allicin on the other,
can be understood if one realizes that quinones can generate O2ꢀꢁ
radicals, which subsequently oxidize cellular thiols, whilst allicin
and related compounds themselves react with the cellular
thiols.20,34e37
Fig. 3. Simple chemogenetic screen of selected compounds (at 100 mM concentration,
16 h incubation time) in a yeast-based cell survival assay. Besides the wild-type strain
BY4742, mutants deficient in sod1, sod2 and cytosolic catalase (ctt1) have been em-
ployed. Whilst most compounds show a moderate cytotoxicity against the wild-type
strain, they are particularly toxic against the sod2 mutant strain, pointing towards
a possible link between the compounds’ biochemical mode(s) of action and the in-
tracellular role(s) of sod2. The latter includes the removal of superoxide radical anions.
Shown are the significances calculated with Student’s t-test compared to control.
biosynthesis show an ambivalent picture: Whilst the gsh1
shows a high sensitivity to most (but not all) of the tested com-
pounds, sensitivity of the gsh2 mutant is much less pronounced.
Overall, data on the gsh1 and gsh2 mutants are fairly in-
D
mutant
The possible involvement of Oꢀ2ꢁ has been confirmed in a simple
cell-based luminescent lucigenin assay indicative of ROS genera-
tion. As part of this assay, the relative luminescence caused by the
various selenium- and tellurium compounds is measured in the
absence and presence of wild-type (BY4742) yeast cells. ROS gen-
eration based on simple chemical reactions (e.g., reactions with
molecular oxygen) may be detected in the absence of cells, whilst
increases in ROS levels due to biochemical processes (e.g., enzy-
matic conversions, inhibition of antioxidant enzymes) only become
apparent in the presence of the cells.
D
D
D
conclusive and at this stage does not point towards a specific cau-
sality between the compounds tested and glutathione synthesis in
general (data not shown).
It should be mentioned that inorganic selenite (SeO23ꢁ) is known
to affect DNA integrity, and hence DNA damage caused by selenium
and tellurium agents may be another major cause of cell death.
Therefore,
a
mutant deficient in homologous recombination
As Fig. 4 indicates, none of the compounds tested generates
significant levels of ROS in the absence of cells under the experi-
mental conditions used in this assay (with the possible exception of
the naphthoquinone 14, which does seem to generate a small
amount of ROS by its own). In contrast, the naphthoquinone
menadione and the naphthoquinone-containing tellurides 8, 14
(rad52 ), the main pathway responsible for repairing DNA double-
D
strand breaks, was also screened. This mutant was not sensitive to
any of the compounds tested. This finding clearly counts against
significant DNA damage or lack of DNA repair caused by the orga-
noselenides and tellurides investigated as part of this study and is
in sharp contrast to the known toxic effects of SeO23ꢁ on DNA in-
tegrity.28 Such issues surrounding selenium and tellurium com-
pounds and their association with DNA damage and repair are of
major interest as far as chalcogen toxicity is concerneddand form
the topic of our ongoing investigations.
Although it is difficult to derive at finite conclusions based on
these still limited results, it nonetheless seems that SOD activity
(predominantly in the mitochondria, i.e., SOD2) is important for the
defence of the cells against the effect(s) of the tellurium com-
pounds tested. Hence, an involvement of superoxide radical anions
(Oꢀ2ꢁ)dpossibly generated by some of the quinone compounds and
exerting its damaging activity at or in the mitochondriadcan be
hypothesized (as various quinones are known to generate ROS). In
contrast, as catalase deficient strains are less affected, H2O2 does
not seem to play a major role in the activity of the compounds,
which is striking because the SOD-dependent superoxide degra-
dation results in the formation of H2O2.29 One should note, how-
ever, that the yeast catalase in question is located in the cytosol, and
therefore the H2O2 generated by sod2 as part of the detoxification of
Oꢀ2ꢁ may be detoxified in a catalase-independent manner (for in-
stance by a mitochondrial peroxiredoxin).30
Nonetheless, one must be careful not to over-interpret these still
preliminary sets of data. Firstly, the mutants lacking SOD or catalase
may behave differently because Oꢀ2ꢁ and H2O2 may possess a dif-
ferent cytotoxicity in yeast (if one of these ROS is less cytotoxic than
the other, then the mutant lacking the corresponding defence en-
zyme(s) may also be less affected). Such issues have formed the
subject of several studies in the past and it seems that a general link
between the SOD and catalase mutants on the one hand and the
Fig. 4. Lucigenin-based luminescent yeast assay to estimate the formation of in-
tracellular ROS in response to the compounds tested (incubation time 16 h). Whilst
none of the compoundsdwith the possible exception of compound 14dgenerates ROS
on its own (white bars), significant levels of ROS can be measured for naphthoquinone
and the naphthoquinone derivatives in the presence of cells (grey bars). Interestingly,
the benzoquinone derivatives (and bq itself) under these conditions do not seem to
generate any significant amounts of ROS. Statistical significances have been calculated
with the Student’s t-test and refer to the respective DMSO control.

M. Doering et al. / Tetrahedron 68 (2012) 10577e10585
10581
and the selenide 7 cause a sharp increase in relative luminescence
in the presence of cells. This increase in ROS levels is particularly
significant at compound concentrations of 100 mM. Interestingly,
the benzoquinones are notably less active than the naph-
thoquinones in this assay.
Fig. 5 illustrates this apparent ROS removing (i.e., ‘antioxidant’)
activity. The relative fluorescence of the macrophages stressed with
H2O2 or stimulated for internal ROS production with PMA evidently
diminishes upon addition of compound 2. This apparent antioxi-
dant activity may have several causes, including a partial removal of
macrophages (see below), inhibition of pro-oxidant enzymes or
a genuine chemical ‘neutralization’ of various ROS. It is also possible
that some ROS are removed catalytically in the presence of cellular
thiols (the latter may become oxidized as part of this process). The
figure allows the estimation of a rough ‘IC50 value’ for the inhibition
It should be emphasized that ROS formation is almost certainly
due to the quinone moiety of the multifunctional compounds
tested, and that the chalcogen (selenium or tellurium) moiety plays
a subordinate role in ROS generationdbut not in the processing of
the quinone-generated ROS, which ultimately explains the differ-
ences in cytotoxicity between the selenium and tellurium com-
pounds observed in the macrophages.24,25,38
The notion of quinones (primarily naphthoquinones) as ROS
generating agents, and the apparent differences between the gen-
erally more active naphthoquinones and the less active benzoqui-
nones, are supported by electrochemical results. Here, cyclic
voltammetry in conjunction with a glassy carbon working electrode
has been used to estimate the electrochemical potentials of the
most active and some of the reference compounds, focussing on the
of ROS formation, which is around 100e120 mM for compound 2.
This IC50 value is quite high when compared to the IC50 cytotoxicity
values obtained for such compounds in the MTT assay (IC50 of
compound 2 is 25 mM). The apparent ‘antioxidant’ effect illustrated
in Fig. 5 therefore may not necessarily result from a true reduction
of ROS levels in intact macrophages, but maydin partdbe due to
secondary effects, such as loss of viable, radical generating mac-
rophages. Nonetheless, the effect can also be observed at lower
concentrations, i.e., when cell survival is less affected.
quasi-reversible
hydroquinone/quinone
redox
pair
(see
Experimental section for more details). Overall, E1/2 values of
around ꢁ190 mV to þ100 mV have been obtained for the benzo-
quinones and (significantly more negative) E1/2 values of ꢁ250 mV
to ꢁ180 mV for the naphthoquinones, confirming the distinct dif-
ferences in redox behaviour, which exist between these two classes
of quinones. For comparison, benzoquinone bq shows an E1/2 value
of ꢁ192 mV, while naphthoquinone nq shows an E1/2 value of
ꢁ270 mV.
ns
ns
1,0
0,8
0,6
0,4
0,2
*
*
***
***
***
**
*
***
***
***
***
***
***
Interestingly, the anodic oxidation potentials Epa, which are
indicative of the ease of oxidation of the reduced hydroquinones,
and hence determine the ease of electron transfer from the hy-
droquinones to O2 and the ease of Oꢀ2ꢁ formation (or H2O2), are
generally lower (i.e., more reducing) for the naphthoquinones in
comparison to the benzoquinones. The ‘chalcogen-free’ naph-
thoquinone nq, for instance, exhibits an Epa of ꢁ190 mV
0
20
40
60
80
100
120
140
160
concentration [µM]
2 + 50 µM H2O2
2 + 1 µM PMA
ROS assay of compound 2
Fig. 5. Compound 2 suppresses or removes ROS added externally (H2O2) or formed
internally upon stimulation of macrophages with PMA. Data show relative fluores-
cence in cells incubated for 40 min with compound 2 in the presence of H2O2 or PMA
compared to H2O2 or PMA treatment alone. Shown are the significances calculated
with the Student’s t-test compared to control.
(IC50¼8.9
m
M), while the corresponding benzoquinone bq shows an
Epa of ꢁ3 mV (IC50¼27.6
mM). Similarly, the most cytotoxic
(naphthoquinone) compounds 8 and 14 exhibit Epa values of
ꢁ167 mV and ꢁ222 mV, respectively. These values are considerably
lower than the ones observed for the (less active) benzoquinones,
such as 4, with an Epa value of þ65 mV.
Confronted with this rather interesting result, the impact of the
selenium compounds on another reactive species, i.e., NO, was
investigated. Nitric oxide plays a major role in the activity and
signalling of immune cells. First of all, NO belongs to the Reactive
ꢀ
Ultimately, these differences in Epa values (and E1/2) may in part
explain the somewhat higher ROS generating ability and biological
activity observed for the naphthoquinones. One should note,
however, that the most active naphthoquinones also contain tel-
lurium, and a direct comparison between the benzoquinones and
naphthoquinones is therefore not straight forward (the tellurium-
containing benzoquinone analogues were not suitable for bi-
ological testing, see section 2.1). Hence, future studies may also
consider anthraquinones as a possible alternative to the benzo- and
naphthoquinones.
ꢀ
Nitrogen Species (RNS), and therefore is often associated with OS.39
Here, it can react with Oꢀ2ꢁ to form peroxynitrite (ONOOꢁ). ꢀNO also
binds to certain metalloproteins, such as soluble guanylyl cyclase
(sGC), and hence is involved in a range of beneficial health effects,
such as vasodilation. Finally, ꢀNO is a signalling molecule involved in
inflammatory processes. It controls, for instance, the activity of T
lymphocytes and signal transduction, and also has a pronounced
effect on mitochondrial events (some of which may result in
apoptosis).40
ꢀ
2.4. Selenium: from traditional antioxidant and GPx mimic to
In order to estimate the impact of our compounds on the NO
production of macrophages, the latter were stimulated with
100 ng/ml lipopolysaccharide (LPS), which results in an increased
production of NO. Fig. 6 illustrates the results obtained by the
Griess assay for compound 3, which are in strong contrast to the
‘selenium-free’ reference benzoquinone bq. Whilst bq does not
seem to interfere with NO production, compound 3, at a concen-
tration of just 2.5
macrophage (at this concentration 3 is not toxic to the cells).
Comparable results were obtained for compound 2, which, at the
same time, is also a good antioxidant in our assays (Fig. 5).
ꢀ
potent interceptor of NO-based cellecell signalling
ꢀ
The results obtained for the tellurium compounds in the MTT
cell survival test and the subsequent assays indicate that the be-
haviour of these compounds differs considerably from the one of
the selenium compounds. In strong contrast to the tellurium
compounds, the selenium analogues are generally considerably less
toxic against macrophages and also do not seem to generate any
significant amounts of ROS in the macrophages or yeast cells. Quite
on the contrary, some of the selenium compounds actually reduce
ROS levels induced by H2O2 or phorbol-12-myristat-13-acetat
(PMA) in a concentration-dependent manner.
ꢀ
ꢀ
m
M, prevents any notable NO production in the
Some compounds, such as (the reduced form of)
are known to react directly with NO and also inhibit apoptosis of
a
-lipoic acid,
ꢀ

10582
M. Doering et al. / Tetrahedron 68 (2012) 10577e10585
detail. There have been reports that the selenium compound
A
120
ebselen inhibits isolated iNOS when employed in the low micro-
molar range. Yet this effect seems to disappear in the presence of
thiols.46 Inside the cell, matters seem to be even more complicated,
as ebselen does not impact on iNOS expression, yet may interfere
with iNOS activity or sequester ꢀNO directly.47 Furthermore, ebselen
may also intercept intracellular redox signalling, hence acting on
the ꢀNO producing system more indirectly.48
100
80
60
40
20
0
ns
***
ns
***
ns
***
From a pharmacological perspective, the rather dramatic effect
exerted by low concentrations of 3, as seen in Fig. 6a, is of special
interest. Whilst macrophages are not killed at these concentrations,
their ability to generate ꢀNO is diminished, and hence some of their
pro-inflammatory actions may also be reduced. If this effect is
sufficient to reduce inflammation itself needs to be studied in more
complex models, such as animals. Nonetheless, other agents able to
control
2.5 µM
3
5 µM
10 µM
3 + 100 ng/ml LPS
Griess assay of compound 3
B
140
ns ***
ns
**
ns
*
ꢀ
interfere with the NO production of macrophages, such as plum-
120
100
80
bagin (5-hydroxy-2-methyl-1,4-naphthoquinone), have recently
attracted considerable interest and are already under discussion in
the context of a possible treatment of inflammatory diseases.49
Future studies need to investigate the potential of these and re-
lated selenium compounds (such as ebselen) as modulators of
60
40
ꢀ
macrophage NO generation in considerably more detail. Available
20
literature on this topic is still sparse and the various interactions of
ꢀ
ꢀ
ꢀ
selenium compounds with NO, NO generation, NO removal and
peroxynitrite (ONOOꢁ) therefore provide a fertile ground for future
studies.46e48,50
0
control
1.25 µM
2.50 µM
5 µM
6
6 + 100 ng/ml LPS
Griess assay of compound bq
In any case, the selenium-based approach of interfering with
ꢀNO generation and cellular signalling represents a milder, and
perhaps more sophisticated strategy towards anti-inflammatory
treatment compared with the tellurium-based approach, which
simply kills the macrophages. Nonetheless, the concentrations re-
quired in both scenarios differ (a couple of 100 nM of the tellurium
agents are required to kill the macrophages compared to low mi-
Fig. 6. Impact of some of the selenium compounds on ꢀNO production of macrophages
stimulated with LPS. Cells were incubated in the absence or presence of LPS with
compounds for 20 h. Panel A illustrates the ability of compound 3 to almost completely
suppress ꢀNO production at concentrations in the low micromolar range. In contrast,
the ‘selenium-free’ benzoquinone bq is unable to influence ꢀNO production, high-
lighting the special role of selenium in this process. Whilst these results point towards
a specific activity of the chalcogen in this process, it has not been possible to dem-
onstrate a similar effect for the tellurium analogues, as these compounds are too cy-
totoxic. Shown are the significances calculated with the Student’s t-test compared to
control.
ꢀ
cromolar concentrations of the selenium agents to suppress NO
production). Therefore statements referring to ‘milder’ effects, to
more specificity and less side-effects are premature and need to be
taken with some caution. Both strategies employ comparably low
concentrations of agents, and both may be quite effective and
selective.
certain cells by a more indirect mode of action.41,42 Therefore, in
order to rule out any simple chemical interactions between ꢀNO and
the compounds tested, and hence to confirm a genuine interference
with cellular processes, cell-free in vitro assays were carried out
3. Conclusions
using diethylamine nonoate diethylammonium (DEA) and S-
Overall, our studies confirm that selenium- and tellurium-
containing agents are not only active against cancer cells but also
bear considerable potential in the field of inflammatory patholo-
gies. The toxicity of some of the tellurium-containing agents
manifests itself in the sub-micromolar range, i.e., at concentrations,
which are considerably lower than the ones required for these
compounds to kill cancer cells and healthy control cells.5 Whether
macrophages are really the prime targets of such compounds, and
whether such an activity may be turned into a pharmaceutical
application, still needs to be shown. At the same time, the un-
derlying chemical interactions and biochemical processes remain
vastly unexplored. Whilst antioxidant enzymes (primarily SOD),
Oꢀ2ꢁ and redox processes appear to play an important role, the
elucidation of such complex signalling pathways requires consid-
erably more in-depth investigations in the future. Ultimately, the
chemical design of these redox-modulating agents may also be
refined further, for instance by exploring anthraquinone derivatives
as possible alternatives.
ꢀ
nitroso-N-acetyl-
D
,L-penicillamine (SNAP) as chemical NO donors.
In these cell-free assays, none of the compounds tested interfered
ꢀ
notably with NO levels, pointing towards an interference of com-
ꢀ
ꢀ
pounds such as 3 with the biosynthesis of NO (and not with NO
itself). Indeed, there are reports in the literature, which point to-
wards an inhibitory effect of quinones, sulfur (such as hydrogen
sulfide) and selenium compounds on iNOS, and it may be possible
ꢀ
that compound 3 acts as inhibitor of iNOS, which is the sole NO
generating synthase in macrophages. As the quinone bq is not ac-
tive, this effect seems to be in large parts due to the presence of
selenium. Indeed, iNOS is a haem-containing enzyme and strong
inhibitory ligand binding effects of sulfur and selenium compounds
at the haem centre are possible.43e45 Interestingly, benzoquinones
generally seem to be more active than naphthoquinones, which is
in contrast to our observations with the tellurium compounds (see
above).
One should note that the tellurium agents employed may also
ꢀ
impact on the synthesis of NO, yet these agents are also cytotoxic
Similarly, the perhaps rather surprising finding that some of the
selenium agents, despite the presence of various quinone moieties
in their respective structures, act as antioxidants and suppressors of
ꢀNO synthesis, bodes well for possible drug design but also poses
some interesting questions. Compounds, such as 2 and 3, which on
the one hand prevent ꢀNO formation by macrophages when used in
and hence such an effect has not been observed. The sulfur com-
ꢀ
pounds tested were generally not particularly active in the NO
assay (data not shown).
If, how, and how strongly such chalcogen-containing com-
pounds inhibit enzymes such as iNOS needs to be studied in more

M. Doering et al. / Tetrahedron 68 (2012) 10577e10585
10583
low micromolar concentrations and, on the other hand, also act as
(978 mg,
3.0 mmol)
and
2,5-dibromo-3,6-dimethyl-1,4-
antioxidants (albeit at higher concentrations), may counteract
some of the pro-inflammatory events associated with macrophages
and the aggressive chemical and signalling molecules they release.
It needs to be shown if this kind of ‘double impact’ on ROS and ꢀNO
levels is particularly beneficial or not.
benzoquinone (949 mg, 3.2 mmol) following the general pro-
cedure. Compound 2 was purified by repeated column chroma-
tography on silica gel using petrol ether/ethyl acetate (95:5 v/v) as
solvent, Rf¼0.50. Yield 23.4%, red solid, mp 112 ꢂC. 1H NMR:
d
¼7.45e7.44 (m, 4H, H-a), 7.24e7.22 (m, 6H, H-b and H-c), 1.99 (s,
Since tellurium compounds are generally more active in bi-
ological systems compared to their selenium analogues, the in-
fluence of (less cytotoxic) tellurium compounds on the macrophage-
6H, CH3). 13C NMR:
d
¼181.5 (2C), 148.1 (2C), 143.9 (2C), 133.7 (4C),
129.6 (2C), 129.5 (4C), 128.1 (2C), 17.8 (2C) ppm. 77Se NMR:
d
¼367.34 ppm. HRMS for C20H16O2Se2 (m/z): calcd: 477.95 (100%),
ꢀ
driven synthesis of NO represents a topic, which also needs to be
445.95 (92.2%), 443.95 (51.8%); found 447.9483 (100%), 445.9556
(96.37%), 443.9646 (55.23%).
addressed in earnest as part of future investigations.
In any case, our studies provide ample opportunities for future
interdisciplinary investigations in the field of selenium and tellu-
rium chemistry and biochemistry, redox modulation, macrophage
targeting and innovative anti-inflammatory drug design.
4.2.3. Synthesis of 2,6-bis(phenylselanyl)-3,5-dimethoxy-1,4-benzoqui-
none (3). Compound 3 was synthesized from diphenyldiselenide
(470 mg,
1.5 mmol)
and
2,6-dibromo-3,5-dimethoxy-1,4-
benzoquinone (441 mg, 1.3 mmol) following the general procedure.
Compound 3 was purified by repeated column chromatography on
silica gel using petrol ether/ethyl acetate (95:5 v/v) as solvent,
4. Experimental section
4.1. General procedure for the synthesis of compounds 1e17
Rf¼0.32. Yield 34.1%, black solid, mp 81 ꢂC. 1H NMR:
¼7.54e7.53 (m,
d
4H, H-a), 7.26e7.21 (m, 6H, H-b and H-c), 3.57 (s, 6H, CH3). 13C NMR:
Compounds 5e14 have been synthesized according to
literature procedures.5 Compounds bq and nq were purchased
from SigmaeAldrich. Compounds 1e4 have not been reported
in the literature. The bromides 2-bromo-3-methyl-1,4-
naphthoquinone, 2,5-dibromo-3,6-dimethyl-1,4-benzoquinone, 2-
bromo-5,6-dimethoxy-3-methyl-1,4-benzoquinone, 2,6-dibromo-
d
¼182.6 (1C), 174.9 (1C), 156.9 (2C), 134.7 (4C), 129.3 (2C), 129.1 (4C),
128.7 (2C), 128.3 (2C), 60.7 (2C) ppm. 77Se NMR:
d
¼355.35 ppm.
HRMS for C20H16O4Se2 (m/z): calcd: 479.94 (100%), 477.94 (92.3%),
475.94 (51.6%); found 479.9597 (100%), 477.9580 (97.56%), 475.9626
(53.00%).
3,5-dimethyl-1,4-benzoquinone
and
2-bromo-5-methyl-1,4-
4.2.4. Synthesis of 2-(phenylselanyl)-5-methyl-1,4-benzoquinone
(4). Compound 4 was synthesized from diphenyldiselenide
benzoquinone were prepared according to a literature procedure.51
(492 mg, 1.5 mmol) and 2-bromo-5-methyl-1,4-benzoquinone
(158 mg, 0.8 mmol) following the general procedure. Com-
pound 4 was purified by repeated column chromatography on
silica gel using petrol ether: ethyl acetate (95:5 v/v) as solvent,
Rf¼0.51. Yield 21.1%, orange solid, mp 112 ꢂC. 1H NMR:
4.2. General procedure
Under argon atmosphere, disulfide (or diselenide or ditelluride)
(1 equiv) was dissolved in a mixture of 100 ml THF and 25 ml water.
NaBH4 (w4 equiv) was added to the yellow or orange solution and
the mixture was stirred vigorously until it turned colourless. The
appropriate haloquinone (1 equiv for dihaloquinone or 2 equiv for
monohaloquinone) in THF (5 ml) was added and the formation of
the desired product was monitored via thin layer chromatography
(TLC). Afterwards the solution was stirred for further 15 min on air.
The violet, dark red or orange coloured reaction mixture
(depending upon the Te, Se or S counterpart of the product) was
diluted with saturated aqueous NH4Cl and extracted with ethyl
acetate. The combined organic extracts were dried over Na2SO4 and
the solvent was evaporated under reduced pressure. The crude
product was purified by silica gel chromatography (mesh size
d
¼7.82e7.80 (m, 2H, H-a), 7.74e7.66 (m, 3H, H-b and H-c), 7.50
(s, 1H), 6.45 (s, 1H), 5.46 (s, 1H), 1.80 (s, 3H, CH3). 13C NMR:
¼185.2 (1C), 184.7 (1C), 147.3 (1C), 137.1 (2C), 132.7 (1C), 130.6
(1C), 130.4 (2C), 130.3 (1C), 16.1 (1C) ppm. 77Se NMR:
d
d
¼414.08 ppm. HRMS for C13H10O2Se (m/z): calcd: 277.98 (100%),
275.99 (50.4%), 273.99 (18.9%); found 277.9823 (100%), 275.9825
(49.87%), 273.9939 (18.49%).
4.3. Cell culture
RAW 264.7 cells (murine macrophages, established from a tu-
mour induced by the Abelson murine leukaemia virus) were cul-
tured in RMPI medium containing 10% FCS, 5% glutamine, 5%
penicillin/streptomycin and kept in an atmosphere containing 5%
CO2 and at a temperature of 37 ꢂC.
40e60 m
m) using mixtures of petrol ether (40e65 ꢂC) and ethyl
acetate as specified for each compound below. Since the com-
pounds might be sensitive to oxidation and light, they were stored
under argon atmosphere and in the dark.
4.4. Statistics
4.2.1. Synthesis of 2,6-bis(phenylselanyl)-3,5-dimethyl-1,4-benzoqui-
none (1). Compound 1 was synthesized from diphenyldiselenide
Unless stated otherwise, all experiments were performed three
times and in triplicate. All graphs represent meansꢃSE as error
bars. Statistical differences were performed using independent
two-sample Student’s t-test in Microsoft Excel. MTT assays were
analyzed using one-way ANOVA at a significance level of 0.001
followed by a Bonferroni test (OriginPro 8.6G Software, OriginLabs,
Northampton, MA, USA). ***: P<0.001, **: P<0.01, *: P<0.05.
(376 mg,
1.2 mmol)
and
2,6-dibromo-3,5-dimethyl-1,4-
benzoquinone (354 mg, 1.2 mmol) following the general pro-
cedure. Compound 1 was purified by repeated column chroma-
tography on silica gel using petrol ether/ethyl acetate (95:5 v/v) as
solvent, Rf¼0.51. Yield 8.6%, red solid, mp 112 ꢂC. 1H NMR:
d
¼7.25e7.24 (m, 4H, H-a), 7.04e7.01 (m, 6H, H-b and H-c), 1.90 (s,
6H, CH3) ppm 13C NMR:
(2C),133.6 (4C),129.6 (2C),129.5 (4C) 128.0 (2C),17.5 (2C) ppm. 77Se
NMR:
d
¼182.5 (1C), 180.0 (1C), 147.8 (2C), 144.1
4.5. MTT assay
d¼371.25 ppm. HRMS for C20H16O2Se2 (m/z): calcd: 477.95
(100%), 445.95 (92.2%), 443.95 (51.8%); found 447.9486 (100%),
445.9583 (93.10%), 443.9655 (55.60%).
The MTT assay was performed as described in the literature.52
Briefly, cells were seeded in a 96-well plate at a density of
40,000 cells/100
treated with test compounds in 2.5, 5, 10, 15, 20, 25 and 100
the absence or presence of 30 M H2O2. Cells were also incubated
m
l and allowed to adhere for 6 h. Then cells were
4.2.2. Synthesis of 2,5-bis(phenylselanyl)-3,6-dimethyl-1,4-benzoqui-
none (2). Compound 2 was synthesized from diphenyldiselenide
mM in
m

10584
M. Doering et al. / Tetrahedron 68 (2012) 10577e10585
with 0.2% DMSO, which was used as solvent for the tested com-
pounds and served as control. After incubation for 24 h the medium
4.8. Yeast strains and cultivation
was removed, 150
incubated for 40 min. Then MTT was removed and cells were lysed
by addition of 200 l DMSO. The absorbance was measured at
ml MTT (0.5 mg/ml in medium) were added and
Yeast strain BY474255 and its mutant derivatives (see table) were
obtained from Euroscarf, University of Frankfurt, Germany (http://
web.uni-frankfurt.de/fb15/mikro/euroscarf/) and grown overnight
in liquid complete synthetic dropout medium (CSM; 7 g/l yeast
nitrogen base; ForMedium, Hunstanton, Norfolk, UK; 0.8 g/l com-
plete dropout, Vista, CA, USA; 40 g/l glucose, supplied by Carl Roth,
Karlsruhe, Germany) with shaking (210 rpm) at 28 ꢂC.
m
a wavelength of 550 nm and 690 nm was used as reference wave-
length. Each concentration was tested in three independent ex-
periments and in triplicate. A preliminary experiment showed that
the appropriate solvent control containing 0.2% DMSO had no
statistically significant effect on the cell viability and thus all cal-
culated cell viabilities are expressed relative to 0.2% DMSO. IC50
values were calculated using OriginPro8.6.
Mutant
ORF
Genotype
; his3
gsh1
D
D
YJL101C
MAT
YJL101c::kanMX4
MAT ; his3 1; leu2
YOL049w::kanMX4
MAT ; his3 1; leu2
YPL091w::kanMX4
MAT ; his3 1; leu2
YJR104c::kanMX4
MAT ; his3 1; leu2
YHR008c::kanMX4
MAT ; his3 1; leu2
YGR088wc::kanMX4
a
D
1; leu2
D
D
D
D
D
D
0, lys2
0, lys2
0, lys2
0, lys2
0, lys2
0, lys2
D
D
D
D
D
D
0, ura3
0, ura3
0, ura3
0, ura3
0, ura3
0, ura3
D
D
D
D
D
D
0;
0;
0;
0;
0;
0;
4.6. Griess assay
gsh2
YOL049W
YPL091W
YJR104C
a
D
The Griess assay was performed according to a literature pro-
glr1D
a
D
cedure.53 Briefly, cells were seeded in a 96-well plate at a density of
sod1
sod2
D
D
a
D
80,000 cells/200 ml. After allowing the cells to adhere for 6 h, cells
were treated with the test compounds in the presence or absence of
100 ng/ml LPS (ultrapure from Escherichia coli, K12 strain. TLR 4
ligand). DMSO in the appropriate concentration was used as con-
YHR008C
YGR088W
a
D
ctt1D
a
D
trol. After incubation for 20 h, 100
ml of the supernatant were
transferred into a 96-well plate and 90
ml of sulfanilamide and 90
ml
of N-(1-naphthyl)-ethylene-diamine were added to quantify nitrite
ꢀ
as a metabolite of NO. NaNO2 was used for the standard curve on
4.9. Lucigenin-assay for ROS accumulation
the same plate. The absorbance at 550 nm was measured using
a microplate reader against the background of 690 nm. For the
determination of cell viability, MTT assay was performed as de-
scribed. In a preliminary experiment different concentrations of LPS
The lucigenin assay was employed as a general method for
quantification of ROS accumulation rather than as a specific probe
for superoxide accumulation, since the specificity of lucigenin for
Oꢀ2ꢁ has been called into question.9,56 Overnight yeast cultures were
adjusted to an OD600¼1 and treated with the test compounds,
(50 ng/ml to 1 mg/ml) were tested to determine the effective con-
centration of LPS. To make sure that the effects observed were not
an interaction of the test compounds with Griess reagents them-
selves, the same assay was performed without cells42 using the ꢀNO
donors diethylamine nonoate diethylammonium salt (DEA) or S-
dissolved in DMSO, at end concentrations of 10 and 100 mM. Con-
trols were treated with DMSO alone. The final concentration of
DMSO in the cultures was 0.6 mM. Cultures were incubated for
a further 16 h (28 ꢂC, 210 rpm). The same treatment was performed
with cell-free medium in order to exclude a chemical and cell-
independent formation of ROS.
Lucigenin (bis-N-methylacridinium nitrate; SigmaeAldrich,
Steinheim, Germany) was dissolved in DMSO to a concentration of
50 mM. Lucigenin-solution (100 ml) was added to a 100 ml aliquot of
nitroso-N-acetyl-
of test compounds were added to a solution containing 100
or 100 M SNAP and allowed to incubate for 15 min (DEA) or 7 h
D
,L-penicillamine (SNAP). Briefly, 10
mM or 100
mM
mM DEA
m
ꢀ
ꢀ
(SNAP). The amounts of NO released by the NO donors were not
statistically significantly changed in the presence of the test
compounds.
yeast culture and the emission of photons by luminiscence was
quantified over an interval of 10 s using a luminometer (Lumat
LB9501, Berthold, Wildbad, Germany).
4.7. ROS assay
The ROS assay was performed as described in the literature.54
Cells were seeded with a density at 80,000 cells/200
well plate and allowed to adhere for 4 h. Then, 100
medium were removed and 100 M of the test compounds were
added to yield final concentrations of 5, 10, 25, 50, 75, 100 and
150
M. Cells were incubated at 37 ꢂC for 25 min. Afterwards the
complete medium was removed, cells were washed with warm
HBSS and 200 l of 20
M 20,70-dichlorodihydrofluorescein diac-
etate in HBSS were added. After incubation for 25 min, cells were
washed with HBSS, 200 l of HBSS were added and cells were
stimulated with 1 M PMA or 50 M H2O2. Cells only treated with
m
l in a 96-
4.10. Quantification of cell survival
m
l of the
m
Yeast cells were grown overnight as described above. The
culture was adjusted to an OD600¼1 and test compounds were
m
added to final concentrations of 10 and 100 mM. Cells were in-
cubated further for 16 h (28 ꢂC, 210 rpm). Subsequently, dilution
series of the yeast cultures were plated onto YPD plates (10 g/l
yeast extract; Carl Roth, Karlsruhe, Germany; 20 g/l pepton;
Duchefa, Haarlem, The Netherlands; 20 g/l glucose; Carl Roth,
Karlsruhe, Germany; 2% agar) and incubated at 28 ꢂC. The number
of colony forming units (cfu) was counted. Cell survival after
treatment with the test substances is expressed as a percentage of
the control colony counts.
m
m
m
m
m
DMSO, dye and stimulus served as control. The fluorescence was
followed for 40 min in a fluorescence reader (Wallac Victor 2) at
a temperature of 37 ꢂC using the excitation filter set at 485 nm
and the emission filter set at 535 nm. In a control experiment,
different concentrations of PMA and H2O2 were tested and 1
m
M
Acknowledgements
of PMA and 50 M of H2O2 were found to differ significantly from
m
the cells treated only with dye. Another control experiment, in
which cells were only incubated with the test compounds and
stimuli in the absence of dye, showed no fluorescence, proving
that neither test compounds nor stimuli induced fluorescence by
themselves.
The authors acknowledge financial support from Saarland Uni-
versity and RWTH Aachen University, the Government of Saarland,
the Interreg IVa Programme (Corena-Network; 35GR11051, to C.J.)
and the European Community’s Seventh Framework Programme
(FP7/2007e2013) under grant agreement 215009 RedCat (to C.J.),

M. Doering et al. / Tetrahedron 68 (2012) 10577e10585
10585
25. Giles, N. M.; Giles, G. I.; Holley, J. E.; Gutowski, N. J.; Jacob, C. Biochem. Phar-
macol. 2003, 66, 2021e2028.
26. Butler-Gralla, E. In Oxidative Stress and the Molecular Biology of Antioxidant
Defense; Scandalios, J. G., Ed.; Cold Spring Harbor: New York, NY, 1998.
27. Auerbach, D.; Arnoldo, A.; Bogdan, B.; Fetchko, M.; Stagljar, I. Curr. Proteom.
2005, 2, 1e13.
the Deutsche Forschungsgemeinschaft (KI702, to A.K.K., JA1741/2-1
to C.J.) and VEGA Grant Agency of the Slovak Republic (2/6082/26,
to M.C.) for financial support. The authors would further like to
thank Ulrike Noll, Danusa Vlasakova and Dr. Josef Zapp for their
technical assistance.
28. Letavayova, L.; Vlasakova, D.; Vlckova, V.; Brozmanova, J.; Chovanec, M. Mutat.
Res. 2008, 652, 198e203.
29. Miller, A. F. FEBS Lett. 2012, 586, 585e595.
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