Novel peptidomimetic compounds containing redox active chalcogens and quinones as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.09.033

Many types of cancer cells are associated with a disturbed intracellular redox balance and oxidative stress (OS). Among the various agents employed to modulate the intracellular redox state of cells, certain redox catalysts containing quinone and chalcoge

Full text of DOI:10.1016/j.ejmech.2012.09.033

European Journal of Medicinal Chemistry 58 (2012) 192e205
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel peptidomimetic compounds containing redox active chalcogens and
quinones as potential anticancer agents
,
Saad Shaaban a c, Randi Diestel a, Bettina Hinkelmann a, Yazh Muthukumar a, Rajeshwar P. Verma b,
,
c,
**
Florenz Sasse a , Claus Jacob
*
a Department of Chemical Biology, Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany
b Department of Chemistry, Pomona College, 645 North College Avenue, Claremont, CA 91711, USA
c Division of Bioorganic Chemistry, School of Pharmacy, Saarland University, Campus B2.1, D-66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Many types of cancer cells are associated with a disturbed intracellular redox balance and oxidative
stress (OS). Among the various agents employed to modulate the intracellular redox state of cells, certain
redox catalysts containing quinone and chalcogen moieties have shown considerable promise. Passerini
multicomponent reaction has been developed for the synthesis of agents combining two, three or even
four redox centers in one molecule in a good yield. When incubated with cancer cells these agents
inhibited cell proliferation and induced apoptotic cell death. Interestingly, some of these redox active
compounds exhibited quite low toxicity with normal cells. The cause was obviously OS, which was re-
flected by significant decrease in reduced glutathione, subsequently cell cycle arrest and induction of
apoptosis.
Received 5 August 2012
Received in revised form
22 September 2012
Accepted 25 September 2012
Available online 4 October 2012
Keywords:
Oxidative stress
Multicomponent reactions
Passerini reaction
Anticancer activity
Chemogenomic assay
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
species (ROS) levels are considerably closer to the critical redox
threshold at which cell death is induced [4]. These biochemical
Therapeutic selectivity and drug resistance are two major issues
in cancer chemotherapy. By targeting the genetic differences
between normal and cancer cells, drugs like Gleevec and Herceptin
show promising therapeutic activity and few side effects [1]. These
gene-targeting strategies however, are still facing problems
because of acquired drug resistance and genetic instability of
cancer cells [2]. Recent studies suggest that targeting the particular
biochemical alterations, biochemical signature, in cancer cells
might be a feasible approach to develop a cancer therapy that does
not lead to the development of drug resistance [3].
Many types of cancer cells show a disturbed intracellular redox
balance, making them different from their healthy counterparts.
Some tumors, such as solid lung carcinoma, are hypoxic, i.e., their
cells are more reducing than normal ones, while others, such as the
cells of prostate and breast cancer are naturally under oxidative
stress (OS). When compared to healthy cells, their reactive oxygen
differences between healthy and malignant tissue are significant,
and can be used to design selective, yet effective redox drugs [5,6].
Several avenues have therefore been explored during the last
ten years to use the naturally occurring OS to selectively kill cancer
cells. Currently explored ROS-inducing strategies can be divided
into three main lines of investigation: i) agents that directly
increase ROS in cancer cells to lethal levels (ROS-generators), ii)
agents that inhibit antioxidant enzymes and hence raise ROS
concentrations to lethal levels, and iii) catalysts that enhance the
toxicity of pre-existing ROS (ROS-users and ROS-enhancers) [4,7].
ROS generators and inhibitors of antioxidant defense systems
(strategies i and ii) add an additional ROS burden without really
discriminating directly between normal and sick cells. They rely
only on the pre-existing differences in basal ROS levels between
cancer and normal cells. Furthermore, the dosage of such agents is
a serious problem. These compounds are not catalytic, and there-
fore need to be administered in rather large quantities, which
might lead to serious side effects. In contrast, catalytic molecules
(strategy iii) that employ ROS as their substrates may not only raise
ROS levels, but they may also exhibit selectively in cells rich in ROS,
without exhibiting the same chemistry in normal cells. Although
some of these agents may exhibit selectivity to cells rich in ROS,
* Corresponding author. Tel.: þ49 531 6181 3428; fax: þ49 531 6181 3499.
** Corresponding author. Tel.: þ49 681 302 3129; fax: þ49 681 302 3464.
E-mail addresses: florenz.sasse@helmholtz-hzi.de (F. Sasse), c.jacob@mx.uni-
saarland.de (C. Jacob).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.033

S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
193
they are not able to efficiently eliminate cancer cells that have
become adapted to stress. This may be due to the fact that
a disturbed redox balance is not just due to one chemical species
but is the result of a combination of various ROS and reactive
nitrogen species, metal ions, and deficiencies in antioxidant
defenses. Having the above facts in mind and in continuation of our
earlier efforts to synthesize agents that increase ROS selectively in
cancer cells to lethal levels, we were interested to synthesize agents
containing both ROS-generating and ROS-using moieties in one
molecule.
A combination of ROS-modulators is required to effectively
eliminate cancer cells and is expected to maximally exploit the
ROS-mediated cell-death mechanism as a therapeutic strategy. This
approach is adopted to increase the catalytic efficiency and might
be particularly useful in cells that have become adapted to stress
and are therefore resistant to traditional anticancer agents.
As part of our present study, we have therefore explored the
effective synthesis of multifunctional redox modulators and used
these agents to perform a comprehensive cell biological analysis of
the potential mode of biochemical action. Combining an in depth
analysis of whole cell behavior (cell morphology, cell cycle arrest,
induction of apoptosis) with biochemical changes (ROS and GSH
levels, caspase activity) and a modern chemogenomic approach, we
are able to confirm the suspected redox link which ultimately is the
most likely explanation for some of our compound’s selective
activity against certain cancer cells under (pre-existing) OS.
as ROS-enhancers. The synthesis of such compounds encounters
increasing difficulties when moving from just one or two to three
or more redox sites. The synthesis of chalcogen based quinone
agents has often been marred by low yields, decomposition of the
products and difficulties to generate compounds in sufficient
quantities and purities. Even the most basic, bi-functional agents
have been difficult to obtain [11e13]. These problems in turn have
hindered the development of multifunctional redox catalysts.
Therefore, the development of
a multicomponent one-step
synthetic method using stable reagents under neutral conditions
has attracted our attention. The Passerini three-component reac-
tion (P-3CR) in the context of diversity-oriented synthesis has been
used to achieve high levels of diversity and brevity. The design of
new combinations of simple and flexible building blocks gave rise
to novel and diverse complex structures by simultaneous forma-
tion of two or more bonds, according to the domino and diver-
gence principles [14,15].
Apart from being able to successfully deliver highly complicated,
multifunctional redox catalysts, the P-3CR employed here is char-
acterized by two additional aspects. From the perspective of orga-
nochalcogen chemistry, the yields obtained by this method vary
from 62% to 93%, i.e., they are generally quite good as far as organo-
selenium compounds are concerned (Table 1).
Furthermore, the P-3CR was performed under different condi-
tions using different solvents. The best results in terms of reaction
time and yield were obtained when the reaction was performed in
the presence of water and under mild conditions (room tempera-
ture) [16,17]. Moreover, not only does the use of water as solvent
permit the reaction to be conducted rapidly, the products are often
insoluble, facilitating their ready isolation. Although mixed
aqueous solvents are popular as a medium for MCRs and related
chemistry, the use of pure water without any co-solvents seems to
be an exception to the rule [16]. This could be attributed to many
factors, including the hydrophobic effect and enhanced hydrogen
bonding in the transition state.
2. Results and discussion
2.1. Chemistry
The desire to increase the efficiency and selectivity for cancer
cells under OS has required the synthesis of complicated redox
modulators which are tailored to recognize the biochemical redox
signature of OS in cancer cells. These agents often combine three or
even more functionalities (redox centers, metal binding sites) in
one molecule and therefore may act efficiently and selectively
against those cells [8]. Recently we have reported compounds
containing two, three or even four redox sites in one molecule.
These molecules were efficient in sub-micromolar concentrations
i.e., at rather low concentrations [9,10]. Based on these results, we
were interested to further conduct an extensive antitumor screen
using a variety of novel multi-redox compounds in order to obtain
evidences regarding the structural requirements underlying the
cytotoxicity/selectivity of ROS-generators, ROS-users and/or ROS-
enhancers when they are combined together.
2.1.2. Building blocks used in the P-3CR
The P-3CR described here required a range of building blocks,
which needed to be designed and synthesized first. Fig. 1 provides
an overview of the various aldehydes, acids, and isonitriles used.
Although the synthesis of appropriate quinone- and chalcogen-
bearing building blocks is not always straight forward, a range of
such compounds have now been reported by us in the literature
[9,10]. Of particular importance is the bifunctionalized quinonee
selenium bearing aldehyde (1c) and the quinoneesulfur bearing
acid (2b). Other building blocks were chosen to incorporate the
amino acid residues of glycine, L-serine, and cysteine to the product
backbone for biological and pharmacological reasons (2ce2e).
In case of the isonitrile building blocks, 3b was used in addition
to the commercially available but expensive tert-butyl isonitrile
(3a) which is frequently used. In most cases 3b offers superior
2.1.1. The multicomponent Passerini reaction as a key to
multifunctional redox agents
Our design strategy relies on the combination of quinones as
ROS-generators, organochalcogens as ROS-users, and/or porphyrin
Table 1
Building blocks, solvents, and yields of the synthesis of compounds 4e18. Building blocks were designed to carry one or two relevant redox or metal binding sites, which could
be combined to larger, highly functionalized molecules with four or more biologically interesting sites. Reactions were mostly performed in water at high concentrations and
under mild conditions.
Cpd. no.
Building blocks
Solvent
Yield (%)
Redox centers
Cpd. no.
Building blocks
Solvent
Yield (%)
Redox centers
4
5
6
7
8
9
10
11
1b, 2b, 3a
1a, 2b, 3a
1a, 2b, 3b
1c, 2b, 3a
1b, 2b, 3b
1c, 2b, 3b
1c, 2d, 3a
1c, 2e, 3a
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
62
68
71
76
75
73
76
54
3
3
3
4
3
4
2
4
12
13
14
15
16
17
18
1c, 2d, 3b
1c, 2c, 3b
1c, 2e, 3b
1c, 2a, 3b
1d, 2f, 3a
1a, 2f, 3a
1c, 2f, 3a
H2O
H2O
H2O
H2O
CHCl3
CHCl3
CHCl3
75
66
85
79
93
89
86
2
2
4
3
1
2
3

194
S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
O
O
Se
Se
H
O
H
O
O
O
O
Se
1a
1b
1c
1d
O
O
O
OH
OH
O
H
N
O
O
OH
O
N
H
Se
OH
S
OH
O
2d
O
2a
2b
2c
O
OH
O
H
N
O
O
O
N HN
NH N
N HN
NH N
O
OH
OH
O
HO
OH
S
S
2e
2g
2f
HO
O
O
N
C
O
N
C
3b
3a
Fig. 1. Building blocks used to synthesize multifunctional redox agents. Acid, aldehyde and isonitrile building blocks used in the Passerini reaction were synthesized following
literature procedures or, in case of hitherto unknown agents, could be synthesized in sufficient yield.
reactivity compared to 3a. Furthermore, 3b adducts can undergo
further derivatization [18].
2.2. Pharmacology
All the building blocks required were synthesized following
literature procedures or, in case of hitherto unknown agents, were
synthesized in sufficient yield and purity. Analytical information is
given in the Supplementary material.
Naphthoquinones, particularly 1,4-naphthoquinone, utilize
several mechanisms to exert a cytotoxic effect [19,20]. Of special
interest is their ability to redox cycle with triplet oxygen, forming
superoxide and peroxide capable of inflicting damage (ROS gener-
ators) [21e23]. In contrast, organoselenium compounds (so called
ROS-users), mimicking the catalytic cycle of the selenoenzyme GPx,
are able to use ROS and speed up reactions with redox-sensitive
proteins and enzymes, ultimately causing malfunction and cell
2.1.3. Synthesis of selected multifunctional redox agents
Our main objectives are to exploit the ‘mix and match’ design
criteria to synthesize
a small library of chemically diverse
compounds able to target cancer cells under oxidative stress using
simple and efficient synthetic procedure. In order to turn the hits
into lead compounds, this library thereafter will be submitted to
in vitro evaluation. Therefore a library of 15 chemically diverse
compounds (4e18) (Fig. 2) was synthesized using P-3CR. Large
libraries accordingly are planned to be synthesized after obtaining
evidences regarding the structural requirements underlying the
cytotoxic/selective behavior of our representative library.
Compounds were synthesized in order to combine diverse redox
centers: A) compounds possessing two redox centers of which one is
quinone and one is selenium (10, 12, 13); B) compounds possessing
three redox centers, either with one quinone, one selenium and one
sulfur (4, 5, 6, 8), or with one quinone and two selenium (15), or with
one quinone, one selenium and disulfide moiety (11,14); C) compounds
possessing four redox centers with two quinones, one selenium and
one sulfur moieties (7, 9). Furthermore, compounds containing
a porphyrin metal binding center with/without chalcogen and quinone
redox sites were also synthesized. These compounds contain: A) only
a metal binding porphyrin center (16); B) a metal binding porphyrin
center and selenium redox center (17); and C) a metal binding
porphyrin center, one quinone and selenium redox center (18).
death [24]. Furthermore, metalloporphyrins (ROS-enhancers) are
ꢀ
able to convert less reactive and damaging ROS, such as O2 ꢁ, to
more damaging species, such as the highly aggressive hydroxyl
(ꢀOH) radical [25,26].
In theory, the ROS-generating and the ROS-using centers
complement to each other. The peroxide formed as a result of redox
cycling of the quinone moiety has the ability to induce cellular
injury and, at the same time, to activate the chalcogen/porphyrin
moieties, which in turn are responsible for the oxidation of redox-
sensitive proteins and enzymes leading to oxidative damage.
Importantly, such multifunctional compounds have not been fully
tested before in a cancer cell screen and therefore may provide
a truly new lead for compound development.
2.2.1. Cytotoxic activity of redox active compounds in cancer cells
and healthy cells
In order to check if any of the multifunctional agents may be
useful for further biochemical investigations, a one-dose screen of
compounds 9,10,12, and 14 was performed at the National Institute
of Health (NIH) in Bethesda, MD, USA. This screen includes 58 cell
lines grouped into breast cancer, renal cancer, colon cancer,

S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
195
O
O
O
Se
O
O
Se
O
Se
O
O
O
O
H
N
H
N
H
N
S
O
S
O
S
O
O
O
5
6
O
O
O
4
5
O
O
O
O
O
Se
O
O
O
O
O
Se
O
Se
O
O
O
O
O
O
H
H
H
N
N
N
S
O
O
S
O
S
O
5
5
O
7
8
9
O
O
O
O
Se
O
O
O
O
H
H
Se
Se
O
O
N
N
O
O
O
O
H
H
H
H
O
N
N
O
N
N
O
O
O
O
S
S
5
O
O
O
OH
OH
10
11
12
O
O
O
O
O
O
Se
O
O
O
O
O
H
H
N
Se
O
N
Se
O
O
O
O
O
O
H
H
H
5
O
N
N
O
N
O
O
S
S
Se
O
5
5
O
14
13
15
O
Se
O
O
O
O
N
N
N
NH
N
NH
N
NH
N
O Se
O
O
O
HN
HN
NH
NH
HN
NH
16
O
17
18
O
Fig. 2. The Passerini multicomponent reaction was used for the synthesis of di-, tri- and tetra-functional redox agents 4e18 containing multiple chalcogen and quinone redox sites.
prostate cancer, ovarian cancer, CNS cancer, non-small-cell lung
cancer, leukemia, and melanoma cell lines.
Based on the above promising results, the cytotoxic activity of
the test compounds was further assayed in details with different
cancer cell lines, i.e., MCF-7 (human breast adenocarcinoma), A-498
(human kidney carcinoma), and A-431 (human epidermoid carci-
noma). Cells were incubated with serial dilutions of the
compounds, and the metabolic activity measured by means of an
MTT assay after 24 h and the IC50 values are estimated from the
doseeresponse curves (Table 2).
Interestingly, we founda correlation betweenchemicalstructures
and toxic activities. Compounds 7 and 9 with four redox centers are
morecytotoxicthantheirhomologswithtwoandthreeredoxcenters
as was expected. Furthermore, the most toxic compounds 7, 9, 10, 12
and 14 posses a direct attachment of the selenium to the quinone, in
contrast to the structurally similar get less toxic compounds 4, 5, 6
and 8, where the selenium atom is located further away from the
Interestingly, all the compounds tested exhibited a significant
cytotoxicity against cancer cell lines and were subsequently
selected for 5-dose testing. GI50 values were in the low to sub-
micromolar range (Supplementary material). COMPARE analyses
were performed using the correlation coefficient method. The
compounds’ pattern activities correlate with cisplatin which is used
to treat various types of cancers, including sarcomas, lymphomas,
and germ cell tumors, and methyl mitomycin C which has an
antitumor antibiotic activity against breast cancer, and last but not
least with anthracycline-based redox agents representing meno-
garil, deoxydoxorubicin, and MX2 HCl, which are used in the
treatment of prostate cancer and leukemia (Supplementary
material).

196
S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
Table 2
Influence of multifunctional redox compounds 4e18 on the viability of human cancer cells and normal cells.
Cpd. no.
MCF-7b
A-498b
A-431b
HUVECa
HFa
Cpd. no.
MCF-7b
A-498b
A-431b
HUVECa
HFa
IC50
(
mM)
IC50 (mM)
4
5
6
7
8
9
10
11
26
14
10
3
19
10
3
>100
>100
>100
3
>100
5
>100
7
>100
1
13
34
23
1
17
7
11
26
50
n.d.
21
5
12
13
14
15
16
17
18
5
4
8
9
5
7
13
4
>100
>100
>100
4
5
4
4
35
36
10
3
>100
>100
>100
27
6
4
6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6
2
2
5
5
3
2
7
4
3
The metabolic activity of the cells was measured after one day of incubation with different concentrations of the test compounds by means of an MTT assay. The IC50 was
determined from the doseeresponse curves as the mean of two parallel experiments; b cancer cell line; a primary cells; n.d. not detected.
quinone moiety. But there were exceptions: compounds 10 and 12
with only two redox centers were also among the most active
compounds. On the other hand, compounds 16, 17 and 18 with
a metal binding porphyrin site showed no cytotoxic effect in the
Therefore, the intracellular levels of reduced glutathione were
estimated by the DTNB assay.
The DTNB assay is fairly specific for GSH and allows the deter-
mination of GSH concentrations in the presence of other intracel-
lular thiols [20]. Fig. 3 shows that the GSH levels were reduced
when the cells were incubated with different redox active
concentration range tested (IC50 > 100 mM) even though compound
18 contains two additional redox centers (quinone and selenium).
When incubated with primary human fibroblasts (HF) and
human umbilical vein endothelial cells (HUVEC) most of the test
compounds had a toxic effect with the exception of compounds 5, 6,
12 and 13 (Table 2). Compound 12 clearly showed lower cytotoxicity
against both primary cells than against the three cancer cell lines
under investigation. The same holds true for 13 with the exception
that it was toxic to HF cells. Compound 5 showed higher activity
against breast and epidermoid cancer cells while compound 6
showed higher toxicity against breast cancer but not against
epidermoid cancer cells. These compounds share almost the same
chemical features and this might be the reason for their selective
anticancer behavior. Compounds 6, 12 and 13 possess the same
hydrolyzable benzylhexanoate ester side chain and have a compa-
rable molecular weight (707, 730, and 700 g/mol, respectively).
Compounds 5 and 6 have three redox centers, compounds 12 and 13
two redox centers each. Importantly, all these compounds combine
a quinone with a selenium redox center. Such specific molecular
features might be the reason for the selectivity of the compounds.
compounds. After 1 h, a concentration of 5 mM of compounds 11, 10,
15, and 13 caused a decrease of the intracellular GSH level in MCF-7
cells by 46%, 59%, 62%, and 76%, respectively. These observations are
consistent with a recently reported study where several quinone-
containing compounds, with anticancer property, exert their
activity via depletion of GSH [27e29].
2.2.4. Measurement of intracellular ROS levels
ꢁꢀ
The mediation of OS via the production of O2 and H2O2 needed
to be further investigated. The DCF and DHE assays are considered
to be general indicators of ROS. DCF reacts with H2O2, ONOOꢁ, and
lipid hydroperoxides. It is cell permeable, and after uptake is
cleaved by intracellular esterases to the non-fluorescent 20,70-
dichlorofluorescin (DCFH) which is trapped within the cells. DCFH
could be oxidized by a variety of ROS to the fluorescent 20,70-
dichlorofluorescein (DCF) which can be detected on fluorescence
spectrophotometer [30].
2.2.2. Assessment of OS induction
Quinones and organochalcogens, each separately, have been
shown to be cytotoxic via various mechanisms. The exact mecha-
nism by which chalcogen based quinones exhibit their cell killing
effect, however, remains to be explored. Previously, we have
investigated the GPx like catalytic activity of the multifunctional
redox agents using a thiophenol-based (PhSH) assay [9]. The results
obtained, at that time, showed that several compounds were even
more active than the benchmark compound ebselen [9]. We also
reported that these compounds had no tangible antioxidant
activity (which may be counter-productive). This was confirmed
with the thiobarbituric acid assay, where most of the compounds
were not particularly active [9].
In continuation to our previous work, the notion that the
multifunctional agents might be toxic via increasing the severity of
OS was further investigated. The GSH level in addition to the
ꢀꢁ
intracellular levels of ROS (e.g., O2 and H2O2), upon treatment
with different concentrations of the test compounds, were assessed
spectrophotometrically with specific probes, i.e., 5,50-dithiobis
(2-nitrobenzoate) (DTNB), 20,70-dichlorodihydrofluorescin diac-
etate (DCF), and dihydroethidium (DHE).
Fig. 3. Assessment of the intracellular levels of reduced glutathione in breast cancer
cells. Depletion of the intracellular levels of reduced GSH in MCF-7 cells treated with
compounds 10, 11, 13, and 15 for 1 h were assessed employing the DTNB assay which is
fairly specific for glutathione.
2.2.3. GSH assay
Low levels of GSH are linked to mitochondrial dysfunction and
induction of apoptosis, thus decreasing chemoresistance [27e29].

S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
197
The DHE assay is close to being a gold standard method for
detecting O2 [31]. DHE is a lipophilic probe that readily diffuses
nucleus, and the morphology of the endoplasmic reticulum of
potoroo cells (PtK2).
ꢀꢁ
across plasmatiꢀcꢁ membranes. Once inside the cell, it is rapidly
oxidized by O2 to the red fluorescent 2-hydroxyethidium. The
later is trapped in the nucleus by intercalating into DNA, leading to
an increase of fluorescence.
Fig. 6 shows that the ER structure is affected by the redox
modulators (typical ER stress) and that the cell morphology in
general is altered; cells are rounded, and detached from each
other. The adhesion of the cells seems to be reduced and actin
stress fibers are barely detectable. On the other hand, no pheno-
typical changes were noticed in case of the microtubular network
(not shown).
The cytoskeleton may represent one of the preferential targets
of ROS whatever OS is applied due to its structure. Indeed, cyto-
skeletal proteins are particularly abundant within the cells and
several protein constituents of the cytoskeleton display highly
reactive residues that can be easily oxidized. Hence, the actin
cytoskeleton is considered to be an early target of cellular OS [35].
The later causes disruption of the normal organization of micro-
filaments essentially due to oxidative modifications of specific
cysteine thiols and methionine sulfides of actin [29,36,37].
Although the interplay between the OS and ER stress is not clear,
both of them have been implicated in the pathogenesis of a wide
variety of diseases, such as neurodegenerative disorders, diabetes
and ischemia reperfusion heart disease [38].
Compounds under investigation were able to significantly
increase the intracellular ROS levels in A-431 cells (Fig. 4, panel
a and panel b). The ROS formed as a result of redox cycling of the
quinone moiety are sufficiently potent to damage cells and have the
potential to activate the chalcogen center(s) of the catalyst. The
chalcogen redox center(s), in turn, are able to oxidize redox-
sensitive thiol groups in proteins and enzymes, ultimately causing
malfunction and cell death. Thiol oxidation catalysis is, however,
highly detrimental to the cell, which could explain the high toxicity
of such chalcogen-based compounds.
In theory, an ideal anticancer agent should be toxic to malignant
cells with minimum toxicity in normal cells. Within this context,
the levels of ROS of A-431 cells were monitored in comparison with
HUVECs. Fig. 5, panel a and panel b show that A-431 cells have
higher basal levels of ROS compared to HUVECs. Importantly,
compounds 4, 8, 9 and 11 were able to increase the intracellular
ROS level in melanoma cells to a greater extent than in HUVECs.
This behavior points to a specific kind of selectivity, supporting
the notion that exploiting the biochemical differences between
normal cells and cancer cells might lead to the development of new
selective anticancer agents able to overcome the challenges facing
current therapeutic strategies [3]. Although it is too early to spec-
ulate about possible uses of such catalytic, multifunctional redox
agents in therapy, their activity and selective cytotoxicity against
certain cancer cells encourages the further investigation of catalytic
agents as potential anticancer drugs.
2.2.6. Cell cycle and apoptosis
Examination of the current literature on the effect of OS on the
cell cycle reveals that increases in ROS-induced DNA damage are
correlated with cell cycle arrest. For instance, Upadhyay et al.
recently described that
a sublethal dose of H2O2 prevents
progression of cell cycle by causing delay in G0/1 [39]. The same
behavior was observed with our multifunctional redox agents. A
significant delay of cell cycle progression was observed when MCF-
7 cells were treated with 10, 11 and 15 at their respective IC50
concentrations for 24 h. A clear reduction of cells in G2/M phase
and an increase in the G0/1 phase compared to methanol treated
cells (negative control) were observed (Fig. 7).
2.2.5. Phenotypical changes in cellular morphology, cytoskeleton,
and endoplasmic reticulum
OS induces cellular injury by the production and accumulation
of highly reactive free radicals. This may cause increased plasma-
membrane permeability and peroxidation, release of cytosolic
components, disassembly of the cytoskeleton, and damage to all
types of cellular biomolecules, including DNA, proteins and lipids
[32e34]. Within this context, we used fluorescence techniques to
look for phenotypical changes in the cytoskeleton architecture, the
It has been reported that high levels of ROS can induce apoptosis
by activating either the ER stress-mediated apoptotic pathway or
the mitochondrial-mediated apoptotic pathway or by activating
both pathways. The elevation of cytosolic Ca2þ, due to ER stress,
may trigger the mitochondrial permeability transition pore
opening, cytochrome c release, caspase cascade activation, and
apoptosis [33,40].
Fig. 4. Assessment of oxidative stress in melanoma and normal human umbilical vein endothelial cells. Using two different methods ROS levels were measured in A-431 in
comparison with HUVECs after 1 h of treatment with different concentrations of multifunctional compounds. a) A DCF assay was run after incubation with 8, 9, and 11, b) a DHE
assay was performed after incubation with 4 and 9.

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S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
Fig. 5. Immunofluorescence investigations of the ER (panel a and b) and the actin cytoskeleton (panel c and d) of PtK2 cells that were incubated with 10 and 9, respectively, in
comparison with control cells. Compared to the control cells (panel a and c), treated cells show holes in the ER (panel b) and reduced stress fibers (panel d). Cells are rounded and
detached from each other.
In general, all apoptotic pathways depend on activation of cas-
pases, in particular effector caspase-3 and -7, for the final execution
of apoptosis. Therefore, it might be reasonable to first check these
two caspases as a sign of apoptosis. Within this context, the
assessment of caspase-3/7 activity in A-431 cells showed a strong
activation after 24 h of treatment with compound 15 at a concen-
resulting in a chemicalegenetic interaction profile. Analyzing such
profiles provides valuable information (e.g., about the pathways
and targets that are addressed by new compounds) which is not
revealed by conventional methods that easily [42]. We applied
a five-step strategy to possibly link the multifunctional compounds
to their target pathways: i. We screened all the multifunctional
compounds using an agar diffusion assay against S. cerevisiae wild
type (BY4741) and four mutants (YJR104C, YHR008C, YHR106W and
YLR011W), which were chosen because we already know that the
genes knocked out in these mutants might be important to coun-
terbalance OS (see Supplementary materials for more details). ii.
The IC50 values of compounds 10 and 15 (the most active
compounds) were determined using a serial-dilution assay against
the sensitive mutants YJR104C and YHR008C. iii. Compounds 10
and 15 were then screened against a set of w4800 viable S. cer-
evisiae deletion strains using the IC90 concentration found in step ii.
iv. We identified a set of mutants which were sensitive in two
chemicalegenetic interaction screening procedures (step iii). v. The
results from the chemicalegenetic interaction profiling were
evaluated and confirmed using an agar diffusion assay in a second
round of analysis.
tration of 2.8 mM (see Supplementary material). The induction of
apoptosis was further investigated by flow cytometry after staining
the cells with Annexin V and PI. Externalization of phosphati-
dylserine (PS) to the outer leaflet from the inner leaflet of the
plasma membrane is a hallmark of early apoptosis. The FITC labeled
Annexin V binds to PS in the presence of Ca2þ, resulting in green
fluorescence of apoptotic cells. In later stages of apoptosis, PI enters
the cells and bind to cellular DNA, resulting in red and intense
green fluorescence with Annexin V. As shown in Fig. 7, treatment of
A-431 cells with 15 for 12 h resulted in an increase in the
percentage of Annexin V (þ) (lower right quadrant) and PI (þ)
(upper right quadrant) cells indicative of apoptosis in a dose
dependent manner. The appearance of late apoptotic cells was
predominantly (12.9e29.3%) seen at the higher concentrations
(13 mM) of 15. These results were similar to the findings of Kalya-
naraman et al., where adriamycin (an anthracycline drug used in
cancer chemotherapy) induces apoptosis [41].
At the concentrations applied the compounds under investiga-
tion had no effect on the wild type strain, BY4741, but striking effects
were observed with ten mutants (Table 3). Superoxide dismutase
(mitochondrial (SOD2) and cytosolic copperezinc (SOD1)), gluta-
thione synthetase (GSH2) and transferase (GTT2), and cytochrome c
oxidase (COX17) deficient strains were among the most sensitive
mutants regarding the test compounds. These enzymes play a major
roleintheantioxidantdefensesystemandarepivotalfortheremoval
of toxic oxidants. Deletion of the respective genes therefore might
cause an OS sensitive phenotype of the mutant. These observations
2.2.7. Chemical genetic interaction approach
Chemogenomic assays using a mutant library of Saccharomyces
cerevisiae have proven to be a powerful means to study and predict
the mode of action of bioactive compounds. These assays rely on
comparing the growth of each gene deletion strain to the wild type
strain in the absence and presence of the compounds. The growth
inhibition of deletion strains upon exposure to compounds is

S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
199
Fig. 6. Cell cycle analysis of MCF-7 breast cancer cells that were treated with methanol (vehicle control; panel a), or with 10 (panel b), 11 (panel c), 15 (panel d) at their respective
IC50s for 24 h. The diagrams show the distribution of the cells according to their DNA content. The inserts give the percentages of cells in different cell cycle phases.
are in excellent agreement with our notion that the compounds
employed affect the redox status of the cell. Redox modulation as the
most likely mode of action of our compounds is also in line with the
other cell-based assays performed as part of this study.
inhibit proteins and enzymes. It is likely that a combination of
a ‘ROS generator’ and a ‘ROS user’ is effective in increasing levels
and severity of OS in cells and, in the case of cancer cells, pushes
them over the critical ROS threshold. In contrast, normal cells with
comparably low intrinsic levels of OS may be less affected. Inter-
estingly, some of the compounds showed no apparent reduction in
cell survival when incubated with normal healthy cells, and it is
therefore possible that such compounds may have a selective
anticancer activity.
Indeed, a chain of biochemical events is emerging, which leads
from our redox modulating compounds to ROS generation, reduc-
tion of GSH levels and OS, and subsequently to cell cycle arrest and
induction of apoptosis. These events seem to occur primarily in
cells with a pre-existing disturbance in their intracellular redox
balance, such as certain cancer cells.
Our studies provide ample opportunities for future research at
the chemistry/biology interface. As far as synthetic chemistry is
concerned, future studies may refine and expand the method
proposed here, building upon more e and more diverse e building
blocks, most of which will be easily accessible. At the same time,
there is a need for more chemically diverse catalytic compounds.
Here, the development of such multifunctional catalysts is not just
dictated by an interest in anticancer drugs. It also poses a real
challenge to synthetic organic chemistry, which needs to employ
a sophisticated arsenal of modern synthetic techniques to deal with
3. Conclusion
This study has shown a promising synthetic avenue able to
generate, with comparable ease, a wide range of tailor-made multi-
functional catalysts designed to target cancer cells under OS. The
use of simple, straight forward, yet effective multi-component
reactions has elegantly cleared the way for the synthesis of tri-
and tetra-functional redox agents containing multiple chalcogen,
porphyrin metal binding, and quinone redox sites. In addition, the
implementation of all combinations of P-3CR with rather flexible
building blocks supports the great potential of this concept toward
the diversity-oriented synthesis of multifunctional agents. Such
agents inhibit proliferation and induce cell death via multifactorial
mechanisms, such as induction of oxidative stress, cell cycle delay,
and apoptosis. Although one can only speculate about the exact
mode(s) of biochemical action of these compounds, the presence of
several redox sites in these molecules points toward a modulation
of the intracellular redox state in these cells.
Quinones are known to increase ROS production in cells, while
selenium compounds may ‘use’ ROS to oxidize and hence impair or

200
S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
Fig. 7. Flow cytometric analysis of apoptosis. A-431 melanoma cells were treated with 0, 8, 11, and 13 mM of compound 15 for 12 h, and stained with FITC annexin V and propidium
iodide. Quadrant I contains the percentage of viable cells (stained neither with PI nor with FITC annexin V); quadrant III shows the percentage of late apoptotic cells (stained with
both PI and FITC annexin V), and quadrant IV the percentage of early apoptotic cells (stained with FITC annexin V only).
the relevant quinone and chalcogen chemistry. The synthesis of
organoselenium and organotellurium compounds has never been
an easy task, and this area of research provides ample opportunities
for further development and expansion. Indeed, this study opens
the door to a range of follow-up studies in the area of synthetic
chemistry, redox chemistry, biochemistry and, ultimately, drug
development. Preliminary cell based studies point toward
a reasonably selective activity of some of these compounds, which
needs to be investigated further by using a considerably wider
arsenal of cells. Furthermore, in order to establish the complete
picture of redox catalysts as possible therapeutics, this line of
investigation will then need to move on to studies in animal
models. In any case, there is plenty of scope for further, multi-
disciplinary studies involving chemistry, biochemistry, cell
biology, and pharmacology in order to develop a strategy to treat
cancer by applying redox active compounds.
Table 3
Activity of the multifunctional compounds against S. cerevisiae wild type and mutant strains. Diameters (mm) of inhibition zones of an agar diffusion assay are provided. In each
case 6 mm disks with 20 g of the test compound were incubated.
m
Cpd. no.
BY4741
Control
YJR104C
SOD1
YHR008C
SOD2
YDR032C
PST2
YHL028W
WSC4
YLL060C
GTT2
YPL188W
POS5
YGL158W
RCK1
YOL049W
GSH2
YLL009C
COX17
YDL190C
UFD2
4
5
6
8
0
0
0
0
0
0
0
0
0
0
0
10
15
18
13
16
22
21
17
20
12
14
0
12
13
12
15
19
18
11
25
0
12
12
12
11
11
11
11
15
11
11
16
11
11
12
11
16
19
20
0
0
12
0
0
9
0
0
9
0
0
9
0
10
0
0
10
0
0
0
0
13
0
0
0
0
17
12
21
20
12
18
12
25
0
0
0
0
9
10
11
11
0
0
0
10
18
14
0
0
0
10
18
14
0
0
0
11
13
14
0
0
0
10
11
12
13
14
15
14
14
26
19
17
15
15
15
15

S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
201
At the end, this is one of the first studies on a vastly unknown
class of compounds and hence it is too early to report on metabolic
studies, pharmacokinetics in animals and enrichment of such
compounds in specific tissues or degradation. Nonetheless, these
issues are clearly important and will form part of future studies in
appropriate animal models.
145.3 (s), 133.8 (d), 133.5 (d), 132.7 (d, 2C), 132.6 (s), 131.9 (s), 129.6
(s), 129.1 (d, 2C), 127.1 (d), 126.8 (d), 126.7 (d), 74.2 (d), 51.5 (s), 35.6
(t), 32.5 (t), 29.1 (t), 28.6 (q, 3C), 22.5 (t), 15.4 (q) ppm. LCeMS (ESI):
m/z calc. 573.21, Rt ¼ 14.76 min, m/z found 574.15 [M þ H]þ. HRMS:
[M þ H] calc. 574.1141, [M þ H] found 574.1161, [M þ Na] calc.
596.0130 [M þ Na] found 596.0980. Isotopic pattern of selenium:
m/z (relative abundance %) 574.1161 (100), 575.1194 (30), 576.1163
(18), 577.1196 (7).
4. Experimental protocols
4.1. Material and methods
4.2.1.2. 1-(Benzylhexanoatecarbamoyl)-2-(phenylselanyl)ethyl-3-
(1,4-dihydro-2-methyl-1,4-dioxonaphthalen-3-ylthio)propanoate (6).
Compound 6 was synthesized from 2-(phenylselanyl)acetaldehyde
All chemical reagents for the synthesis of compounds were
purchased from SigmaeAldricheFluka and used without further
purification unless stated otherwise. Reactions under inert atmo-
sphere were carried out under argon (4.6) using standard Schlenk
(200
2-ylthio)propanoic acid (331.6 mg, 1.2 mmol) and benzyl 6-
isocyanohexanoate (346.9 L, 1.5 mmol). Its formation was moni-
mL, 1 mmol), 3-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-
m
techniques. Silica gel 60 (MachereyeNagel, 50e200
m
m) was used
tored by TLC petrol ether: ethyl acetate ¼ 4:1, Rf ¼ 0.35, purified by
column chromatography on silica gel with petrol ether: ethyl
acetate ¼ 3:1. Yield ¼ 71%. 1H NMR (CDCl3, 500 MHz): dppm ¼ 8.02e
7.97 (m, 2H), 7.65e7.61 (m, 2H), 7.45e7.41 (m, 2H), 7.29e7.21 (m,
5H), 7.17e7.12 (m, 3H), 6.41 (br t, 1H), 5.37e5.33 (dd, J ¼ 4.4, 5.8 Hz,
1H), 5.00 (s, 2H), 3.41e3.37 (dd, J ¼ 4.4, 13.5 Hz, 1H), 3.28e3.11 (m,
5H), 2.56e2.50 (m, 1H), 2.46e2.40 (m, 1H), 2.27e2.41 (m, 5H),
for column chromatography. Unless noted otherwise, the dimen-
sions of columns used were 2.5 cm (diameter) and 25e30 cm
(height of silica gel). TLC plates (silica gel 60 F254, 0.20 mm) were
purchased from Merck. NMR Spectroscopy: 1H NMR spectra were
recorded at 500 MHz, 13C NMR spectra at 125 MHz on a Bruker DRX
500 or Avance 500 spectrometer. Chemical shifts are reported in
d
(ppm), expressed relative to the solvent signal at 7.26 ppm (CDCl3,
1.59e1.53 (m, 2H), 1.48e1.42 (m, 2H), 1.31e1.25 (m, 2H) ppm. 13
C
1H NMR) and at 77.16 ppm (CDCl3, 13C NMR), as well as 3.31 ppm
(1H NMR, CD3OD) and 49.00 ppm (13C NMR, CD3OD). Coupling
constants (J) are given in Hz. LCeMS/MS analysis: Analyses were
performed using a TSQ Quantum mass spectrometer equipped with
an ESI source and a triple quadrupole mass detector (Thermo Fin-
nigan). HRMS: High-resolution mass spectrometry was performed
on an Accela UPLC-system (Thermo-Fisher) coupled to a linear
trap-FT-Orbitrap combination (LTQ-Orbitrap), operating in positive
ionization mode. These spectra indicated the ꢂ95% purity of the
prepared compounds.
NMR (CDCl3, 125.79 Hz): dppm ¼ 182.3 (s), 181.5 (s), 173.5 (s), 170.2
(s), 168.2 (s), 148.3 (s), 145.5 (s), 136.3 (s), 134.1 (d), 133.7 (d), 133.1
(d, 2C),132.9 (s), 132.2 (s),129.8 (s),129.4 (d, 2C),128.8 (d, 2C),128.4
(d), 128.3 (d, 2C), 127.5 (d), 127.6 (d), 126.9 (d), 73.9 (d), 66.4 (t), 39.5
(t), 35.4 (t), 34.2 (t), 29.3 (t, 2C), 29.1 (t), 26.5 (t), 24.6 (t), 15.7 (q)
ppm. LCeMS (ESI): m/z calc. 707.146, Rt ¼ 15.16 min, m/z found
708.08 [M þ H]þ. HRMS: [M þ H] calc. 708.1456, [M þ H] found
708.1529, [M þ Na] calc. 730.1456 [M þ Na] found 730.1348.
Isotopic pattern of selenium: m/z (relative abundance %) 708.1529
(100), 709.1562 (40), 710.1530 (20), 711.1564 (5), 730.1348 (100),
731.1382 (38), 732.1350 (20), 733.1383 (5).
4.2. Synthesis and characterization
4.2.1.3. 1-(Benzylhexanoatecarbamoyl)-3-(phenylselanyl)propyl-3-
(1,4-dihydro-2-methyl-1,4-dioxonaphthalen-3-ylthio)propanoate (8).
Compound 8 was synthesized from 3-(phenylselanyl)propanal
4.2.1. General procedure for the preparation of compounds via the
three-component Passerini reaction 4e18
As a general procedure, a mixture of aldehyde (1 mmol),
carboxylic acid(1.2 mmol) and isonitrile (1.5 mmol) in 5 mL degassed
water was stirred at room temperature overnight. Upon completion
(monitored by TLC), 10 mL CH2Cl2 was added to dissolve the sticky
product. The aqueous layer was washed with CH2Cl2 three times, the
organic layers were combined, dried over Na2SO4 and concentrated
to yield a sticky product which was purified by chromatography on
silica gel, usually with petrol ether: ethyl acetate (4:1) as eluent.
Analytical information for individual compounds is given in the
Supplementary material.
(213.1
2-ylthio)propanoic acid (331.6 mg, 1.2 mmol) and benzyl 6-
isocyanohexanoate (346.9 L, 1.5 mmol). Its formation was moni-
mL, 1 mmol), 3-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-
m
tored by TLC petrol ether: ethyl acetate ¼ 4:1, Rf ¼ 0.33, purified by
column chromatography on silica gel with petrol ether: ethyl
acetate ¼ 2.5:1. Yield ¼ 75%.1H NMR (CDCl3, 500 MHz): dppm ¼ 7.99e
7.93 (m, 2H), 7.63e7.57 (m, 2H), 7.39e7.35 (m, 2H), 7.27e7.20 (m,
5H), 7.16e7.12 (m, 3H), 6.33 (br s,1H), 5.19e5.16 (m,1H), 4.96 (s, 2H),
3.71e3.69 (t, J ¼ 5.4 12.1 Hz, 2H), 3.34e3.30 (t, J ¼ 5.7, 12.4 Hz, 2H),
2.86e2.81 (t, J ¼ 8.0, 15.9 Hz, 2H), 2.76e2.65 (m, 2H), 2.29e2.24 (m,
5H), 2.15e2.09 (m, 2H),1.56e1.50 (q, J ¼ 7.2,15.25 Hz, 2H),1.44e1.38
(q, J ¼ 7.2, 14.8 Hz, 2H), 1.26e1.19 (m, 2H) ppm. 13C NMR (CDCl3,
125.79 Hz): dppm ¼ 181.9 (s), 181.3 (s), 173.2 (s), 170.1 (s), 168.6 (s),
147.9 (s), 145.2 (s), 135.9 (s), 133.8 (d), 133.4 (d), 132.7 (d, 2C), 132.5
(s), 132.1 (s), 129.5 (s), 129.1 (d, 2C), 128.4 (d, 2C), 128.1 (d), 128.0 (d,
2C), 127.0 (d), 127.7 (d), 126.6 (d), 73.9 (d), 66.0 (t), 39.0 (t), 35.4 (t),
33.9 (t), 32.5 (t), 29.2 (t), 29.0 (t), 26.1 (t), 24.3 (t), 22.5 (t), 15.3 (q)
ppm. LCeMS (ESI): m/z calc. 721.16, Rt ¼ 15.10 min, m/z found 721.94
[M þ H]þ. HRMS: [M þ H] calc. 722.1556, [M þ H] found 722.1685,
[M þ Na] calc. 744.1456 [M þ Na] found 744.1505. Isotopic pattern of
selenium: m/z (relative abundance %) 722.1685 (100), 723.1719 (39),
724.1687 (18), 725.1721 (6), 744.1505 (100), 745.1538 (36), 746.1506
(16), 747.1540 (5).
Compounds number 5, 7, 11, 15, and 18 were synthesized
according to the literature [9,10].
4.2.1.1. 1-(tert-Butylcarbamoyl)-3-(phenylselanyl)propyl3-(1,4-
dihydro-2-methyl-1,4-dioxonaphthalen-3-ylthio)propanoate
Compound 4 was synthesized from 3-(phenylselanyl)propanal
(213.1 L, 1 mmol), 3-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-
2-ylthio)propanoic acid (331.6 mg, 1.2 mmol) and tert-butyl iso-
cyanide (124.6 L, 1.5 mmol). Its formation was monitored by TLC
(4).
m
m
petrol ether: ethyl acetate ¼ 4:1, Rf ¼ 0.63, purified by column
chromatography on silica gel with petrol ether: ethyl acetate ¼ 5:1.
Yield ¼ 62%. 1H NMR (CDCl3, 500 MHz): dppm ¼ 8.03e7.97 (m, 2H),
7.66e7.61 (m, 2H), 7.41e7.38 (m, 2H), 7.17e7.14 (m, 3H), 5.84 (br s,
1H), 5.08e5.06 (t, J ¼ 5.4, 11.8 Hz, 1H), 3.37e3.34 (t, J ¼ 7.3, 13.9 Hz,
2H), 2.86e2.83 (t, J ¼ 7.9, 15.5 Hz, 2H), 2.74e2.71 (m, 2H), 2.27 (s,
3H), 2.18e2.12 (m, 2H), 1.27 (s, 9H) ppm. 13C NMR (CDCl3,
125.79 Hz): dppm ¼ 182.0 (s), 181.3 (s), 170.2 (s), 167.7 (s), 147.5 (s),
4.2.1.4. 1-(Benzylhexanoatecarbamoyl)-2-(1,4-dihydro-2-methyl-
1,4-dioxonaphthalen-3-ylselanyl)ethyl3-(1,4-dihydro-2-methyl-1,4-
dioxonaphthalen-3-ylthio)propanoate
(9). Compound
9
was

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S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
synthesized from 2-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-
ylselanyl)acetaldehyde (293 L, 1 mmol), 3-(3-methyl-1,4-dioxo-
1,4-dihydronaphthalen-2-ylthio)propanoic acid (331.6 mg,1.2 mmol)
and benzyl 6-isocyanohexanoate (346.9 L, 1.5 mmol). Its formation
(346.95 mL, 1.5 mmol). Its formation was monitored by TLC petrol
m
ether: ethyl acetate ¼ 2.5:1, Rf ¼ 0.18, purified by column chroma-
tography on silica gel with petrol ether: ethyl acetate ¼ 2:1.
Yield ¼ 75% of the two isomers. We separated the two isomers by
column chromatography for analytical reasons. Isomer 1: 1H NMR
(CDCl3, 500 MHz): dppm ¼ 8.02e7.99 (m, 2H), 7.65e7.60 (m, 2H),
7.29e7.24 (m, 5H), 7.15 (br s, 1H), 5.50e5.45 (m, 2H), 5.05 (s, 2H),
4.25e4.15 (m, 1H), 4.05e3.95 (m, 1H), 3.85e3.65 (m, 2H), 3.45e3.40
(m, 1H), 3.25e3.15 (m, 2H), 2.31e2.38 (m, 5H), 1.57e1.39 (m, 4H),
1.35 (s, 9H), 1.27e1.21 (m, 2H) ppm. 13C NMR (CDCl3, 125.79 Hz):
dppm ¼ 182.1 (s), 181.3 (s), 174.3 (s), 169.9 (s), 168.1 (s), 153.7 (s), 145.0
(s),133.8 (d),133.5 (d),132.6 (s),132.0 (s),130.8 (s),128.8 (s),128.6 (d,
2C), 128.3 (d), 128.2 (d, 2C), 127.1 (d), 126.8 (d), 80.4 (s), 74.5 (d), 66.5
(t), 63.7 (t), 56.1 (d), 39.2 (t), 33.9 (t), 28.8 (t), 28.5 (t), 28.3 (q, 3C),
26.0 (t), 24.2 (t), 17.5 (q) ppm. Isomer 2: 1H NMR (CDCl3, 500 MHz):
dppm ¼ 8.03e7.98 (m, 2H), 7.65e7.60 (m, 2H), 7.30e7.25 (m, 5H),
7.15 (br s, 1H), 5.51e5.41 (m, 2H), 5.05 (s, 2H), 4.30e4.17 (m, 1H),
4.08e4.01 (m, 1H), 3.78e3.71 (m, 2H), 3.50e3.38 (m, 1H), 3.26e3.15
(m, 2H), 2.31e2.25 (m, 5H), 1.56e1.43 (m, 4H), 1.33 (s, 9H), 1.27e1.21
(m, 2H) ppm. 13C NMR (CDCl3, 125.79 Hz): dppm ¼ 181.9 (s), 181.3 (s),
174.2 (s), 169.9 (s), 168.0 (s), 155.8 (s), 145.0 (s), 133.8 (d), 133.5 (d),
132.5 (s), 131.9 (s), 130.8 (s, 2C), 128.5 (s), 128.3 (d, 2C), 128.2 (d),
128.2 (d, 2C), 127.1 (d), 126.8 (d), 80.3 (s), 74.5 (d), 66.5 (t), 63.7 (t),
56.1 (d), 39.1 (t), 33.9 (t), 28.9 (t), 28.8 (t), 28.2 (q, 3C), 26.0 (t), 24.2
(t), 17.5 (q) ppm. LCeMS (ESI): m/z calc. 730.20, Rt ¼ 13.53 min, m/z
found 730.96 [M þ H]þ. HRMS: [M þ H] calc. 731.2097, [M þ H]
found 731.2077, [M þ Na] calc. 753.1807 [M þ Na] found 753.1897.
Isotopic pattern of selenium: m/z (relative abundance %) 731.2077
(100), 732.2111 (37), 733.2079 (21), 734.2113 (4), 753.1897 (100),
754.1930 (28), 755.1899 (15), 756.1932 (4).
m
was monitored by TLC petrol ether: ethyl acetate ¼ 4:1, Rf ¼ 0.30,
purified by column chromatography on silica gel with petrol ether:
ethyl acetate ¼ 2.5:1. Yield ¼ 73%. 1H NMR (CDCl3, 500 MHz):
dppm ¼ 8.02e7.95 (m, 4H), 7.66e7.56 (m, 4H), 7.30e7.21 (m, 5H), 6.48
(br s, 1H), 5.49e5.46 (m, 1H), 5.01 (s, 2H), 3.69e3.65 (dd, J ¼ 4.5,
13.0 Hz, 1H), 3.50e3.46 (dd, J ¼ 6.2, 13.0 Hz 1H), 3.31e3.23 (m, 2H),
3.19e3.15 (dt, J ¼ 6.7, 13.0 Hz, 2H), 2.67e2.64 (t, J ¼ 6.2, 13.0 Hz, 2H),
2.29e2.24 (m, 8H), 1.58e1.52 (m, 2H), 1.48e1.42 (m, 2H), 1.30e1.18
(m, 2H) ppm. 13C NMR (CDCl3, 125.79 Hz): dppm ¼ 182.0 (s), 181.6
(s), 181.3 (s), 181.2 (s), 173.3 (s), 170.0 (s), 167.7 (s), 148.9 (s), 148.0 (s),
145.5 (s), 145.2 (s), 135.9 (s), 133.8 (d), 133.7 (d), 133.5 (d), 133.4 (d),
132.7 (s),132.6 (s), 131.9 (s),131.8 (s), 128.5 (d, 2C),128.2 (d),128.1 (d,
2C),127.0 (d),126.7 (d),126.6 (d, 2C), 74.1 (d), 66.1 (t), 39.2 (t), 35.4 (t),
34.0 (t), 29.1 (t), 29.0(t), 28.8(t), 26.3(t), 24.4 (t),17.5(q),15.4 (q) ppm.
LCeMS (ESI): m/z calc. 801.151, Rt ¼ 17.44 min, m/z found 823.89
[M þ Na]þ. HRMS: [M þ H] calc. 802.1576, [M þ H] found 802.1584,
[M þ Na] calc. 824.1406 [M þ Na] found 824.1403. Isotopic pattern of
selenium: m/z (relative abundance %) 802.1584 (100), 803.1617 (43),
804.1585 (17), 805.1619 (7), 808.1610 (2), 824.1403 (100), 825.1437
(41), 826.1405 (5), 827.1438 (3).
4.2.1.5. tert-Butyl(R)-1-((1-(tert-butylcarbamoyl)-2-(1,4-dihydro-2-
methyl-1,4-dioxonaphthalen-3-ylselanyl)ethoxy)carbonyl)-2-
hydroxyethylcarbamate (10). Compound 10 was synthesized from
2-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-ylselanyl)acetal-
dehyde (293
ypropanoic acid (246 mg, 1.2 mmol) and tert-butyl isocyanide
(124.6 L, 1.5 mmol). Its formation was monitored by TLC petrol
mL, 1 mmol), 2-(tert-butoxycarbonylamino)-3-hydrox-
m
4.2.1.7. tert-Butyl((1-(benzylhexanoatecarbamoyl)-2-(1,4-dihydro-2-
methyl-1,4-dioxonaphthalen-3-ylselanyl)ethoxy)carbonyl)methyl-
carbamate (13). Compound 13 was synthesized from 2-(3-methyl-
ether: ethyl acetate ¼ 4:1, Rf ¼ 0.54, purified by column chroma-
tography on silica gel with petrol ether: ethyl acetate ¼ 2.5:1.
Yield ¼ 76% of the two isomers. We separated the two isomers by
column chromatography for analytical reasons. Isomer 1: 1H NMR
(CDCl3, 500 MHz): dppm ¼ 8.05e8.02 (m, 1H), 7.99e7.97 (m, 1H),
7.68e7.62 (m, 2H), 6.64 (br s,1H), 5.37e5.30 (m, 2H), 4.28 (br d,1H),
4.16 (br s, 1H), 3.74e3.66 (m, 2H), 3.50 (br t, 1H), 3.31e3.26 (dd,
1,4-dioxo-1,4-dihydronaphthalen-2-ylselanyl)acetaldehyde (293
mL,
1
mmol), 2-(tert-butoxycarbonylamino)acetic acid (210 mg,
1.2 mmol) and benzyl 6-isocyanohexanoate (346.9 mL, 1.5 mmol). Its
formation was monitored by TLC petrol ether: ethyl acetate ¼ 2.5:1,
Rf ¼ 0.54, purified by column chromatography on silica gel with
petrol ether: ethyl acetate ¼ 2:1. Yield ¼ 66%. 1H NMR (CDCl3,
500 MHz): dppm ¼ 8.03e8.00 (m, 2H), 7.66e7.60 (m, 2H), 7.30e7.23
(m, 5H), 6.79 (br s, 1H), 5.53e5.51 (m, 1H), 5.03 (s, 2H), 4.79
(br t, 1H), 3.80e3.63 (m, 3H), 3.50e3.46 (dd, J ¼ 6.9, 13.8 Hz, 1H),
3.21e3.07 (m, 2H), 2.30e2.28 (m, 5H), 1.61e1.55 (m, 2H), 1.50e1.44
(m, 2H), 1.35 (s, 9H), 1.30e1.24 (m, 2H) ppm. 13C NMR (CDCl3,
125.79 Hz): dppm ¼ 181.6 (s), 181.3 (s), 173.3 (s), 168.8 (s), 167.6 (s),
156.5 (s), 148.9 (s), 136.4 (s), 133.7 (d), 133.5 (d, 2C), 132.7 (s), 131.9
(s), 128.5 (d, 2C), 128.2 (d, 2C), 126.9 (d), 126.8 (d), 80.6 (s), 74.4 (d),
66.1 (t), 42.3 (t), 39.3 (t), 34.1 (t), 28.9 (t), 28.8 (t), 28.2 (q, 3C), 26.2
(t), 24.4 (t), 17.5 (q) ppm. LCeMS (ESI): m/z calc. 700.19,
Rt ¼ 13.83 min, m/z found 701.15 [M þ H]þ. HRMS: [M þ H] calc.
701.1976, [M þ H] found 701.1972, [M þ Na] calc. 723.1707 [M þ Na]
found 723.1791. Isotopic pattern of selenium: m/z (relative abun-
dance %) 701.1972 (100), 702.2005 (36), 703.1974 (19), 704.2007 (2),
723.1791 (100), 724.1825 (39), 725.1825 (20), 726.1827 (2).
J ¼ 8.3, 13.5 Hz, 1H), 2.29 (s, 3H), 1.37 (s, 9H), 1.29 (s, 9H) ppm. 13
C
NMR (CDCl3, 125.79 Hz): dppm ¼ 182.6 (s), 181.2 (s), 170.3 (s), 167.5
(s), 148.9 (s), 144.8 (s), 134.0 (d), 133.5 (d), 132.6 (s), 132.0 (s), 126.9
(d), 126.8 (d), 80.3 (s), 63.7 (t), 56.1 (s), 52.0 (s), 75.3 (d), 77.3 (d),
28.9 (t), 28.6 (q, 3C), 28.3 (q, 3C), 17.3 (q) ppm. LCeMS (ESI): m/z
calc. 582.148, Rt ¼ 12.58 min, m/z found 583.04 [M þ H]þ. HRMS:
[M þ H] calc. 583.1523, [M þ H] found 583.1553, [M þ Na] calc.
605.1306 [M þ Na] found 605.1373. Isotopic pattern of selenium: m/
z (relative abundance %) 583.1553 (100), 584.1587 (34), 585.1555
(20), 586.1588 (4), 605.1373 (100), 606.1406 (26), 607.1374 (16),
608.1408 (4).
Isomer 2: 1H NMR (CDCl3, 500 MHz): dppm ¼ 8.09e8.02 (m, 1H),
8.04e8.02 (m, 1H), 7.79e7.59 (m, 2H), 6.46 (br s, 1H), 5.55e5.32 (m,
2H), 4.25 (br d, 1H), 4.07 (br s, 1H), 3.88e3.70 (m, 2H), 3.50 (br t,
1H), 3.25e3.40 (dd, J ¼ 7.6, 13.2 Hz, 1H), 2.35 (s, 3H), 1.42 (s, 9H),
1.34 (s, 9H) ppm. 13C NMR (CDCl3, 125.79 Hz): dppm ¼ 182.4 (s), 181.1
(s), 170.5 (s), 167.1 (s), 148.5 (s), 144.7 (s), 133.9 (d), 132.5 (d), 132.4
(s), 131.9 (s), 127.0 (d), 126.8 (d), 80.8 (s), 75.3 (d), 77.3 (d), 62.9 (t),
56.1 (s), 51.9 (s), 29.7 (t), 28.9 (q, 3C), 28.5 (q, 3C), 17.5 (q) ppm.
4.2.1.8. (2S)-1-(Benzylhexanoatecarbamoyl)-2-(1,4-dihydro-2-methyl-
1,4-dioxonaphthalen-3-ylselanyl)ethyl 3-(2-tert-butyldisulfanyl)-2-(piv-
alamido)propanoate (14). Compound 14 was synthesized from 2-(3-
methyl-1,4-dioxo-1,4-dihydronaphthalen-2-ylselanyl)acetaldehyde
4.2.1.6. tert-Butyl(R)-1-((1-(benzylhexanoatecarbamoyl)-2-(1,4-
dihydro-2-methyl-1,4-dioxonaphthalen-3-ylselanyl)ethoxy)carbonyl)-
2-hydroxyethylcarbamate (12). Compound 12 was synthesized from
2-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-ylselanyl)acetalde-
(293
sulfanyl)propanoic acid (370.8 mg, 1.2 mmol) and benzyl 6-
isocyanohexanoate (346.9 L, 1.5 mmol) following the general
mL, 1 mmol), 2-(tert-butoxycarbonylamino)-3-(tert-butyldi-
m
hyde (293
mL, 1 mmol), 2-(tert-butoxycarbonylamino)-3-hydroxyp-
procedure described above. Its formation was monitored by TLC
ropanoic acid (246 mg, 1.2 mmol) and benzyl 6-isocyanohexanoate
petrol ether: ethyl acetate ¼ 2.5:1, Rf ¼ 0.18, purified by column

S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
203
chromatography on silica gel with petrol ether: ethyl acetate ¼ 2:1.
Yield ¼ 85% of the two isomers. We were unable to separate the two
isomers by simple column chromatography. 1H NMR (CDCl3,
500 MHz): dppm ¼ 8.03e7.99 (m, 2H), 7.65e7.61 (m, 2H), 7.30e7.22
(m, 5H), 7.04 (br s, 1H), 5.48e5.48 (m, 1H), 5.23 (s, 2H), 5.03 (s, 2H),
3.14e3.02 (m, 3H), 2.30e2.26 (m, 6H), 1.60e1.54 (m, 2H), 1.50e1.43
(m, 3H), 1.37 (s, 3H), 1.33 (s, 9H), 1.24 (s, 9H) ppm. 13C NMR (CDCl3,
125.79 Hz): dppm ¼ 181.46 (s), 181.3 (s), 170.1 (s), 169.6 (s), 167.6 (s),
148.7 (s), 136.1 (s), 133.7 (d), 133.5 (d), 133.6 (d), 132.8 (s), 132.7 (s),
131.9 (s), 128.5 (d, 2C), 128.2 (d, 2C), 126.9 (d), 126.7 (d), 74.8 (d), 74.5
(d), 66.5 (t), 53.9 (s), 53.4 (s), 48.6 (s), 39.4 (t), 33.3 (t), 34.1 (t), 29.8 (q,
3C), 28.9 (t), 28.3 (q, 3C), 26.3 (t), 24.5 (t, 2C), 17.5 (q) ppm. LCeMS
with 1%
(Lonza), A-431 in RPMI 1640 (Gibco), HUVEC in EBM-2 (Lonza) and HF
in MEM (Gibco) supplemented with 1% -glutamine. All media were
L-glutamine and 1% non essential amino acids, A-498 in MEM
L
supplemented with 10% fetal calf serum (Lonza or Gibco).
MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bro-
mide] (Sigma) was used to measure the metabolic activity of cells
which are capable of reducing it by dehydrogenases to a violet
colored formazan product. Briefly, 120
mL aliquots of a cell
suspension (50,000 cells mLꢁ1) in 96-well microplates were incu-
bated at 37 ꢃC and 10% CO2 and allowed to grow for two days. Then
60
m
L of serial dilutions of the test compounds were added. After
24 h of incubation at 37 ꢃC and 10% CO2, 20
mL MTT in phosphate
(ESI): m/z calc. 818.20, Rt
¼
16.13 min, m/z found 835.02
buffered saline (PBS) were added to a final concentration of
0.5 mg mLꢁ1. After 2 h the precipitate of formazan crystals was
centrifuged and the supernatant discarded. The precipitate was
[M þ NH4]þ. HRMS: [M þ H] calc. 818.1196, [M þ H] found 835.2196,
[M þ NH4] calc. 857.2017 [M þ K] found 857.2015. Isotopic pattern of
selenium: m/z (relative abundance %) 835.2196 (100), 836.2229 (47),
837.2197 (20), 840.2189 (2).
washed with 100
mL PBS and dissolved in 100 mL isopropanol
containing 0.4% hydrochloric acid. The resulting color was
measured at 590 nm using an ELISA plate reader. All investigations
were carried out in two parallel experiments. The IC50 values were
determined as the concentrations of tested materials, which
showed 50% of the absorbance of untreated control cells as esti-
mated from the doseeresponse curves.
4.2.1.9. 21H,23H-Porphine,5-(4-((tert-butylcarbamoyl)methylformate)
phenyl)-10,15,20-triphenyl (16). Compound 16 was synthesized from
formaldehyde (75
nylporphyrin (789.0 mg, 1.2 mmol) and tert-butyl isocyanide
(124.6
L, 1.5 mmol). TLC, Rf ¼ 0.39 (petroleum ether: ethyl acetate
mL,1 mmol), 5-(4-carboxyphenyl)-10,15,20-triphe-
m
4:1). It was purified by column chromatography on silica gel with
petrol ether: ethyl acetate ¼ 5:2. The compound was obtained as
a purple solid. Yield ¼ 93%. 1H NMR (DMSO-d6, 500 MHz):
dppm ¼ 8.90e8.88 (m, 6H), 8.80e8.75 (m, 2H), 8.53e8.49 (m, 2H),
8.39e8.25 (m, 2H), 8.24e8.19 (m, 6H), 7.79e7.71 (m, 9H), 6.12 (s, 1H),
4.92 (s, 2H), 1.50 (s, 9H), ꢁ2.75 (s, 2H) ppm. 13C NMR (DMSO-d6,
125.79 Hz): dppm ¼ 167.44 (s), 166.0 (s), 149.60 (s), 148.19 (s), 145.72
(s), 142.25 (s, 8C), 134.81 (d), 134.53 (d, 10C), 128.02 (d, 2C), 127.87 (d,
2C), 126.73 (d, 10C), 120.99 (s), 120.70 (s, 4C), 118.17 (s), 77.02 (d, 2C),
52.08 (s), 29.36 (t), 28.79 (q, 3C) ppm. LCeMS (ESI): m/z calc. 771.32,
Rt ¼ 16.99 min, m/z found 772.18 [M þ H]þ. HRMS: [M þ H] calc.
772.3377, [M þ H] found 772.3265.
4.3.2. In vitro studies
4.3.2.1. DTNB assay as indicative of intracellular GSH concentrations.
MCF-7 cells (106) were treated with different concentrations of
selected test compounds (final concentration 5e20 mM) for 1 h at
37 ꢃC. The cells were removed by mild trypsinizing, centrifuged
at 800 rpm for 5 min, washed twice with cold phosphate
buffered saline and lysed using 5% w/v chilled metaphosphoric
acid at 4 ꢃC for 2 h to extract the cellular GSH. The suspension
was then centrifuged at 13,000 rpm for
5 min and GSH
determined by the following method. The supernatant was mixed
with 0.2 M sodium phosphate buffer (pH 8.0) and 0.04 mM DTNB
(5,50-dithiobis(2-nitrobenzoic acid)) and kept at room tempera-
ture for 10 min. The absorbance of the samples was recorded
against reagent blank at 412 nm in a UVevis double beam spec-
trophotometer (Shimadzu-1640). The GSH levels were calculated
from a standard curve prepared with known concentrations of GSH
under similar conditions.
4.2.1.10. 21H,23H-Porphine, 5-(4-(1-(tert-butylcarbamoyl)-2-(phenyl-
selanyl)ethyl formate)phenyl)-10,15,20-triphenyl (17). Compound 17
was synthesized from 2-(phenylselanyl)acetaldehyde (200
1 mmol), 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin (789.0 -
mg, 1.2 mmol) and tert-butyl isocyanide (124.6 L, 1.5 mmol). TLC,
mL,
m
Rf ¼ 0.51 (petroleum ether: ethyl acetate 4:1). It was purified by
column chromatography on silica gel with petrol ether: ethyl
acetate ¼ 5:2. The compound was obtained as a purple solid.
Yield ¼ 89%. 1H NMR (DMSO-d6, 500 MHz): dppm ¼ 8.98e8.90 (m,
6H), 8.85e8.75 (m, 2H), 8.20e8.35 (m, 10H), 7.82e7.75 (m, 10H),
7.68e7.60 (m, 2H), 7.29e7.35 (m, 2H), 6.15 (s, 1H), 5.75 (t, J ¼ 5.7,
4.3.2.2. Detection of ROS generation. The ability of multifunctional
redox compounds to induce intracellular ROS formation was
determined using DHE and DCF assays. Cells were seeded in 96
wells plates at a density of 105 cells per well and treated with
different concentrations of the test compounds for 1 h. The cells
(180
probes for 30 min in the dark. The fluorescence was immediately
read in a fluorescence spectrophotometer (DHE, lex
lem ¼ 540 ꢄ 25/
mL) were then incubated with 20 mL of 10 mM DHE or DCF
12.4 Hz, 1H), 3.75e3.40 (m, 2H), 1.45 (s, 9H), ꢁ2.85 (s, 2H) ppm. 13
C
NMR (CDCl3, 125.79 Hz): dppm ¼ 167.43 (s), 165.35 (s), 165.30 (s),
147.80 (s), 142.01 (s, 10C), 134.66 (d, 4C), 134.53 (d, 10C), 132.99 (d,
2C),129.74 (s),129.29 (d, 6C),128.02 (d),127.82 (d),127.32 (s),126.73
(d, 8C), 120.71 (s, 2C), 120.45 (s, 2C), 74.4 (d), 51.81 (s), 29.08 (t),
28.79 (q, 3C) ppm. LCeMS (ESI): m/z calc. 941.28, Rt ¼ 18.40 min, m/z
found 942.43 [M þ H]þ. HRMS: [M þ H] calc. 942.2937, [M þ H]
found 942.2917. Isotopic pattern of Se: m/z (relative abundance %)
942.2917 (100), 943.2950 (63), 944.2984 (20), 945.2952 (12).
/
600 ꢄ 40 nm; DCF, lex lem ¼ 485 ꢄ 20/528 ꢄ 20 nm). Results were
/
expressed as arbitrary units per 105 cells.
4.3.2.3. Determination of caspase-3/7 activity. The caspase activity
was assessed in A-431 cells with the caspase-Glo 3/7 kit (Promega)
according to supplier’s instruction. Briefly, melanoma cells were
seeded in 384-well plates at a density of 20,000 cells per well and
treated with 2.8 mM of test compound 15 and allowed to incubate
4.3. Biological assays
for 1, 2, 4, 6, 12, and 24 h in the dark (37 ꢃC, at 5% CO2). Caspase-Glo
3/7 reagent was then added and kept at room temperature for
further 30 min in the dark. Luminescence of each sample was
measured in a plate-reading luminometer.
4.3.1. Cytotoxicity assay
All cell lines were from the DSMZ (Braunschweig, Germany).
Human umbilical vein endothelial cells (HUVEC) were from Lonza,
human fibroblasts (HF) isolated from foreskin were a generous gift of
Dr. Thierauch, Bayer-Schering PharmaAG. All cells were grownat 37ꢃC
and 10% CO2 in the following media: MCF-7 in DMEM supplemented
4.3.2.4. Monitoring phosphatidylserine translocation. Apoptosis was
also analyzed by FITC annexin V: propidium iodide staining (BD
Biosciences). A-431 cells (106) were treated with different

204
S. Shaaban et al. / European Journal of Medicinal Chemistry 58 (2012) 192e205
concentrations (0, 8, 11, and 13
m
M) of test compound 15 for 12 h.
Appendix A. Supplementary material
Cells were then trypsinized and washed with PBS. 106 cells were
suspended in 1 mL binding buffer, then 100
stained with 5 L FITC annexin V and 5 L PI for 15 min. At the end
of incubation 400 L of binding buffer was added and the samples
were analyzed by FacScan.
m
L were transferred and
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.09.033.
m
m
m
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