Sulfur, selenium and tellurium pseudopeptides: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2014.05.019

A new series of sulfur, selenium and tellurium peptidomimetic compounds was prepared employing the Passerini and Ugi isocyanide based multicomponent reactions (IMCRs). These reactions were clearly superior to conventional methods traditionally used for organoselenium and organotellurium synthesis, such as classical nucleophilic substitution and coupling methods. From the biological point of view, these compounds are of considerable interest because of suspected anticancer and antimicrobial activities. While the sulfur and selenium containing compounds generally did not show either anticancer or antimicrobial activities, their tellurium based counterparts frequently exhibited antimicrobial activity and were also cytotoxic. Some of the compounds synthesized even showed selective activity against certain cancer cells in cell culture. These compounds induced a cell cycle delay in the G0/G1 phase. At closer inspection, the ER and the actin cytoskeleton appeared to be the primary cellular targets of these tellurium compounds, in line with some of our previous studies. As most of these peptidomimetic compounds also comply with Lipinski's Rule of Five, they promise good bioavailability, which needs to be studied as part of future investigations.

Full text of DOI:10.1016/j.bmc.2014.05.019

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Sulfur, selenium and tellurium pseudopeptides: Synthesis
and biological evaluation
c
c,
Saad Shaaban a,b,c, Florenz Sasse b, , Torsten Burkholz , Claus Jacob
⇑
⇑
a Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhoria Street, Mansoura 35516, Egypt
b Department of Chemical Biology, Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany
c Division of Bioorganic Chemistry, School of Pharmacy, Saarland University, D-66123 Saarbruecken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of sulfur, selenium and tellurium peptidomimetic compounds was prepared employing the
Passerini and Ugi isocyanide based multicomponent reactions (IMCRs). These reactions were clearly
superior to conventional methods traditionally used for organoselenium and organotellurium synthesis,
such as classical nucleophilic substitution and coupling methods. From the biological point of view, these
compounds are of considerable interest because of suspected anticancer and antimicrobial activities.
While the sulfur and selenium containing compounds generally did not show either anticancer or anti-
microbial activities, their tellurium based counterparts frequently exhibited antimicrobial activity and
were also cytotoxic. Some of the compounds synthesized even showed selective activity against certain
cancer cells in cell culture. These compounds induced a cell cycle delay in the G0/G1 phase. At closer
inspection, the ER and the actin cytoskeleton appeared to be the primary cellular targets of these tellu-
rium compounds, in line with some of our previous studies. As most of these peptidomimetic compounds
also comply with Lipinski’s Rule of Five, they promise good bioavailability, which needs to be studied as
part of future investigations.
Received 18 March 2014
Revised 9 May 2014
Accepted 12 May 2014
Available online xxxx
Keywords:
Isonitrile based multicomponent reactions
(IMCR)
Tellurium
Redox-modulation
Anticancer activity
Antimicrobial activity
Lipinski’s Rule of Five
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
are legion and become immediately apparent.3 Furthermore, many
of these compounds exhibit good solubility, permeability—and
The efficient synthesis of drug-like molecules is a major theme
in the field of pharmaceutical chemistry. In this context, multicom-
ponent reactions (MCRs) offer an interesting approach to achieve
structural diversity and molecular complexity through comparably
straight-forward chemical transformations. Indeed, the easy access
to a wide variety of starting materials, comparably mild reaction
conditions and the wide spectrum of attainable products place
such reactions at the heart of many combinatorial approaches
and also form the bedrock for the generation of small product
libraries.1,2
In pharmaceutical chemistry, isocyanide-based MCRs (IMCRs),
such as the Ugi and Passerini reactions, are of particular interest
as they allow the synthesis of peptidomimetic compounds, that
is, compounds which ‘resemble’ natural, peptide-like substances.
Many peptides possess signaling and regulatory functions, and
therefore the potential applications of such peptide-like structures
hence bioavailability—and are also rather stable chemically and
metabolically. In fact, among the various MCRs, the Ugi and Passe-
rini reactions can be considered a breakthrough as far as diversity
of products, chemo-, regio- and stereoselectivity and ease of oper-
ation are concerned.4–7
At the same time, chalcogen containing peptoids often also
exhibit pronounced—yet also selective—biological activities,8–10
especially in the context of redox-modulation. Although, several
preparative methods are nowadays available for the preparation
of simple chalcogen-containing molecules, there have been only
a few reports on the synthesis of more sophisticated compounds,
such as larger, multifunctional selenium- or tellurium compounds
or biologically ‘more amenable’ structures, such as redox-active
seleno- and telluropeptoids. This may be due to the fact that orga-
noselenium and organotellurium chemistry are not always straight
forward. Furthermore, decomposition of the products and difficul-
ties to generate compounds with sufficient purity are often
observed.
⇑
Corresponding authors. Tel.: +49 531 6181 3428; fax: +49 531 6181 3499 (F.S.);
Recently, we have therefore applied the potential provided by
IMCRs to the synthesis of biologically useful chalcogen-compounds,
reporting the synthesis of selenium- and tellurium-containing
tel.: +49 681 302 3129; fax: +49 681 302 3464 (C.J.).
E-mail addresses: florenz.sasse@helmholtz-hzi.de (F. Sasse), c.jacob@mx.
uni-saarland.de (C. Jacob).
http://dx.doi.org/10.1016/j.bmc.2014.05.019
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Shaaban, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.019

2
S. Shaaban et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
HO
O
HO
O
O
O
HO
O
O
H
N
O
O
OH
S
S
OH
O
1a
1b
1c
1d
1e
Te
N
Se
N
N
C
C
C
2a
2b
2c
NH2
O
HCHO
Figure 1. Acid, aldehyde and isonitrile building blocks used in the Passerini and Ugi multicomponent reactions.
peptidomimetic compounds using IMCRs.11,12 Although the
method appears to work well, the scope of the method is rather lim-
ited by the accessibility to the isonitrile building block. Chalcogens
(sulfur, selenium and tellurium) bearing isonitriles have therefore
been synthesized accordingly to overcome this sophisticated limi-
tation.13 Based on these results, we are now interested to further
explore the reactivity of these isonitriles and to generate a library
of diverse and highly functionalized organochalcogens of expected
biological activity.
Additionally, the use of water as solvent permitted the reaction to
be conducted rapidly, and as the products are often insoluble in
water, it also facilitated their isolation as precipitates. In contrast,
low yields and the formation of unfavourable by-products were
observed in case of the tellurium-containing isonitrile. Methanol,
ethanol and trifluoroethanol, described in the literature as good sol-
vents for MCRs, were used instead; nonetheless, the yield remained
low. The best result in case of tellurium isonitrile was achieved
using chloroform as a solvent which in turn led to an improvement
of yield and a minimization of side products (yields were in the
range of 64–96%).
2. Results and discussion
Besides the mild reaction conditions employed and the good
yields obtained, this synthetic strategy has additional advantages
over conventional methods. Firstly, it is now possible to insert phe-
nyl chalcogenides (especially phenyl telluride) into molecules via
IMCRs. Secondly, by keeping the isonitriles fixed whilst varying
the other synthetically accessible building blocks as part of a
‘mix and match’ approach, it has been possible to assemble a
diverse arsenal of chalcogen-based compounds, that is, a small
library of potential redox modulators which was subsequently
tested in biologically relevant in vitro assays.
Our present study aimed at the synthesis of novel peptidomi-
metic redox modulating substances with potential biological activ-
ity, which was subsequently investigated in different biological
assays. As before, the IMCR-based approach turned out to be rather
successful for the synthesis of a range of chemically diverse sulfur-,
selenium- and tellurium-containing peptidomimetic compounds
(3–20). These compounds were tested in various cancer cell lines
and, in the case of the tellurium compounds, showed considerable
activity in many of these cell cultures, possibly by selectively dam-
aging the ER and actin cytoskeleton, delaying the cell cycle and
inducing apoptosis. Some of these peptidomimetic compounds
even exhibited antimicrobial activity which needs to be investi-
gated further. These results will now be presented and discussed
in more detail.
2.2. Biological evaluation
2.2.1. Preliminary screen for biological activity
As part of this biological evaluation, a preliminary in vitro anti-
tumor screening was performed under the Developmental Thera-
peutics Program (DTP) offered by National Cancer Institute (NCI)
of the National Institute of Health (NIH) in Bethesda, Maryland,
United States. Here, compounds1 5, 6, 9 and 13 were exposed to
58 different cancer cell lines including leukemia cells (CCRF-CEM,
HL-60 (TB), K-562, MOLT-4 and SR), non-small cell lung cancer cells
(A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-23, NCI-
H322M, NCI-H460 and NCI-H522), colon cancer cells (COLO 205,
HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620), melanoma cells
(LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-ME-28,
SK-MEL-5, UACC-257 and UACC-62), ovarian cancer cells (IGROV1,
OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI/ADR-RES and SK-OV-
3), renal cancer cells (786-0, A498, ACHN, CAKI-1, RXF 393, SN
12C, TK-10 and UO-31), prostate cancer cells (PC-3 and DU-145)
2.1. Chemistry
As far as the chemical synthesis is concerned, readily available
building blocks, including isonitriles were used in the Passerini
and Ugi MCRs for the synthesis of 18 peptidomimetic compounds
(3–20) (Fig. 2). The individual building blocks used are shown in
Figure 1, whereby the isonitriles were prepared in a good yield
(up to 82%) by dehydration of their corresponding formamides
using phosphoryl chloride (POCl3).
In order to optimize the reaction conditions and the yield, the
reaction was performed in different solvents. In the case of the sul-
fur- and the selenium-containing isonitriles, a significant enhance-
ment of the reaction rate and yields (75–94%) was achieved when
using water as solvent instead of the classical organic solvents. This
could be attributed to various factors, including the hydrophobic
effect and enhanced hydrogen bonding in the transition state.14–16
1
The compounds were chosen according to the National Cancer Institute (NCI)
guidelines.
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S. Shaaban et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
O
O
O
O
O
O
O
O
O
O
H
N
H
N
H
N
S
Se
Te
S
N
O
S
N
O
S
N
O
O
H
N
O
O
O
O
Te
S
O
O
3
4
5
6
O
O
O
H
H
H
H
H
N
H
O
N
N
S
O
N
N
Se
O
N
Te
N
N
N
O
H
N
H
N
O
O
O
O
O
O
O
Te
O
O
O
O
O
O
7
8
9
10
O
O
O
O
H
H
H
N
H
N
N
S
N
Se
Te
S
N
O
N
N
O
N
O
O
O
O
O
O
11
12
13
14
O
O
H
H
N
Se
N
Te
O
N
O
N
O
O
H
H
N
N
S
O
O
Te
N
O
O
O
O
15
16
17
18
O
H
N
Se
N
O
O
H
N
O
Te
O
O
19
20
Figure 2. Influence of multifunctional redox compounds on the viability of human cancer and primary cell lines.
and breast cancer cells (MCF7, MDA-MB-231/ATCC, HS 578T, BT-549
and T-47D). As part of this routine screening programme, five differ-
ent doses were used and cells were incubated with compounds for
48 h. The LC50 values obtained for the compounds tested generally
were listed in Supporting information and were below 10 mM,
pointing towards a general and quite significant biological activity
(see below).
Based on these results, the compounds under investigation
could be clustered into three different groups: (1) sulfur- and sele-
nium-based compound; (2) tellurium-based compounds and (3)
quinone-based compounds. Compounds falling in the first group,
that is, containing sulfur (7, 11, 14 and 18) or selenium (8, 12, 15
and 19), were unable to reduce the survival of cancer cells and pri-
mary cells.
Furthermore, a COMPARE analysis was performed for each com-
pound, which revealed that there are some general similarities in
the mode of action with known anti-cancer compounds such as cis-
platin, methyl mitomycin C and anthracycline-based redox agents
(e.g., menogaril, deoxydoxorubicin, and MX2 HCl) which are used
to treat various types of cancers (see Supporting information).
In contrast, tellurium-containing compounds (9, 10, 13, 16, 17
and 20) exhibited a significant cytotoxic effect, whereby the tellu-
rium-containing Passerini products were more toxic than their cor-
responding Ugi analogues. This may attributed to the particular
nature of the Passerini products, that is, structures based on an
a-acyloxy amide. Indeed, these compounds contain a readily cleav-
able ester bond which could be broken enzymatically, hence
resulting in potentially more active metabolites. A comparable
cytotoxic behaviour was observed in case of the quinone-contain-
ing compounds, and regardless if these compounds incorporate
sulfur, selenium and/or tellurium atoms.
When comparing the activity of compounds against cancer cells
and HF ‘normal’ cell controls, certain selectivity could be noticed.
Compounds 3 and 4 (containing a quinone-redox centre) 6 (con-
taining a quinone-redox centre and tellurium) and 9, 10, 17 and
20 (containing tellurium) showed apparent lower toxicities when
incubated with HF, pointing to a selective cytotoxic activity against
the cancer cells used in this study. The selectivity was more pro-
nounced in case of MCF-7 cells when compared to A-431 cells.
Compound 10, for instance, exhibited considerable selectivity
against MCF-7 cells compared to A-431 cells and HF control cells.
2.2.2. Cytotoxicity in selected cancer cells and human fibroblast
control cells
Based on the above results, the cytotoxic activities were evalu-
ated further and in considerable more detail against human breast
adenocarcinoma (MCF-7) and human epidermoid carcinoma
(A-431) cancer cell lines, whilst primary human fibroblasts (HF)
were used as ‘healthy cell’ controls. These cell lines were chosen,
as they allow a basic evaluation of the activity of the compounds
and, in conjunction with PtK2 cells from Potorous tridactylis (see
below) also an investigation into the possible modes of action. In
the first step, cytotoxicity of the compounds was determined using
an MTT assay and 24 h of incubation. The IC50 values were esti-
mated from the respective dose response curves and are summa-
rized in Table 1.
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4
S. Shaaban et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 1
Influence of multifunctional redox compounds on the viability of human cancer and primary cells and the partition coefficient values (logPow) in the octanol–water system
b
b
Compound
IC50
(l
M)
LogPow
Compound
IC50
(lM)
LogPow
MCF-7
A-431
HF
MCF-7
A-431
HF
a
a
a
3
4
5
6
7
8
9
10
11
16
8
7
17
17
1
26
24
4
4.20
4.23
1.97
3.80
3.12
3.30
4.20
3.83
3.10
12
13
14
15
16
17
18
19
20
3.10
1.72
4.00
3.70
1.31
3.01
3.40
2.60
0.90
27
5
7.5
a
a
a
a
a
a
12
1
13
a
a
a
2
6
5
a
a
a
1
6
11
a
a
a
5
5
25
a
a
a
0.3
10
19
a
a
a
1
7
7
a
The measured IC50 was P100
The lipophilicity was determined by mean of HPLC.
l
M.
b
Whilst these findings seem to confirm a considerable, yet also
selective activity of some of these compounds, it should be noted
that only a limited number of cell types have been tested and a
generalization regarding selectivity is not yet feasible. As an alter-
native explanation, MCF-7 cells may be particularly prone to cell
death whilst HF cells are simply normally more resilient.
In the first step, an analysis of the cell cycle was performed in
order to indentify irregularities and possible causes of apoptosis
(Fig. 3). Indeed, flow cytometric analysis of MCF-7 cells treated with
3, 10 and 13 at their respective IC50s for 24 h revealed a significant
delay in cell cycle progression. Figure 3 reflects a clear increase in
the G0/G1 phase and reduction in G2/M phase compared to the
methanol control. For example, cell cycle analysis of MCF-7 cells
treated with 10, 3 and 13 showed that 92%, 89% and 85% cells were
in G0/G1 phase, respectively, compared to 68% in the methanol
treated cells (Table 2). Whilst the precise relation between cell cycle
arrest and apoptosis is still not fully understood, it is possible that
cell cycle delay may induce an apoptotic response.
Interestingly, an arrest in G0/G1 phase is rather unusual for
such compounds, as our previous studies have frequently pointed
towards an arrest in the G2/M phase. These issues and their poten-
tial implications for the activity and possible uses of the tellurium
compounds need to be addressed in more detail as part of subse-
quent studies.
2.2.3. Mechanistic studies
In order to shed some light on the processes underlying effi-
ciency—and possibly also selectivity—a more detailed mechanistic
study has been performed employing a combination of intracellu-
lar analytical techniques (so-called ‘intracellular diagnostics’).
Compared to extensive screening in different cancer and normal
cell lines (which has in part already been performed at the NCI,
see above), this approach has two advantages. Firstly, it identifies
some of the intracellular events responsible for activity, and sec-
ondly, it may well provide some information if and why certain
compounds are acting selectively.
Figure 3. MCF-7 cells were treated with (panel b), 10 (panel c), and 13 (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.
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S. Shaaban et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Table 2
unable to disturb the ROS levels in A-431 melanoma cells over a
1 h treatment period. In contrast, compounds containing tellurium
decreased ROS levels in A-431 cells in a concentration-dependent
manner (see Supporting information).
Cell cycle distribution of MCF-7 cells treated with different compounds and methanol
as negative control
Cell cycle phase in%
In order to evaluate if this decrease in ROS is a cause or
consequence of other major cellular events, including the arrest
in G0/G1 phase—and if specific cellular components may be
affected—immunofluorescence investigations on the cell adher-
ence and associated ER and actin cytoskeletal network were per-
formed, using potorous PtK2 cells as a suitable model (PtK2 cells
are an excellent model to study most aspects related to mitosis)
(Fig. 4). Interestingly, treatment of PtK2 cells with compound 10
resulted in major morphological changes. Upon treatment, the
PtK2 cells begun to lose their typical shape and became round.
At the same time, a distinct loss of cell adhesion, partial lysis of
the ER and disappearance of the actin stress fibers were also
observed (Fig. 4).
It is possible that these modifications have a major impact on
the cell and its ability to function properly and ultimately to divide.
Whilst speculative at time, damage to the ER and cytoskeleton are
possible targets of the tellurium agents and/or the ROS disturbance
as a result of their presence. These issues require more detailed cell
biological investigations in the future.
G0/G1
S
G2/M
Methanol
5
10
13
68
89
92
85
19
1.6
5.8
8.8
8.8
5.5
6.2
6.2
We have already shown that the quinone-containing chalcogen
compounds (3, 4, 5 and 6) exert their main cytotoxic effects at least
in part via modulation of the intracellular redox state.13 Conse-
quently, these compounds were able to inhibit cancer cell prolifer-
ation by induction of Oxidative Stress, cell cycle delay, alteration of
the cytoskeleton and apoptosis. Furthermore, we have also shown
that the tellurium-containing compounds 6 and 5 are generally
able to enhance the oxidation of thiols in the presence of H2O2,
using a simple in vitro thiophenol oxidation assay.11,12
In continuation of our previous work, the ROS levels in (the
more robust) A-431 cells were monitored using the standard
fluorescent 20,70-dichlorodihydrofluorescin diacetate (DCHFA)
assay, which can be considered as a general ROS indicator, as
DCHFA reacts readily with most common ROS, including H2O2,
lipid hydroperoxides and ONOOꢀ.17 As suspected, in the concentra-
tion range used, sulfur- and selenium-based compounds were
So far, we have considered the impact of the peptidomimetic
compounds on cultured human cells. There is, of course, also the
chance that such agents may be toxic against lower organisms,
such as fungi and bacteria. The antimicrobial activity of the
a
b
c
d
Figure 4. Immunofluorescence investigations of the ER (panel a and b) and the actin cytoskeleton (panel c and d) of PtK2 cells in the absence (panel a and c) and presence of
compound 10 (panel b and d). PtK2 cells were incubated with 10 in comparison with control cells. Treated cells show that the ER is affected in general (panel b) and actin
stress fibres are barely detectable (panel d). Adhesion seems to be reduced and cells become rounded and detached from each other.
Table 3
Diameters (in mm) of inhibition zones of agar diffusion assays against a variety of fungi and bacteria (growth was quantified after 1 and 2 days)a
Compound
S. cerevisiae
C. albicans
As. niger
M. phlei
M. luteus
S. aureus
K. pneumonia
P. aeruginosa
E. coli tolC
3
4
5
6
7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
19
0
0
0
0
0
18
10
0
0
0
0
0
19
0
0
0
15
16
0
0
7
8
11
0
0
7
7
0
0
13
0
0
8
7
0
0
9
0
0
11
13
0
0
0
12
13
0
0
0
0
12
0
0
10
0
0
0
9
0
0
9
0
0
0
0
0
0
12
11
0
8
9
0
0
0
0
12
14
0
10
16
0
0
12
0
0
11
0
0
0
12
15
0
0
12
0
0
13
0
0
0
12
12
0
0
11
0
0
11
0
0
0
10
11
12
13
14
15
16
17
18
19
20
0
17
15
0
12
0
0
0
15
0
0
0
a
Diameters (mm) of zones of inhibition (agar diffusion assay) are provided. In each case, 6-mm disks with 20 lg of the test compounds were incubated.
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6
S. Shaaban et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
compounds was therefore evaluated against three fungal strains,
namely Saccharomyces cerevisiae, Candida albicans and Aspergillus
niger, and against six bacterial strains, including Mycobacterium
phlei, Micrococcus luteus, Staphylococcus aureus, Klebsiella pneumo-
nia, Pseudomonas aeruginosa and Escherichia coli tolC. A standard
agar diffusion assay was used and the diameters (mm) of inhibition
zones are summarized in Table 3.
libraries of such peptidomimetic compounds with comparable
ease will clearly benefit such pharmaceutical and cell biological
investigations.
4. Experimental
4.1. Chemistry
Overall, none of the compounds investigated were particularly
toxic against S. cerevisiae and C. albicans. Indeed, most of sulfur
and selenium compounds at the concentrations used showed little
toxicity against any of the organisms. In contrast, compounds 6, 19,
12 and 13 showed moderate to high toxicity against A. niger (with
inhibition zone diameters of 19, 19, 18 and 10 mm, respectively).
4.1.1. Material and methods
All chemical reagents for the synthesis of compounds were pur-
chased from Sigma–Aldrich or Fluka (Germany) and used without
further purification unless stated otherwise. Reactions under inert
atmosphere were carried out under argon (4.6) using standard
oxygen and water free conditions. Silica gel 60 (Machereye-Nagel,
50–200 mm) was used for column chromatography. Unless noted
otherwise, the dimensions of columns used were 2.5 cm (diameter)
Nonetheless,
confirmed.
a convincing antifungal activity could not be
In the case of bacteria, most of the compounds showed low to
moderate toxicity against Mycobacterium phlei and Micrococcus
luteus. Tellurium compounds were rather toxic against Staphylo-
coccus aureus and Klebsiella pneumonia, while most of the qui-
none-containing compounds were not. The same behavior was
noticed in case of Pseudomonas aeruginosa and Escherichia coli tol
C., yet these two strains generally were less affected.
Whilst the spectrum of activities associated with the peptidom-
imetic compounds against cancer cells, fungi and bacteria
therefore bodes rather well, especially for the tellurium com-
pounds, it should also be noted that these compounds follow the
traditional Lipinski’s Rule of Five. Here, most of the compounds
studied have less than 10 hydrogen bond acceptors and only one
or two hydrogen bond donors. The molecular mass in most cases
is below 500 Dalton (or slightly above). The experimental partition
coefficient values (logPow) of the compounds in the octanol–water
system were measured by the high performance liquid chromatog-
raphy (HPLC) technique and are found to be below 5 (see Support-
ing information).
and 25–30 cm (height of silica gel). TLC plates (silica gel 60 F254
,
0.20 mm) were purchased from Merck (Germany). NMR spectros-
copy: 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, 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. LC–MS/MS analysis: Analyses were performed using a TSQ
Quantum mass spectrometer equipped withan ESI source and a
triple quadrupole mass detector (Thermo Finnigan). 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 a P95% purity of the prepared compounds.
4.1.2. General procedure for the preparation of chalcogen
containing isocyanide building blocks (2a–2c)
A mixture of 1 g of appropriate amine, 1.0–1.2 equiv of formic
acid and 1.5–2.0 equiv of acetic anhydride were mixed and heated
for 2 h. The progress of the reaction was monitored by TLC, and
after the starting material had disappeared, the solvent was evap-
orated from the reaction mixture to yield the crude N-formyl com-
pound, essentially as an oily product which could be purified by
chromatography on silica gel, usually with dichloromethane/meth-
anol (10:1).
The N-formyl compound (6.05 g, 50.0 mmol) and diisopropyl-
amine (DIPA) (19 ml, 0.14 mol) were dissolved in CH2Cl2 (50 ml)
and cooled to 0 °C. POCl3 (5.0 ml, 55 mmol) was added slowly
and stirring was continued at 0 °C for another 30 min. Sodium car-
bonate (10 g) in H2O (50 ml) was added at room temperature in a
rate so that the temperature was maintained between 25 and
35 °C. The mixture was stirred for 90 min at room temperature.
H2O and CH2Cl2 (25 ml each) were added. The organic layer was
separated, washed with H2O (3 ꢁ 25 ml), dried over MgSO4 and
purified by chromatography on silica gel, with petrol ether/ethyl
acetate (8:1) as eluent.
3. Conclusion
A
novel library of sulfur-, selenium- and tellurium-based
pseudopeptides has been prepared using the isocyanide-based
Passerini and Ugi multicomponent reactions and a variety of
selected building blocks. The resulting products were obtained in
moderate to good yields, turning this approach to a promising syn-
thetic avenue able to provide a rather straight-forward access to
many biologically interesting chalcogen-based redox modulating
agents. Notably, this strategy is superior to the classical methods
as it only requires mild conditions, can be performed easily and
relies on a plethora of readily available starting materials. The
compounds synthesized were tested for their cytotoxic and
antimicrobial activities. Whilst the sulfur and selenium containing
compounds generally did not show any major activity, virtually all
of the tellurium-containing compounds exhibited both cytotoxic
and antimicrobial activities. Interestingly, some of the compounds
investigated even showed lower toxicity in HF primary cells
compared to cancer cells, appointing towards a possible selective
cytotoxic effect. Whilst it is too early to speculate on selectivity
and underlying mechanisms, it seems that the tellurium agents
are interrupting the ER structure and also actin cytoskeleton, hence
causing G0/G1 cell cycle arrest and subsequently inducing
apoptosis. The intracellular ROS levels also seemed to be disturbed
upon treatment, yet it is still unclear if this is a cause or a rather a
consequence of the ER and cytoskeleton damage and cell cycle
arrest observed. Additional cell biological studies are clearly
required to investigate the exact impact of such peptidomimetic
compounds on human and microbial cells and to study the targets
and underlying mechanisms involved. The ability to create small
4.1.3. General procedure (A) for the preparation of
amide via the three-component Passerini reaction
a-acyloxy
As a general procedure, a mixture of aldehyde (1 mmol), car-
boxylic acid (1.2 mmol) and isocyanide (1.5 mmol) in 5 ml solvent
(H2O was used in most cases) was stirred at room temperature
overnight. Upon completion (monitored by TLC), 10 ml CH2Cl2
were added to dissolve the sticky product.
The aqueous layer was extracted three times with CH2Cl2, the
organic layers were combined, dried over Na2SO4 and concentrated
to yield a sticky product which was purified by chromatography on
silica gel, with petrol ether/ethyl acetate (4:1) as eluent.
Please cite this article in press as: Shaaban, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.019

S. Shaaban et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
4.1.4. General procedure (B) for the preparation of
amide via the four-component Ugi reaction
a
-aminoacyl
2.80–2.77 (t, J = 7.66, 15.15 Hz, 2H), 1.97–1.91 (p, J = 6.95, 14.24,
21.66, 28.65 Hz, 2H), 1.43 (s, 9H) ppm; 13C NMR (125.79 Hz, CDCl3)
d 170.25, 168.30, 159.72, 155.92, 138.45, 133.58, 129.23, 128.68,
127.70, 115.26, 111.46, 79.80, 55.25, 54.22, 43.05, 41.22, 31.52,
28.30, 4.75 ppm; LC–MS (ESI): m/z calcd 585.15, Rt = 1.54 min, m/
z found 608.15 [M+Na]+; HRMS: [M+H] calcd 586.1482, [M+H]
found 586.1555, [M+H+O] calcd 602.1431 [M+H] found 602.1504,
[M+Na] calcd 608.1380 [M+Na] found 608.1656. Isotopic pattern
of Te: m/z (relative abundance %) 586.1555 (100), 587.1589 (29),
588.1622 (5), 602.1504 (100), 603.1538 (26), 604.1571 (5),
608.1656 (1).
As a general procedure, a mixture of aldehyde (1 mmol), amine
(1 mmol), carboxylic acid (1.2 mmol), and isocyanide (1.5 mmol) in
5 ml solvent (H2O was used in most cases) was stirred at room
temperature overnight. Upon completion (monitored by TLC),
10 ml CH2Cl2 were added to dissolve the sticky product. The water
layer was extracted three times with CH2Cl2, the organic layers
were combined, dried over Na2SO4 and concentrated to yield a
sticky product which was purified by chromatography on silica
gel, with petrol ether/ethyl acetate (5:2). The synthesis of individ-
ual products, including building blocks, yield and analytical data
are provided as a part of the individual experimental sections.
Compounds 2a–c, 3, 4, 5, and 6 were synthesized according to
literature.13
4.1.8. tert-Butyl (((3-(phenyltellanyl)propylcarbamoyl)
methoxy)carbonyl)methylcarbamate (10)
Following general procedure A, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product 10 as colourless
oil (yield = 83%). 1H NMR (500 MHz CDCl3) d 7.66–7.63 (m, 2H),
7.23–7.20 (m, 1H), 7.15–7.11 (m, 2H), 6.70 (br s, 1H), 5.05 (br s,
1H), 4.56 (s, 2H), 3.83–3.82 (d, 2H), 3.30–3.26 (q, J = 6.68, 12.91,
19.81 Hz, 2H), 2.81–2.78 (t, J = 7.50, 15.11 Hz, 2H), 1.99–1.93 (p,
J = 7.26, 14.53, 21.55, 29.05 Hz, 2H), 1.37 (s, 9H) ppm; 13C NMR
4.1.5. tert-Butyl ((2-(N-(4-methoxyphenyl)-N-amino)-N-(3-
(phenylthio)propyl)acetamide)carbonyl)methylcarbamate (7)
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product 7 as colourless
oil (yield = 77%). 1H NMR (500 MHz, CDCl3) d 7.31–7.24 (m, 6H),
7.17–7.14 (m, 1H), 6.99 (br s, 1H), 6.88–6.86 (d, J = 8.90 Hz, 2H),
5.48 (s, 1H), 4.26 (s, 2H), 3.78 (s, 3H), 3.66–3.64 (d, J = 5.07 Hz,
2H), 3.37-3.33 (q, J = 6.82, 13.00, 19.77 Hz, 2H), 2.92–2.89 (t,
J = 7.24, 14.49 Hz, 2H), 1.86–1.81 (p, J = 7.24, 13.96, 21.11,
28.00 Hz, 2H), 1.49 (s, 9H) ppm; 13C NMR (125.79 Hz, CDCl3)
d 170.13, 168.18, 159.39, 155.84, 136.00, 133.46, 128.82, 128.69,
128.54, 125.71, 114.94, 79.44, 55.25, 53.71, 42.80, 38.22, 30.70,
28.61, 28.08 ppm; LC–MS (ESI): m/z calcd 487.21, Rt = 1.61 min,
m/z found 488.2 [M+1]+; HRMS: [M+Na] calcd 510.2039, found
510.2033 [M+Na]. Isotopic pattern of S: m/z (relative abundance
%) 488.2214 (100), 489.2247 (25), 490.2172 (5), 510.2033 (100),
511.2067 (25), 512.1991 (5).
(125.79 Hz, CDCl3)
d 168.99, 166.87, 156.48, 138.42, 129.21,
127.68, 111.50, 80.79, 63.13, 42.88, 40.96, 31.44, 28.30,
4.70 ppm; LC–MS (ESI): m/z calcd 480.09, Rt = 7.21 min, m/z found
497.59 [M+H+O], 497.44 [M+H+O]+; HRMS: [M+H+O] calcd
497.0849, [M+H+O] found 497.0926, [M+Na] calcd 503.0904,
[M+Na] found 503.1078. Isotopic pattern of Te: m/z (relative abun-
dance %) 495.0908 (93), 497.0926 (100), 498.0959 (22), 499.0993
(7), 503.1078 (2).
4.1.9. N-((3-(Phenylthio)propylcarbamoyl)methyl)-N-(4-
methoxyphenyl)benzamide (11)
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product 11 as colourless
oil (yield = 75%). 1H NMR (500 MHz, CDCl3) d 7.25–7.22 (m, 4H),
7.19–7.17 (m, 2H), 7.11–7.07 (m, 3H), 6.94–6.91 (m, 2H), 6.64–
6.61 (m, 3H), 4.38 (s, 2H), 3.63 (s, 3H), 3.36–3.32 (q, J = 6.48,
12.34, 19.75 Hz, 2H), 2.87–2.84 (t, J = 7.35, 18.85 Hz, 2H), 1.82–
1.76 (p, J = 6.98, 13.85, 20.83, 27.92 Hz, 2H) ppm; 13C NMR
4.1.6. tert-Butyl ((2-(N-(4-methoxyphenyl)-N-amino)-N-(3-
(phenylselanyl)propyl)acetamide)carbonyl)methylcarbamate
(8)
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product 8 as colourless
oil (yield = 86%). 1H NMR (500 MHz, CDCl3) d 7.49–7.46 (m, 2H),
7.27–7.21 (m, 5H), 6.89–6.87 (d, J = 8.92 Hz, 2H), 6.77 (br s, 1H),
5.40 (br s, 1H), 4.24 (s, 2H), 3.79 (s, 3H), 3.66–3.64 (d, J = 5 Hz,
2H), 3.37–3.33 (q, J = 6.71, 12.71, 19.56 Hz, 2H), 2.89–2.87 (t,
J = 7.37, 14.35 Hz, 2H), 1.93–1.87 (p, J = 7.00, 13.73, 21.00,
27.95 Hz, 2H), 1.41 (s, 9H) ppm; 13C NMR (125.79 Hz, CDCl3) d
170.18, 168.20, 159.52, 155.88, 133.52, 132.39, 129.89, 128.95,
128.61, 126.73, 115.07, 79.59, 55.37, 53.91, 42.90, 39.21, 29.69,
28.18, 24.68 ppm; LC–MS (ESI): m/z calcd 535.16, Rt = 1.73 min,
m/z found 558.10 [M+Na]+; HRMS: [M+H] calcd 536.1585, [M+H]
found 536.1658, [M+Na] calcd 558.1478 [M+Na] found 559.1511.
Isotopic pattern of Se: m/z (relative abundance %) 536.1658
(100), 537.1692 (26), 538.1660 (15), 539.1694 (5).
(125.79 Hz, CDCl3)
d 171.49, 168.18, 158.52, 136.70, 136.30,
135.09, 130.26, 129.52, 129.38, 129.15, 129.06, 128.52, 128.04,
126.28, 114.70, 55.57, 38.62, 31.32), 29.06 ppm; LC–MS (ESI): m/z
calcd 434.17, Rt = 15.69 min, m/z found 435.2 [M+H]+; HRMS:
[M+H] calcd 435.1764, [M+H] found 435.1737, [M+Na] calcd
457.1562 [M+Na] found 457.1556. Isotopic pattern of S: m/z (rela-
tive abundance %) 435.1737 (100), 436.1770 (29), 437.1695 (5),
457.1556 (100), 458.1590 (25), 459.1514 (5).
4.1.10. N-((3-(Phenylselanyl)propylcarbamoyl)methyl)-N-(4-
methoxyphenyl)benzamide (12)
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product as colourless
oil (yield = 79%). 1H NMR (500 MHz, CDCl3) d 7.40–7.38 (m, 2H),
7.24–7.22 (m, 2H), 7.16–7.14 (m, 4H), 7.11–7.07 (m, 2H), 6.94–
6.91 (m, 2H), 6.64–6.61 (m, 2H), 4.36 (s, 2H), 3.63 (s, 3H), 3.33–
3.29 (q, J = 6.57, 13.14, 19.41 Hz, 2H), 2.83–2.81 (t, J = 7.06,
14.52 Hz, 2H), 1.86–1.81 (p, J = 6.98, 13.96, 21.31, 28.29 Hz, 2H)
ppm; 13C NMR (125.79 Hz, CDCl3) d 171.22, 168.88), 158.26,
136.44, 134.84, 132.60, 130.00, 129.86, 129.06, 128.79, 128.27,
127.79, 126.89, 114.44, 55.31, 55.28, 39.23, 29.78), 24.78 ppm;
HRMS: [M+H] calcd 483.1109, [M+H] found 483.1181, [M+Na]
calcd 505.1007 [M+Na] found 505.1001. Isotopic pattern of Se:
4.1.7. tert-Butyl ((2-(N-(4-methoxyphenyl)-N-amino)-N-(3-
(phenyltellanyl)propyl)acetamide)carbonyl)methylcarbamate
(9)
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product 9 as colourless
oil (yield = 70%). 1H NMR (500 MHz, CDCl3) d 7.66–7.64 (m, 2H),
7.19 (s, 1H), 7.14–7.10 (m, 4H), 6.82–6.79 (d, J = 8.9 Hz, 2H), 6.32
(br s, 1H), 5.20 (br s, 1H), 4.13 (s, 2H), 3.73 (s, 3H), 3.58–3.57 (d,
J = 5.28 Hz, 2H), 3.28–3.24 (q, J = 6.55, 13.41, 19.32 Hz, 2H),
Please cite this article in press as: Shaaban, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.019

8
S. Shaaban et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
m/z (relative abundance %) 483.1181 (100), 484.1215 (30),
485.1183 (20), 486.1217 (8), 505.1001 (100), 506.1034 (30),
507.1003 (20), 508.1036 (5).
(q, J = 6.42, 12.84, 19.69 Hz, 2H), 2.80–2.77 (t, J = 7.59, 15.19 Hz,
2H), 1.96–1.91 (p, J = 6.94, 14.52, 21.47, 28.88 Hz, 2H) ppm; 13C
NMR (125.79 Hz, CDCl3)
d 169.30, 167.42, 159.43, 143.25,
138.64, 135.13, 135.03, 130.10, 129.42, 129.02, 128.95, 128.21,
127.87, 117.97, 115.15, 111.66, 55.74, 55.06, 41.33, 31.78,
4.95 ppm; LC–MS (ESI): m/z calcd 558.12, Rt = 17.19 min, m/z
found 558.80 [M+H], 582.1 [M+Na]; HRMS: [M+H+O] calcd
575.1162, [M+H+O] found 575.1184. Isotopic pattern of Te: m/z
(relative abundance %) 575.1184 (100), 576.1218 (30), 577.1251
(5), 578.1285 (2).
4.1.11. N-((3-(Phenyltellanyl)propylcarbamoyl)methyl)-N-(4-
methoxyphenyl)benzamide (13)
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product as colourless
oil (yield = 69%). 1H NMR (500 MHz, CDCl3) d 7.78–7.75 (m, 2H),
7.38–7.30 (m, 5H), 7.24–7.22 (m, 3H), 7.06–7.04 (m, 2H), 6.77–
6.75 (m, 2H), 4.49 (s, 2H), 3.78 (s, 3H), 3.43–3.38 (p, J = 6.68,
13.09, 19.56, 25.44 Hz, 2H), 2.93–2.90 (t, J = 7.52, 15.16 Hz, 2H),
2.08–2.03 (p, J = 6.84, 14.37, 21.49, 28.74 Hz, 2H) ppm; 13C NMR
4.1.15. (E)-(3-(Phenyltellanyl)propylcarbamoyl)methyl
cinnamate (17)
Following general procedure A, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product as colourless
oil (yield = 90%). 1H NMR (500 MHz, CDCl3) d 7.73–7.64 (m, 3H),
7.57–7.45 (m, 2H), 7.37–7.33 (m, 2H), 7.22–7.18 (m, 2H), 7.11–
7.15 (m, 2H), 6.50–6.35 (m, 1H), 6.12 (br s, 1H), 4.60 (s, 2H),
3.35–3.31 (q, J = 6.63, 13.07, 19.96 Hz, 2H), 2.82–2.79 (t, J = 7.28,
14.82 Hz, 2H), 2.00–1.95 (p, J = 7.09, 14.27, 21.45, 28.81 Hz, 2H)
ppm; 13C NMR (125.79 Hz, CDCl3) d 167.23, 165.42, 146.79,
138.51, 133.96, 130.87, 129.28, 129.02, 128.28, 127.81, 116.38,
111.33, 63.07, 40.80, 31.47, 4.60 ppm; LC–MS (ESI): m/z calcd
453.06, Rt = 9.01 min, m/z found 469.85 [M+O]+; HRMS: [M+H+O]
calcd 470.0584, [M+H+O] found 470.0606. Isotopic pattern of Te:
m/z (relative abundance %) 470.0606 (100), 471.0639 (23),
472.0673 (4).
(125.79 Hz, CDCl3)
d 171.24, 170.80, 168.90, 158.30, 138.48,
136.46, 134.85, 130.04, 129.22, 128.82, 128.27, 127.82, 127.69,
114.47, 55.36, 55.35, 41.11, 31.52, 4.67 ppm; LC–MS (ESI): m/z
calcd 532.10, Rt = 1.54 min, m/z found 533.0 [M+H]+; HRMS:
[M+H+O] calcd 549.1006, [M+H+O] found 549.1028. Isotopic pat-
tern of Te: m/z (relative abundance %) 549.1028 (100), 550.1061
(30), 551.1095 (6).
4.1.12. (E)-N-((3-(Phenylthio)propylcarbamoyl)methyl)-N-(4-
methoxyphenyl)cinnamamide (14)
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product as colourless
oil (yield = 87%). 1H NMR (500 MHz, CDCl3) d 7.61–7.58 (m, 1H),
7.24–7.04 (m, 11H), 6.88–6.71 (m, 3H), 6.30–6.27 (m, 1H), 4.28
(s, 2H), 3.73 (s, 3H), 3.33–3.29 (q, J = 6.73, 12.95, 19.68 Hz, 2H),
2.87–2.84 (t, J = 7.16, 14.50 Hz, 2H), 1.81–1.75 (p, J = 6.94, 13.99,
21.04, 27.98 Hz, 2H) ppm; 13C NMR (125.79 Hz, CDCl3) d 169.02,
167.10, 159.11, 142.92, 136.08, 134.87, 134.73, 129.78, 129.13,
128.82, 128.75, 128.64, 127.90, 125.90, 117.71, 114.83, 55.42,
54.71, 38.33, 30.99, 28.82 ppm; LC–MS (ESI): m/z calcd 460.18,
Rt = 14.10 min, m/z found 461.2 [M+H]+; HRMS: [M+H] calcd
461.1821, [M+H] found 461.1893. Isotopic pattern of Se: m/z (rel-
ative abundance %) 461.1893 (100), 462.1927 (30), 463.1851 (5),
464.1885 (2).
4.1.16. N-((3-(Phenylthio)propylcarbamoyl)methyl)-N-(4-
methoxyphenyl)-3-phenylpropiolamide (18)
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product as colourless
oil (yield = 86%). 1H NMR (500 MHz, CDCl3) d 7.29–7.24 (m, 4H),
7.19–7.16 (m, 5H), 7.10–7.06 (m, 3H), 6.84–6.81 (m, 2H), 6.47 (br
s, 1H), 4.27 (s, 2H), 3.74 (s, 3H), 3.35–3.31 (q, J = 6.77, 12.89,
19.49 Hz, 2H), 2.88–2.85 (t, J = 7.09, 14.34 Hz, 2H), 1.83–1.77 (p,
J = 7.09, 13.86, 20.94, 27.71 Hz, 2H) ppm; 13C NMR (CDCl3,
125.79 Hz) d 168.15, 159.44, 155.33, 136.02, 134.81, 132.51,
130.20, 129.29, 128.98, 128.92, 128.35, 126.03, 120.03, 114.39,
92.68, 81.96, 55.53, 53.62, 38.51, 31.08, 28.79 ppm; LC–MS (ESI):
m/z calcd 458.17, Rt = 1.56 min, m/z found 459.2 [M+H]+; HRMS:
[M+H] calcd 459.1664, [M+H] found 481.1734, [M+Na] calcd
481.1562 [M+Na] found 481.1556. Isotopic pattern of S: m/z (rela-
tive abundance %) 459.1734 (100), 460.1770 (30), 461.1695 (4),
481.1556 (100), 482.1590 (30), 483.1514 (5).
4.1.13. (E)-N-((3-(Phenylselanyl)propylcarbamoyl)methyl)-N-
(4-methoxyphenyl)cinnamamide (15)
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product as colourless
oil (yield = 92%). 1H NMR (500 MHz, CDCl3) d 7.62–7.59 (m, 1H),
7.39–7.37 (m, 2H), 7.24–7.08 (m, 9H), 6.88–6.64 (m, 3H), 6.30–
6.27 (m, 1H), 4.27 (s, 2H), 3.75 (s, 3H), 3.31–3.27 (q, J = 7.10,
13.37, 20.20 Hz, 2H), 2.83–2.80 (t, J = 7.25, 14.87 Hz, 2H), 1.86–
1.81 (p, J = 6.96, 14.21, 21.31, 28.42 Hz, 2H) ppm; 13C NMR
4.1.17. N-((3-(Phenylselanyl)propylcarbamoyl)methyl)-N-(4-
methoxyphenyl)-3-phenylpropiolamide (19)
(125.79 Hz, CDCl3)
d
169.03, 167.14, 159.15, 142.96, 134.88,
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product as colourless
oil (yield = 90%). 1H NMR (500 MHz, CDCl3) d 7.42–7.39 (m, 2H),
7.27–7.22 (m, 3H), 7.18–7.13 (m, 5H), 7.11–7.07 (m, H), 6.84–
6.82 (m, 2H), 6.46 (br s, 1H), 4.26 (s, 2H), 3.74 (s, 3H), 3.32–
3.28 (q, J = 6.94, 13.23, 19.84 Hz, 2H), 2.84–2.80 (t, J = 7.07,
14.40 Hz, 2H), 1.87–1.81 (p, J = 6.91, 13.92, 20.72, 27.74 Hz, 2H)
ppm; 13C NMR (125.79 Hz, CDCl3) d 168.08, 159.40, 155.28,
134.79, 132.61, 132.48, 130.18, 129.87, 128.06, 128.98, 128.33,
126.86, 120.01, 114.36, 92.62, 81.97, 55.51, 53.54, 39.34, 29.76,
24.77 ppm; LC–MS (ESI): m/z calcd 506.11, Rt = 11.45 min, m/z
found 507.54 [M+H]+; HRMS: [M+H] calcd 507.1109, [M+H] found
507.1181. Isotopic pattern of Se: m/z (relative abundance %)
507.1181 (100), 508.1215 (33), 509.1183 (18), 510.1217 (7),
511.1250 (1).
134.76, 132.56, 129.91, 129.81, 129.02, 128.76, 128.67, 127.93,
126.82, 117.71, 114.83, 55.46, 54.78, 39.22, 29.82, 24.78 ppm;
LC–MS (ESI): m/z calcd 508.13, Rt = 1.71 min, m/z found 509.10
[M+H]+; HRMS: [M+H] calcd 509.1265, [M+H] found 509.1338. Iso-
topic pattern of Se: m/z (relative abundance %) 509.1338 (100),
510.1371 (30), 511.1340 (17), 512.1373 (7), 513.1407 (2).
4.1.14. (E)-N-((3-(Phenyltellanyl)propylcarbamoyl)methyl)-N-
(4-methoxyphenyl)cinnamamide (16)
Following general procedure B, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product as colourless
oil (yield = 77%). 1H NMR (500 MHz, CDCl3) d 7.64–7.61 (m, 3H),
7.40–7.3 (m, 5H), 7.25–7.2 (m, 3H), 6.71–7.02 (m, 3H), 6.75 (br
s, 1H), 6.30–6.27 (m, 1H), 4.26 (s, 2H), 3.76 (s, 3H), 3.28–3.24
Please cite this article in press as: Shaaban, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.019

S. Shaaban et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
4.1.18. N-((3-(Phenyltellanyl)propylcarbamoyl)methyl)-3-
phenylpropiolamide (20)
(DCHFA, kex/kem = 485 20/528 20 nm). Results were expressed
as arbitrary units per 105 cells.
Following general procedure A, the crude residue was purified
by column chromatography on silica gel, eluting with petrol
ether/ethyl acetate (5:1), afforded desired product as colourless
oil (yield = 86%). 1H NMR (500 MHz, CDCl3) d 7.70–7.65 (m, 2H),
7.55–7.51 (m, 2H), 7.36–7.31 (m, 3H), 7.22–7.21 (m, 1H), 7.15–
7.10 (m, 2H), 6.60 (br s, 1H), 4.65 (s, 1H), 4.60 (s, 2H), 3.64–3.61
(m, 2H), 2.75–2.72 (m, 2H), 1.72–1.67 (m, 2H) ppm; 13C NMR
4.2.3.2. Cell cycle analysis.
Treated MCF-7 cells (106) were
fixed with cold (ꢀ20 °C) methanol (70%) at 4 °C for one day. Cells
were washed with PBS and then treated with saponin (0.1% in
PBS). Finally, propidium iodide (500 ll, 20 l
g mlꢀ1) and RNAse
(1 mg mlꢀ1) were added. After 30 min, samples were analyzed by
FacScan.
(125.79 Hz, CDCl3)
d 167.40, 166.55, 152.53, 133.43, 133.37,
131.44, 131.39, 128.97, 128.93, 126.85, 119.28, 88.87, 79.81,
63.97, 43.75, 36.63, 32.89 ppm; HRMS: [M+O] calcd 466.0427,
[M+O] found 466.0449. Isotopic pattern of Te: m/z (relative abun-
dance %) 468.0449 (100), 469.0483 (21), 470.0516 (5).
4.2.3.3. Immunofluorescence microscopy.
Cells were grown
on cover slips in 4-well plates, compounds were added after the
cells had become semi-confluent and were incubated for 24 h.
Subsequently, cells were fixed with 3.7% paraformaldehyde
(followed by Triton-X 100 (0.1%) treatment for 5 min) or ice cold
methanol/acetone (50:50) for 10 min and then washed with phos-
phate-buffered saline (PBS). Primary antibodies were added and
incubated for 45 min and washed with PBS. Secondary antibodies
were then added to the cells and incubated for further 45 min.
After washing with PBS, 40,6-diamidino-2-phenylindole (DAPI)
was added and kept at room temperature for 5 min. Cover slips
were mounted in anti-fade mounting medium (Molecular Probes).
Images were taken with a CCD camera attached to a fluorescence
microscope. The following antibodies were used: anti-GRP94,
4.2. Biological assays
4.2.1. Cancer cell line screening at the National Cancer Institute
The 58 cancer cell line screen was performed at the National
Cancer Institute (NCI) at the NIH (US). These single dose tests (at
10 lM) were performed for cell lines clustered in cells representing
leukemia, non-small cell lung cancer, colon cancer, cancer of the
central nervous system, melanoma, ovarian cancer, renal cancer,
prostate cancer and breast cancer. All compounds were selected
for five dose testing. These cell culture based investigations fol-
lowed the NCI’s standard protocol for cytotoxicity screens, details
of which can be obtained directly from the NCI website at http://
dtp.nci.nih.gov.
anti-a-tubulin, anti-mouse Alexa Fluor 488 and anti-rat Alexa
Fluor 488. The actin filaments were stained with phalloidin Alexa
Fluor 594 for 45 min.
4.2.3.4. Lipophilicity measurements.
High performance
4.2.2. Cytotoxicity assays
liquid chromatography (HPLC) was used as a rapid method for
the determination of lipophilicity. In order to correlate the mea-
sured HPLC data of a compound with its P value, a calibration
graph of logP versus chromatographic data using at least six refer-
ence points has to be established. The retention times (tR), of the
compounds were determined using a Hewlett Packard series
1090 HPLC fitted with a diode array detector and UV detection
with maximum absorbance at k = 230 and k = 245 nm. Chromato-
graphic conditions were as follows: column EC 125/2 mm and pre-
All cell lines required for these studies were obtained from the
DSMZ (Braunschweig, Germany). Human fibroblasts (HF) isolated
from human male foreskin were a generous gift of Dr. Thierauch,
Bayer-Schering Pharma AG. All cells were grown at 37 °C and
10% CO2 in the following media: MCF-7 in DMEM supplemented
with 1%
RPMI 1640 (Gibco), and HF in MEM (Gibco) supplemented with
1% -glutamine. All media were supplemented with 10% fetal calf
L-glutamine and 1% non essential amino acids, A-431 in
L
serum (Lonza or Gibco).
column, Nucleosil 120-5-C18; flow rate 0.5 ml/min. Isocratic
MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bro-
mide] (Sigma) was used to measure the metabolic activity of cells
(alive cells are able to reduce MTT to a violet coloured formazan
product which can be quantified spectrophotometrically). Briefly,
elution was performed with water/methanol at a volume ratio of
(3:1) as the mobile phase. The dead time (t0) of the system was
determined using thiourea (as unretained substance) with a t0
value of 0.728 min.
The retention factors under isocratic conditions, k, were calcu-
lated according to the equation k = (tR ꢀ t0)/t0. The calibration lines
for the RP-HPLC retention factors (Y = A + B * X, with Y = logk and
120
microplates were incubated at 37 °C and 10% CO2 and allowed to
grow for two days. Then 60 l of serial dilutions of the compounds
were added. After 24 h of incubation at 37 °C and 10% CO2, 20
l
l aliquots of a cell suspension (50,000 cells mlꢀ1) in 96-well
l
l
l
X = logPow) were established by linear regression with the
MTT in phosphate buffered saline (PBS) were added to a final
MTT concentration of 0.5 mg mlꢀ1. After 2 h the precipitate of for-
mazan crystals was centrifuged and the supernatant discarded. The
reference data.
Acknowledgments
precipitate was washed with 100 ll PBS and dissolved in 100 ml
isopropanol containing 0.4% hydrochloric acid. The resulting colour
was quantified at 590 nm using an ELISA plate reader. All investi-
gations were carried out in two parallel experiments. The IC50 val-
ues were determined from the dose–response curves as the
concentrations of compounds, which resulted in 50% of the absor-
bance of untreated control cells.
The authors thank the Egyptian Ministry of Higher Education,
the University of Saarland, the Ministry of Economics and Science
of Saarland, and the National Cancer Institute for assistance and
financial support.
Supplementary data
4.2.3. In vitro studies
4.2.3.1. Detection of ROS.
intracellular ROS formation was determined using DCHFA assays.
Cells were seeded in 96 wells plates at a density of 105 cells per
well and treated with different concentrations of the test com-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.05.019.
The ability of compounds to induce
References and notes
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