Multifunctional redox catalysts as selective enhancers of oxidative stress

Article abstract of DOI:10.1039/b502197a

Certain cancer cells proliferate under conditions of oxidative stress (OS) and might therefore be selectively targeted by redox catalysts. Among these catalysts, compounds containing a chalcogen and a quinone redox centre are particularly well suited to r

Full text of DOI:10.1039/b502197a

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A R T I C L E
Multifunctional redox catalysts as selective enhancers of oxidative
stress
a
a
a
a
a
Fiona H. Fry,† Andrea L. Holme,† Niroshini M. Giles, Gregory I. Giles, Catriona Collins,
a
a
b
b
c
Kim Holt, Sandra Pariagh, Thomas Gelbrich, Michael B. Hursthouse, Nick J. Gutowski
and Claus Jacob*
School of Biological and Chemical Sciences, University of Exeter, Stocker Road, Exeter,
a,d
a
UK EX4 4QD
b
School of Chemistry, University of Southampton, Highfield, Southampton, UK SO17 1BJ
Peninsula Medical School, Universities of Exeter and Plymouth and The Royal Devon and
c
Exeter Hospital, Exeter, UK EX2 5DW
Fachbereich 8.2 Pharmazeutische und Medizinische Chemie, Universit a¨ t des Saarlandes,
d
Postfach 151150, D-66041, Saarbr u¨ cken, Germany. E-mail: c.jacob@mx.uni-saarland.de;
Fax: +49 (0)681 302 4386; Tel: +49 (0)681 302 2424
Received 11th February 2005, Accepted 20th May 2005
First published as an Advance Article on the web 9th June 2005
Certain cancer cells proliferate under conditions of oxidative stress (OS) and might therefore be selectively targeted
by redox catalysts. Among these catalysts, compounds containing a chalcogen and a quinone redox centre are
particularly well suited to respond to the presence of OS. These catalysts combine the specific electrochemical
features of quinones and chalcogens. They exhibit high selectivity and efficiency against oxidatively stressed rat PC12,
human Jurkat and human Daudi cells in cell culture, where their mode of action most likely involves the catalytic
activation of existent and the generation of new reactive oxygen species. The high efficiency and selectivity shown by
these catalysts makes them interesting for the development of anti-cancer drugs.
Introduction
detailed account of the synthesis and electrochemical analysis
of the compounds. Importantly, we also show that the sensor–
effector properties of such redox catalysts are not limited to
PC12 cells, but are also observed in human Jurkat and Daudi
cell lines and that the probable mode of action of the catalysts
involves an increase in levels of intracellular OS.
Several cancer cells, such as the cells of lung, kidney and
prostate cancer, possess significantly elevated intracellular levels
of reactive oxygen species (ROS). The latter include, among
1
–2
•
−
others, the superoxide anion radical (O
2
), the hydroxyl radical
•
(
OH), hydrogenperoxide (H
2
O ), organic peroxides and radicals
2
3
and singlet molecular oxygen. In healthy cells, the concentration
of these chemical species is low and increases in the level of
ROS, that are within the micromolar range for some stressors,
Results
Selection and synthesis of multifunctional catalysts
4–5
can be seen as good diagnostic markers of OS related diseases.
Compounds 1–4 (Fig. 1) were chosen since they combine two
individual redox systems, i.e. a quinone and a chalcogen, in one
molecule, which should allow them to participate in a broad
spectrum of redox reactions. The compounds were then studied
in a range of assays, with the aim to gain a more comprehensive
understanding of their chemical and biochemical properties,
biological activities and potential mode(s) of action inside cells.
While the electrochemical studies confirmed the presence of
two individual redox centres in these molecules, the in vitro
catalytic assays indicated the ability of 2–4 to participate in
catalytic peroxidation and radical generation reactions. The
activity of the catalysts was therefore evaluated in cell culture and
the anticipated cytotoxic effects in the presence of peroxide were
observed not only in the rat PC12 cell line, but also in human
Jurkat and Daudi cell lines. Further evaluation with intracellular,
fluorescent redox indicators resulted in preliminary biochemical
results pointing towards a (catalytic) increase of intracellular
OS, with apoptotic cell death as the probable mode of action of
these catalytic compounds.
We have recently proposed that compounds able to use these
naturally occurring ROS markers to kill cells could possess
an interesting combination of sensor and effector properties
that might make them selective in targeting oxidatively stressed
6–8
cells. Within this context, we have shown that catalytic agents
mimicking redox enzymes such as glutathione peroxidase (GPx)
are particularly good candidates, since they combine selectivity
for their natural (ROS) substrate(s) with high (catalytic) effi-
6–8
ciency at low doses.
Among such catalysts, multifunctional compounds able to
respond to a range of oxygen species are of special interest,
since they can take full account of the complex cellular redox
environment existing during OS. Preliminary studies in cultured
mammalian PC12 cells have already shown that compounds
integrating two redox groups, i.e. a redox active quinone and
a chalcogen moiety, are particularly suited to discriminate
between oxidatively stressed and unstressed cells in culture and
6–8
that some catalysts are active in concentrations below 1 lM.
At the time, however, the ability of such catalysts to effectively
and selectively kill cells under OS was only demonstrated for
undifferentiated rat PC12 cells and the precise redox behaviour
and mode of action of these compounds remained largely
unexplored.
Here we present a more comprehensive study of the activities
of such catalysts in vitro and in different bioassays, including a
The following paragraphs describe the results obtained in the
structural, electrochemical, biochemical and cell culture studies
of 1–4 in more detail.
Structural and electrochemical analysis
Structural analysis was employed to ensure the presence and
correct configuration of the quinone and chalcogen redox
centres. The X-ray crystal structure of tellurium compound 3
†
These authors contributed equally.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 7 9 – 2 5 8 7
2 5 7 9

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6
Table 1 Electrochemical parameters of 1–5. Potentials for the quasi-reversible quinone redox couple (Epa
oxidation (Epa
1
and Epc) and the irreversible chalcogen
2
) at pH = 7.4 are provided. pH-Dependence of the oxidation and reduction potentials plays a major role in the pH control of the
redox activity of the compounds. DE/DpH values were determined between pH 4 and pH 9. Experimental details are given in the text. Experimental
error ± 10 mV
Quinone
Epa /mV
Quinone
Epc/mV
Quinone
Chalcogen
Epa /mV
Compound
1
E
1
2
/mV
2
DEpa
1
/DpH/mV
DEpc/DpH/mV
2
DEpa /DpH/mV
1
2
3
4
5
−70
−57
−302
−328
−341
−341
−277
−186
−193
−228
−229
−222
+1348
+1128
+823
+760
—
−89
−71
−73
−75
−76
−89
−42
−40
−40
−38
+13
+7
a
−115
−116
−166
a
—
a
For 3 and 4, Epa
2
decreases from pH 4 to 7.4 with DEpa
2
2
/DpH = −41 mV and −62 mV, respectively. Epa increases from pH 7.4 to 9 with
DEpa
2
/DpH = +120 mV and +187 mV, respectively.
The two redox centres in 1–4 were then analysed by cyclic
voltammetry. The latter provides insight into the oxidation
and reduction processes such compounds might participate in,
6
,11,12
both in vitro and in vivo.
We have recently communicated
6
preliminary electrochemical results for 1–4. In short, these
compounds exhibited a combination of two distinct redox
behaviours indicative of one quinone and one chalcogen centre
(
Table 1). At pH 7.4, the quinone redox couple (Epa
appeared at around −200 mV vs. SSE, with quasi-reversible
characteristics, such as a peak current ratio (|Ipa /Ipc|) ≈ 0.7
and transfer coefficients for a between 0.9 (3) to 1.0 (4) and
for a between 0.6 (3) to 0.7 (4). Assuming a (standard) value
of 0.5 for a, this would imply n values of 1.8 for the reduction
1
and Epc)
1
c
n
a
a
n
a
a
peak and 1.4 for the oxidation peak, i.e. approximately two
electrons transferred during reduction of the quinone to the
dihydroquinone and between one and two during subsequent
6
oxidation to the semiquinone and quinone, respectively.
The quinone redox centre in 1–4 was under pH control
(
Table 1). Both Epa
1
and Epc values of 1–5 consistently
/DpH ≈ −75 mV
decreased between pH 4 and 9, with DEpa
1
for 1–5 and DEpc/DpH ≈ −40 mV for 1–5. These results
imply a shift of the oxidation and reduction potentials of the
quinone moiety to more reducing potentials upon an increase
of the pH. From a chemical perspective this is hardly surprising,
since the reduction of the quinone to semi- and dihydroquinone
involves a protonation step at oxygen, while oxidation to
quinone involves a deprotonation step. Deprotonation of the
OH-group at higher pH values therefore makes the semi- and
dihydroquinone forms more reducing.
Fig. 1 Synthesis and chemical structures of multifunctional com-
pounds used in this study. For E = Se: (i) Bu P–THF–argon; (ii) NaOH.
–EtOH–argon; (ii) not applicable. Details of
3
For E = S, Te: (i) NaBH
4
the chemical synthesis are described in the text. The structure of ebselen
is given as a comparison.
The chalcogen centre resulted in an irreversible oxidation peak
6
(
Epa
2
) between +760 mV (4) and +1350 mV (1). In agreement
with previous studies, the sulfur compound (1) exhibited the
highest oxidation potential and the tellurium compounds 3 and
4
the lowest. Electron withdrawing effects of the quinone led to
more positive oxidation potentials for the chalcogen atoms in 2
and 4 when compared to their previously studied symmetrical
ꢀ
ꢀ
4
,4 -dimethoxydiphenyl selenide (Epa = +753 mV) and 4,4 -
6
dimethoxydiphenyl telluride (Epa = +368 mV) analogues.
Chalcogen oxidation in 1–4 was also under pH control. Unlike
the quinone oxidation and reduction potentials for 1 and 2,
chalcogen oxidation potentials increased with increasing pH,
Fig. 2 Molecular structure of 3. Selected bond lengths ( A˚ ) and angles
◦
A˚ .
(
): Te1–C1 = 2.124(3) and Te1–C7 = 2.119(3) A˚ ; C1–Te–C7 = 96.64(13)
albeit with considerably smaller changes (DEpa
mV for 1 and 7 mV for 2). The pH-dependency of Epa
the tellurium compounds 3 and 4 was more complex, with an
initial decrease in Epa with between pH 4 and 7.4 followed by
a sharp increase between pH 7.4 and 9 (Table 1). This somewhat
different redox behaviour of tellurium compounds 3 and 4,
when compared with the sulfur and selenium analogues, might
be related to their high activity in the PhSH and MT assays.
2
/DpH = 13
2
of
was successfully solved‡ and is shown in Fig. 2. This structure
confirms the close proximity of the quinone and tellurium redox
2
˚
centres. The Te–C bond lengths (Te1–C1 = 2.124(3) A and Te1–
˚
C7 = 2.119(3) A) and the bond angle centred on the tellurium
◦
(
C1–Te–C7 = 96.64(13) ) are in good agreement with literature
values, indicating a normal fusion of the two parts of the
9–10
molecule.
Tentatively, an Epa minimum at around 7.4 would imply highest
2
peroxidation activity of 3 and 4 at physiological pH.
Overall, cyclic voltammetry confirmed the presence of two
individual redox centres in 1–4, both of which were under pH
control. While the centres influenced each other, e.g. via electron
‡
CCDC reference number 269419. See http://www.rsc.org/suppdata/
ob/b5/b502197a/ for crystallographic data in CIF or other electronic
format.
2
5 8 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 7 9 – 2 5 8 7

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withdrawing effects, there was little enhancement or synergy
between them. This was not surprising, as there was a substantial
difference between the chalcogen and quinone redox potentials.
Redox behaviour was further evaluated in bioassays indicative
of redox catalysis.
order of activity of 2–4 in the PhSH assay corresponded to the
oxidation potential of the chalcogen (Epa
2
), with 4 having the
lowest Epa value and highest activity and 2 having the highest
2
potential and lowest activity.
The PhSH assay was complemented by the MT assay.
6
Compared to the PhSH assay, the latter is less prone to
interference and can be carried out in buffered solution at pH 7.4
Thiophenol and metallothionein peroxidation assays
(avoiding the use of methanol as solvent). As would be expected
Based on the electrochemical results, 2–4 in particular were
expected to exhibit catalytic quinone and chalcogen redox
behaviour in vitro (Fig. 3). The latter was measured in two
independent assays; the thiophenol (PhSH) based glutathione
for tellurium-based GPx mimics, 3 and 4 effectively enhanced
cysteine protein oxidation in the presence of peroxide (Table 2).
Compounds 1 and 2 had a similar, yet lower activity, in line
with the results obtained in the PhSH assay and confirming
a previously reported correlation between chalcogen oxidation
potential and zinc release, and a generally lower activity of sulfur
and selenium compounds when compared to their tellurium
13
14
peroxidase (GPx) assay and the metallothionein (MT) assay.
The PhSH assay is a simple catalytic assay measuring the
peroxidation of PhSH by hydrogen peroxide (Table 2). The
results obtained showed significant increases in the rate of
PhSH oxidation in the presence of selenium and tellurium
compounds 2–4, with 13- to 25-fold increases over the control
11–15
analogues.
It should be noted that although the ranking of compounds
is similar in the PhSH and MT assays, relative activities of
compounds differ in the two assays. This is hardly surprising,
since both assays employ significantly different parameters, such
as different solvents, oxidants and thiol species.
−
1
level (5.38 nM s ). In stark contrast, ebselen, used as a known
benchmark, and the menadione 5 control showed no significant
activity under the experimental conditions. Interestingly, the
Fig. 3 Schematic view of the multifunctional properties of 1–4, as illustrated on the most active compound 3. Quinone-like radical generating
activity, biologically important in generating oxidative stressors, has been indicated by the CR assay. GPx-like peroxidation catalysis, biologically
6
important in using oxidative stressors to inflict cell damage, has been confirmed independently in the PhSH and MT assays.
Table 2 Activity of 1–5 in protein-based in vitro assays. Chalcogen-based peroxidation activity is shown in the GPx and MT assays. The CR assay is
indicative of quinone-based NADH consumption and superoxide generation in the presence of dioxygen. Together with the electrochemical findings,
this information has been used to select compounds for testing in cell culture. Experimental details are given in the text
−
9
Initial reduction rate of
MT assay rate zinc
release ± 5%/10 Ms
Zinc release from MT after
60 min with BuOOH ± 5% (%)
CR rate/10
Ms
CR number of
turnovers after 2 h
−
9
−1
−10
−1b
a
−1
c
Compound
H
2
O
2
± 5%/10 Ms
a
1
nd
67
84
0.8
0.8
2.2
2.0
—
12
12
28
28
9
8.6
5.4
8.7
2.7
29.2
—
7.2
6.4
6.3
2.8
7.8
—
2
3
4
129
5
5
Ebselen
10
1.3
7
a
b
c
Not determined. Values have been corrected for background reaction, i.e. reaction without any test compound present. Turnover numbers were
established using the consumption of 1 mole eq. of NADH as 1 turnover. CR concentration in this assay was 6.5 lM.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 7 9 – 2 5 8 7
2 5 8 1

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Cytochrome C reductase assay for quinones
Redox activity of the quinone moiety was evaluated in the
cytochrome C reductase (CR) assay. The results shown in
Table 2 indicate that the quinone compounds are recognised as
substrates by CR. Although there was some variation in activity,
all compounds tested resulted in CR-catalysed oxidation of
NADH. The initial rates observed for 1–4 were somewhat lower
than the one of the parent menadione 5, with 1 and 3 being the
most active and 4 the least.
This assay also indicated that the quinones were acting as
coenzymes in a coupled system with CR, as the consumption of
NADH continued beyond the oxidation of 1 eq. of NADH per
quinone in the presence of a 10-fold excess of NADH. Again,
menadione 5 was most active, and consumed 8.6 eq. of NADH
over a 120 min period (out of a theoretical maximum of 10).
Similarly, sulfur compound 1 turned over 7.2 eq. of NADH,
while 2 and 3 turned over 6.4 and 6.3 eq. of NADH, respectively.
In line with the lowest initial rate observed among 1–5, 4 only
consumed 2.8 eq. of NADH over the 120 min period.
The oxidation of NADH beyond 1 mole eq. per quinone is
incompatible with a stoichiometric oxidation, as only 2 electrons
are required to fully reduce the quinone to the dihydroquinone.
Rather, it suggests that the dihydroquinone moiety is not the final
reduction product and that the reaction involves the generation
of the superoxide radical anion, a process frequently observed
for quinones such as plumbagin under aerobic conditions
6,16
in vitro (Fig. 3). Control incubations of CR with either NADH
or catalysts individually showed no change in absorbance at
3
40 nm, neither did incubation of catalyst and NADH in the
absence of CR.
Fig. 4 Cytotoxicity assays for 3 in the absence ( ) and presence ( ) of
5
2 2
0 lM H O . Fig. 4a show the results for human Jurkat and Fig. 4b for
human Daudi cell culture. Experimental details are given in the text.
Cytotoxicity in response to increased peroxide concentrations in
PC12, Jurkat and Daudi cell lines
although additional cell lines need to be considered before a full
generalisation can be made.
In order to evaluate the effect of the compounds on cells in the
absence and presence of hydrogen peroxide, rat PC12, human
Jurkat (J16) and human Daudi cell lines were chosen. These
cell lines are representative of cancer cells that proliferate in vivo
under OS conditions. Compound 3 was selected for these assays
because it exhibited the highest activity in the MT and CR assay.
At low concentrations (100 nM), 3 was non-toxic to cultured
Preliminary mechanistic studies
The notion that catalysts like 3 might become toxic by increasing
the severity of OS, either by generating additional ROS or by
catalysing reactions with existing ROS, was then investigated in a
series of preliminary mechanistic studies. It should be noted from
the outset that the results obtained so far provide an indication
of a likely mode of action, but not conclusive proof.
PC12 cells that were not challenged by the addition of H
2
2
O .
In the presence of this oxidative stressor however, cell survival
6
dramatically dropped to 45%. While the parent quinone 5
also promoted cell death under oxidative stress conditions, a
The 2,7-dichlorodihydrofluorescein acetate (DCDHF) assay
was chosen to measure the overall levels of intracellular OS.
17–19
significantly higher concentration (3.2 lM) was required to
ꢀ
obtain the same effect. Compound 3 was also far superior to 4,4 -
Although this assay is not specific for individual stressors
and can be affected by hydrolysis of the dye, it is useful in
obtaining an indication of intracellular stress levels during
application of the catalysts. Fig. 5 shows the kinetic profile
of oxidative stress generation in Jurkat and Daudi cells upon
addition of 2 and 3, as assessed by the formation of fluorescent
carboxydichlorofluorescein from carboxydichlorodihydroflores-
cein. This figure indicates that both compounds, in the absence of
added peroxide, were able to significantly increase intracellular
OS conditions inside the two cell lines over a 2 h period. In this
respect, 2 and 3 had similar activity, with 3 being slightly more
active than 2 in the Daudi cell line. In the Jurkat cell line, 2
increased fluorescence by 2.2-fold over the control, compared
to 2.4-fold for 3 (Fig. 5a). In the Daudi cell line, 2 increased
fluorescence by 1.6-fold over the control, compared to 2.1-fold
for 3 (Fig. 5b).
dihydroxydiphenyl telluride, the most active GPx mimic studied
8
earlier. Administration of 10 lM of this compound reduced cell
survival to 45%, whereas the same effect was observed for 3 at
1
00 nM concentration, an increase in efficacy of two orders of
magnitude.
Selectivity for cells under OS was confirmed in the human
Jurkat and Daudi cell lines (Fig. 4). For Jurkat cells exposed to
1
6
00 nM of 3 in the absence of H
7.4%, while the addition of 50 lM H
2
O
2
the survival observed was
(in itself not toxic at
2
O
2
this concentration) decreased this to 27.8%, as shown in Fig. 4a.
Compound 2 had a similar overall effect, where the survival
of Jurkat cells was decreased from 115% to just 54.5% in the
presence of 100 nM of this selenium compound. In contrast,
2
and 3 were not particularly toxic on their own, i.e. towards
the cells not under OS, for catalyst concentrations below 1 lM
Fig. 4). Catalyst concentrations of 1 lM and above were toxic.
(
In order to investigate the relationship between 3 and OS
further, the monochlorobimane (MCB) method was used to
estimate changes in intracellular levels of reduced glutathione
(GSH). As with the DCDHF assay, this method is not without
drawbacks but has been extensively used in the past to estimate
A similar effect was observed for 3 in Daudi cells (Fig. 4b).
Overall, these cell culture experiments indicated that the
catalysts (at low concentrations) reduced cell survival in cells
under OS, but at these concentrations had little toxicity on their
own, i.e. in the absence of peroxide. This effect was not limited
to PC12 cells but instead seemed to be of a more general nature,
20–21
GSH levels.
As expected for an enhancer of OS, 3 also
caused a significant decrease of the intracellular GSH level in
2
5 8 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 7 9 – 2 5 8 7

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might become an important feature of these compounds, since
increases of OS are frequently associated with changes in pH.
In contrast, the significant differences in the chalcogen
oxidation potentials of 1–4 allow an electrochemical ranking,
with compounds with the lowest Epa values, i.e. 3 and 4 at
2
around neutral pH, expected to exhibit the highest activity in the
chalcogen-centred peroxidation assays (GPx and MT assays).
Peroxidation catalysis and superoxide generation in vitro
Building on the electrochemical studies, the catalytic in vitro
assays have confirmed that 1–4 are multifunctional and can
respond to different oxidative stressors. For example, the se-
lenium and tellurium atoms in 2–4 form the centre for effective
peroxidation (Fig. 3). Although the activity of compounds
in the MT assay was generally reduced when compared to
other, non-quinone selenium and tellurium catalysts, the two
oxidation assays demonstrate the ability of these compounds to
use peroxide in order to oxidise thiol proteins, i.e. to act as en-
hancers of OS. We have already shown that related catalysts are
able to attack different, essential cysteine proteins, such as MT
8
and zinc finger proteins, and recent, independent studies have
demonstrated the ability of similar chalcogen compounds to
22
attack zinc finger proteins involved in DNA repair.
The quasi-reversible, one and two electron transfer behaviour
of the quinone moiety enables oxidation reactions. The reduced
activity of 1–4 in the CR assay, when compared to 5, might
be explained by steric effects in the active site of CR, caused
by the presence of the chalcogen atom and its attached phenyl
ring system. The X-ray crystal structure of 3‡ shows the close
proximity of the tellurium atom and its phenyl ring system
to the quinone redox centre, making a steric effect likely.
The surprisingly high quinone-activity of sulfur compound 1,
when compared to the selenium and tellurium analogues, could
therefore be due to a decrease in hindrance from tellurium to
sulfur. Nevertheless, electronic effects, which also change from
sulfur to tellurium, cannot be ruled out.
Fig. 5 Fluorescent DCDHF assays indicative of intracellular OS stress
levels. Fig. 5a shows the results obtained for 2 and 3 in Jurkat cells and
Fig. 5b in Daudi cells (both cell lines were treated with compounds in
the absence of other external OS). In all cases, the basal OS stress level in
untreated compounds and an increased OS stress level caused by added
5
2 2
0 lM H O are given as negative and positive controls, respectively.
Experimental error ± 5%.
As mentioned above, the consumption of excess of NADH,
ranging from 7.2 mole eq. for 1 to 2.8 mole eq. for 4 within
Jurkat cells to 59%. GSH depletion by 3 could be abrogated,
however, by the addition of the antioxidant N-acetyl-L-cysteine
1
20 min, implies that CR-catalysed quinone reduction to
(
NAC). In these experiments, NAC slightly increased the GSH
dihydroquinone cannot be the only redox process in the assay.
The turnover numbers observed in the CR assay should therefore
be tentatively interpreted as a coupled redox process, where
dioxygen re-oxidises the semi- or dihydroquinone (Fig. 3). Such
a process, which is well known for menadione 5, results in
the formation of the superoxide radical anion and hydrogen
level to 125% of control and this level was then maintained
in spite of the presence of 3, supporting the notion that the
3
was acting via a redox pathway, rather than by inhibiting
GSH synthesis. Further evidence of a link between 3, increases
in intracellular OS levels and apoptotic cell death was obtained
using a combination of DCDHF, PI and DAPI staining in Jurkat
and Daudi cells (results not shown). The precise mode of cell
death is important for drug development and is currently being
investigated in considerably more detail.
6,23
peroxide.
The latter might be used as substrate by the
chalcogen moiety of the compound. Although further studies of
the quinone moiety would be required for a deeper interpretation
of these results, radical generation might play a major role
in the biological activity of 1–4. Superoxide, or some of its
metabolic products (e.g. hydroxyl radicals), can lead to serious
cell damage, especially in cells already under OS or with an
impaired antioxidant defence.
Discussion
Redox catalysts have the ability to influence intracellular redox
7
processes, either as pro- or antioxidants. Since catalysts rely on
The presence of two or more independent redox sites, each
of which can be fine-tuned in its activity, is a characteristic
important for the future development of potential catalytic
drugs. Additional functional groups, such as metal binding sites
or an amine anchor for attachment to transport proteins, might
also be included into the design and added with comparable
the presence of their substrates, such as ROS, for activity, they
combine important sensor with effector functions that might
be used to specifically target cells, such as cancer cells, under
OS. The results presented here support the notion that catalysts
develop selective toxicity in response to existing ROS.
24–25
ease.
Redox properties and pH control
The electrochemical results have confirmed the presence of two
distinct redox couples in 1–4. The quasi-reversible electrochemi-
cal behaviour of the quinone-moiety in these compounds, which
is comparable to the one of the parent menadione 5, points
towards a similar biochemical activity of multi-functional com-
pounds 1–4 and menadione. Within a biochemical context, the
pH-dependency of this redox behaviour implies a higher radical
generating activity of the quinone in regions of higher pH. This
Enhancement of OS in Jurkat and Daudi cells
In the light of the electrochemical and in vitro findings, it is
hardly surprising that the activity of 3 in cell culture also points
towards intracellular processes involving oxidative stressors. We
have recently shown that the toxicity of such compounds is
significantly increased in the presence of H
8
2
O
2
. The results
presented here show that this effect is not limited to cultured
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 7 9 – 2 5 8 7
2 5 8 3

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rat PC12 cells, but can also be observed in other cell types, such
as human Jurkat and Daudi cells.
The mechanistic studies, although of a preliminary nature,
support the notion that 3 and, to a somewhat lesser extent, 2 are
able to aggravate OS in the Daudi and Jurkat cell lines. This effect
is likely to be the result of quinone-based superoxide formation
presented here lend experimental support to this notion by
presenting compounds that are more cytotoxic to oxidatively
stressed cells than to cells under normal redox conditions. They
also show that it is possible to use chemical design criteria, such
as electronic effects and the combination of individual catalytic
centres in one molecule, to obtain highly active catalysts with
the expected activities.
More thorough biochemical and in vivo investigations are
now underway to determine the precise mode of action of the
compounds and possible metabolic conversions. These studies
also need to address the complex redox behaviour seen for
compounds such as 2–4 in vitro. Ultimately, animal testing
will be required to evaluate efficiency, toxicity and to study
pharmacokinetic parameters. On the other hand, the notion
of catalysts as biochemical sensor–effector compounds is not
limited to 1–4 and a wide range of multifunctional catalysts, with
two or even more redox centres combined in chemically simple
molecules, is possible. The work presented here should therefore
be seen as providing the impetus for follow-on investigations,
including synthetic chemistry and cell biology.
(
the latter will be metabolised by superoxide dismutase enzymes
to H and dioxygen) and peroxidation catalysis that greatly
enhances the physiological impact of H
2
O
2
26
2
O . The link between
2
the catalysts and an intracellular increase of OS is also reflected
by a significant decrease in GSH levels in the MCB assay, with
GSH a major determinant of redox status in mammalian cells.
Considered together, the biochemical findings support the
notion of a catalytic generation or conversion of ROS that
leads to cell damage and ultimately cell death, probably via
an apoptotic process. An apoptotic mode of cell death is not
too surprising in this context, since hydrogen peroxide induced
stress has already been linked to apoptosis in PC12 and Jurkat
27–28
cells.
Although it is difficult at this point to determine an
exact mechanistic relationship between the compounds and cell
death, modulation of underlying redox processes provides a
good working hypothesis (Fig. 6).
Experimental
Materials
Buffers, thiol reagents, methanol, hydrogen peroxide, catalase
(
bovine liver), cytochrome C reductase (porcine heart), N-
ꢀ
acetyl-L-cysteine (NAC), propidium iodide (PI), 4 ,6-diamidino-
2
-phenylindole (DAPI), 2,7-dichlorodihydrofluorescein ac-
etate (DCDHF), ZnSO (99.999%), and 2-methyl-1,4-
4
naphthoquinone (5) were purchased from Sigma-Aldrich (Poole,
UK). Tissue culture grade phosphate buffered saline (PBS),
foetal bovine serum, L-glutamine, trypsin and gentamicin were
TM
purchased from Gibco (Paisley, UK) or BioWhittaker (Berk-
shire, UK). RMPI 1640 medium with and without Phenol Red,
horse serum, nutrient mixture F-12 HAM medium, fibronectin,
water, endothelial cell growth supplement and heparin were
TM
all purchased from Sigma-Aldrich or BioWhittaker . All
commercial chemicals were analytical or cell culture grade
and needed no further purification. Solutions and buffers were
prepared in MilliQ water and nitrogen flushed prior to use.
Cd,Zn-metallothionein was purchased from Sigma and Zn7–
MT was reconstituted and purified according to a standard
Fig. 6 Proposed mechanism by which redox catalysts cause selective
cell death. This working model shows the sensing ability of catalysts and
the aggravation of intracellular OS conditions that favour cell death via
an apoptotic process.
30
method. Rat adrenal phaeochromocytoma PC12 cell lines
were obtained from the European Collection of Cell Cul-
tures (ECACC, No. 88022401). The human T-lymphoblastic
leukaemia Jurkat J16 cell line and the human Epstein–Barr virus
positive B-lymphoblastoid Daudi cell line were kind gifts from
Dr C. Pourzand, University of Bath, UK and Dr H. Martin,
University of Bristol, UK, respectively.
UV–Vis spectra were recorded on a CARY 50Bio spec-
trophotometer (Varian). Cyclic voltammetry was performed
on a 100B/W workstation (BAS). Cells were incubated in a
Since natural, physiological levels of H
2
O and other ROS
2
vary greatly between healthy and diseased cells, the synergistic
effects between these stress markers and the catalysts provide a
reasonable degree of specificity. This is particularly interesting
in the context of cancer research, since the intracellular redox
state of some cancer cells is more oxidizing compared to healthy
cells and apoptosis would be a preferred mode of cancer cell
death. Interestingly, recent work on the selective cytotoxicity
of superoxide dismutase mimics has led to similar results,
Sanyo CO incubator and cell viability measured using either a
2
29
supporting the possible use of catalysts in cancer therapy.
Dynex technologies MRX microplate reader (MTT assays) or an
EL800 Universal Microplate Reader from Bio-Tek Instruments
Inc. (sulforhodamine B assays). Fluorescent microscopy was
performed using a Carl Zeiss Axioskop2 Mot Plus microscope
with imaging software Axio Vision. A FLUOstar OPTIMA
fluorescent microplate reader, kindly provided by BMG Lab
Technologies, UK, was used for the measurements of intracel-
lular levels of OS. A Spectronic unicam Aminco Bowman Series
Based on the chemistry of the catalytic process, it is likely
that most cell types under OS will be affected. This implies that
other cells suffering from OS, such as inflammatory cells, might
also be affected by the compounds. Further cell culture studies
are therefore required to determine how widely applicable the
catalytic effects observed here are. Such studies will also be able
to shed more light on the exact mode of action of the compounds
inside cells.
2
Luminescence spectrometer was used for the MCB assay.
Conclusions
Methods
Compounds that aggravate, rather than simply generate phys-
iological conditions such as OS, are able to sense intracellular
conditions of existing OS and use these markers to affect cell
survival. In theory, this sensor–effector combination provides
an elegant, catalytic approach towards drug design. The studies
The redox catalysts were synthesised and studied with a
combination of chemical, biochemical and cell culture assays
to evaluate their redox behaviour, interactions with proteins,
cytotoxicity against cells in the absence/presence of OS and
possible mode of action inside cells. Unless stated otherwise,
2
5 8 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 7 9 – 2 5 8 7

View Article Online
−
1
all experiments described (except synthetic chemistry) were
performed in triplicate.
Z = 4, l(Mo–Ka) = 2.084 mm , 9936 reflections measured,
2793 unique (Rint= 0.046) which were used in all calculations.
2
The final wR(F ) was 0.0849 (all data).‡
Synthesis of compounds
Cyclic voltammetry
2
-Methyl-3-bromo-1,4-naphthoquinone was synthesised ac-
31
cording to an established procedure. Compound 2 was synthe-
Cyclic voltammograms of organochalcogens (50–100 lM) were
recorded in potassium phosphate buffer (50 mM) containing
30% methanol, due to the limited solubility of the organochalco-
gens in aqueous media. The pH range included pH 4, 5, 6, 7.4,
sised using an analogous procedure to that outlined by Sakak-
32
ibara et al. using 2-methyl-3-bromo-1,4-naphthoquinone as
the starting reagent (Fig. 1).
8
and 9. A standard Ag/AgCl reference electrode (SSE) and
2
-(Phenylsulfuryl)-3-methyl-[1,4]naphthoquinone (1). The
a glassy carbon (GCE) working electrode were used, with an
internal ferrocene reference standard to compensate eventual
changes in SSE potential. Counter and working electrode were
thoroughly cleaned and polished (Al
voltammograms were recorded at scan rates between 100 to 500
reaction was performed under argon and in the dark (flask
wrapped in foil). Absolute ethanol was degassed with argon
for 30 min prior to use. 4,4 -dimethoxydiphenyl disulfide
748 mg, 2.69 mmol) was dissolved in absolute ethanol (15 ml).
NaBH (193 mg, 5.105 mmol) was added to the stirring
ꢀ
2
3
O ) after each scan. Cyclic
(
4
−1
mV s . The potential range was between −600 mV to +1400
solution, which turned colourless. This was allowed to stir
for 20 min. 2-Bromo-3-methyl-[1,4]naphthoquinone (1.079 g,
6–7
mV vs. SSE.
4
.30 mmol) was added, which turned the mixture dark red
Protein assays
immediately. Stirring continued for 3 h, after which time the
solvent was removed. The resulting brown solid was purified on
a silica column eluting the orange product with 50% Et
Thiophenol peroxidation assay. The thiophenol oxidation
assay (PhSH) is a spectrophotometric assay that measures GPx-
like catalytic activity by monitoring the formation of PhSSPh
2
O–pet.
◦
Reducing the solvent and leaving the solution stand at −20 C
1
13
for 48 h produced orange crystals. Yield 32 mg (2%). H NMR
from PhSH in the presence of hydrogen peroxide. Briefly,
(
2
300 MHz, CDCl
H), 7.38 (m, 2H), 6.83 (m, 2H), 3.78 (s, 3H), 2.31 (s, 3H).
3
): d 8.07 (m, 1H), 7.99 (m, 1H), 7.67 (m,
100 lM of compound was added to a 1 mM methanolic solution
1
3
C
of PhSH, the reaction initiated by addition of 2 mM H
monitored at 305 nm for 20 min at 25 C. Initial rates were
obtained for the interval of 1 and 2 min.
2
O and
2
◦
NMR (CDCl
3
) d 182.99, 180.72, 159.41, 147.35, 146.44, 133.55,
1
33.44, 131.97, 126.84, 126.44, 123.90, 114.70, 55.25, 15.57.
Elemental analysis: calcd for C17 S: C 69.66; H 4.55%.
Found: C 69.48; H 4.85%. Electronic spectra kmax(MeOH)/nm
H
12
O
2
Metallothionein zinc release assay. Since the PhSH assay is
frequently disturbed (e.g. if coloured catalysts are used), the
more robust and also biologically more relevant metallothionein
3
−1
−1
(
(
e/dm mol cm ): 426 (1700), 332 sh (3250), 252 (26 460), 203
47 800).
(
MT) zinc release assay was used to complement the PhSH
14,33
2-(Phenyltelluryl)-3-methyl-[1,4]naphthoquinone (3). Com-
assay.
This assay measures the ability of agents to catalyse
pound
3
was synthesised in exactly the same man-
the reaction of tert-butyl hydroperoxide (t-BuOOH) with the
biologically abundant zinc–sulfur protein MT, whose zinc ions
are released upon oxidation of the protein’s cysteine ligands;
a process that can be monitored spectrophotometrically in the
ꢀ
ner as described directly above using 4,4 -diphenyl ditel-
luride (107 mg, 0.26 mmol) and 2-bromo-3-methyl-
[
1,4]naphthoquinone (137 mg, 0.55 mmol). The resulting purple
solid was purified on a silica column, eluting the starting telluride
with 7% Et O–pet and eluting the purple product with 10%
2
+
presence of a Zn binding dye. In these assays, MT (0.5 lM)
was incubated with t-BuOOH (500 lM) and 4-(2-pyridylazo)-
2
+
Et
2
O–pet. Reducing the solvent and leaving the solution stand
resorcinol monosodium salt (PAR, 100 lM) in 20 mM Na –
◦
◦
at −20 C for 48 h produced dark purple crystals suitable
HEPES buffer (pH 7.4, 25 C) in the absence and presence of
1
for X-ray diffraction studies. Yield 17 mg (9%). H NMR
various concentrations of catalysts. Compounds were evaluated
according to the initial rate of zinc release and the total extent of
zinc release after 60 min. Maximum zinc release was measured
using ebselen as standard and activities are expressed as a
percentage of this value.
(300 MHz, CDCl ) d 8.02 (m, 2H), 7.78 (m, 2H), 7.63 (m, 2H),
3
13
7
1
1
.30 (m, 1H), 7.20 (m, 2H), 1.90 (s, 3H). C NMR (CDCl ) d
84.29, 181.27, 153.48, 142.11, 139.72, 139.56, 139.42, 133.82,
33.28, 131.99, 131.57, 129.54, 127.08, 126.95, 114.23, 20.19.
3
Elemental analysis: calcd for C17
H
12
O
2
Te: C 54.32; H 3.22%.
Cytochrome C reductase assay. To characterise and quantify
the prospective oxidant effects of the quinone moiety of each
catalyst an assay was established based on the ability of
the compounds to function as a coenzyme for porcine heart
Found: C 54.08; H 3.04%. Electronic spectra kmax(MeOH)/nm
3
−1
−1
(
2
e/dm mol cm ): 517 (1100), 326 sh (2806), 269 sh (12 060),
40 sh (20 400), 221 sh (23 000), 204 (38 150).
34
2
-(4-Methoxy-phenyltelluryl)-3-methyl-[1,4]-naphthoquinone
cytochrome C reductase (CR). CR is a widely promiscuous
enzyme, which catalyses the reduction of a variety of substrates
by NADH and therefore serves as a model for the interactions of
the compounds with redox enzymes within the cell. CR (5 lM),
NADH (500 lM) and compound (50 lM), were incubated
together in 10 mM phosphate buffer, pH 7.5, for 10 min. By
monitoring at 340 nm, the rate at which NADH was oxidised to
(
4). Compound 4 was synthesised in exactly the same manner
ꢀ
as described directly above using 4,4 -dimethoxydiphenyl
ditelluride (514 mg, 1.09 mmol) and 2-bromo-3-methyl-
[
(
1,4]naphthoquinone (575 mg, 2.29 mmol). Yield 332 mg
75%). H NMR (300 MHz, CDCl
1
3
) d 8.06 (m, 2H), 7.78 (m,
2
H), 7.68 (m, 2H), 6.80 (m, 2H), 3.83 (s, 3H), 1.92 (s, 3H).
1
3
+
C NMR (CDCl
41.87, 133.77, 133.20, 132.02, 131.59, 126.98, 126.92, 115.44,
03.33, 55.16, 19.39. Elemental analysis: calcd for C18 Te:
3
) d 184.56, 181.27, 160.29, 153.13, 142.19,
NAD was derived from the slope of the kinetic trace.
1
1
H
14
O
3
Cell culture
C 53.26; H 3.48%. Found: C 53.32; H 3.40%. Electronic spectra
3
−1
−1
Cell culture tests were performed in the following cell lines:
rat PC12 cells, human Jurkat (J16) and human Daudi cells.
Undifferentiated PC12 cells were cultured in suspension in
RPMI 1640 supplemented with 2 mM L-glutamine, 10% horse
serum, 5% heat inactivated foetal bovine serum and 250 U per ml
k
(
max(MeOH)/nm (e/dm mol cm ): 517 (1275), ∼325 sh
2730), ∼271 sh (10 800), 243 (25 900), 204 (37 500).
X-Ray measurement of 3
Crystal data. C17
◦
35
H
12
O
2
Te, M = 375.87, monoclinic, a =
gentamicin at 37 C, 5% CO
2
and 95% air. For experimentation,
◦
5
˚
1
1.8427(9), b = 6.0131(3), c = 20.0259(11) A, b = 90.914(5) ,
undifferentiated cells were plated at 10 cells per well into 96 well
tissue culture plates.
3
˚
U = 1425.89(15) A , T = 120 K, space group P2
1
/n (no. 14),
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 7 9 – 2 5 8 7
2 5 8 5

View Article Online
The Jurkat and Daudi cell lines were maintained in RPMI
640 medium supplemented with 10% heat inactivated foetal
the microplate fluorimeter, using an excitation wavelength of
485 nm and an emission wavelength of 538 nm.
1
bovine serum, 2 mM L-glutamine, 100 U per ml penicillin, and
−
1
◦
100 lg ml streptomycin at 37 C, 5% CO
2
and 95% air. Cells
Monochlorobimane GSH assay indicative of intracellular GSH
concentrations
of no lower than 90% viability as determined by the trypan
blue exclusion assay were used. For all experimentation, cells
The MCB assay is fairly specific for GSH and allows the
determination of GSH concentrations in the presence of
4
were plated at 10 cells per well in phenol red free medium
supplemented with 10% heat inactivated foetal bovine serum,
other intracellular thiols. The assay was based on a literature
−
1
2
mM L-glutamine, 100 U per ml penicillin and 100 lg ml
20–21
procedure,
with the following modifications: after treating
streptomycin.
cells with or without 10 lM compounds and with or without
6
2
00 mM N-acetyl-L-cysteine (NAC) for 24 h, samples of 10
Measurement of cell survival by the MTT assay
cells were resuspend in 2 ml of PBS containing 50 lM MCB and
incubated for 30 min in the dark at rt. A 1 ml aliquot of the
cell suspension was transferred into a fluorescent cuvette and
fluorescence emission measured at an excitation wavelength of
395 nm and an emission wavelength of 482 nm. PBS incubated
with 50 lM MCB was used as control. Means of % GSH
compared to the untreated control sample were calculated (as
an average of two samples).
Cell viability of PC12 cells was measured using the MTT (3-[4,5-
3
6
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.
−
1
MTT (0.5 mg ml , in PBS) was added to the 96 well plate (final
−
1
concentration, 0.1 mg ml ) and incubated for a period of 3 h.
The reduced MTT crystals were solubilised in 30% DMSO and
the colourimetric evaluation of reduced MTT was determined
spectrophotometrically at 540 nm.
In order to test the activity of compounds on PC12 cell
survival, cells were plated and then pre-incubated for 1 h with
varying concentrations (1 nM–25 lM) of catalyst 3 at 37 C, 5%
Acknowledgements
◦
This work was financially supported by the Wellcome Trust, the
Leverhulme Trust, DAART, Exeter Antioxidant Therapeutics
Ltd. and the University of Exeter (UK). C. J. acknowledges a
Visiting Professorship from the Universit e´ de Metz (France).
The authors are grateful to Prof. Winyard (Exeter) and Prof.
Kirsch (Metz) for helpful discussions and wish to thank BMG
for the use of their fluorescent microplate reader. The also
acknowledge Drs Pourzand (Bath) and Martin (Bristol) for
donating cell cultures.
CO
2
and 95% air, followed by the addition of H
2
O (200 lM)
2
and overnight incubation. In a separate experiment, cells were
pre-incubated with 3 (10 lM) followed by addition of varying
H
2
O
2
concentrations (50–500 lM). A H
2
2
O dose response curve
in the absence of catalyst was established as a control (data
not shown). In addition, analogue experiments were performed
with the chalcogen-free, parent quinone 5 as a control. All
compounds were initially solubilised in methanol and diluted
in medium to the appropriate concentration with 2% methanol
to maintain solubility. Methanol and also catalyst alone (no
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