2016 Eur J Med Chem

Accepted Manuscript
Combinatorial synthesis, in silico, molecular and biochemical studies of tetrazole-

derived organic selenides with increased selectivity against hepatocellular carcinoma
Saad Shaaban, Amr Negm, Abeer M. Ashmawy, Dalia M. Ahmed, Ludger A.

Wessjohann
PII: S0223-5234(16)30482-2
DOI: 10.1016/j.ejmech.2016.06.005
Reference: EJMECH 8666
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 March 2016

Revised Date: 8 May 2016
Accepted Date: 4 June 2016
Please cite this article as: S. Shaaban, A. Negm, A.M. Ashmawy, D.M. Ahmed, L.A. Wessjohann,
Combinatorial synthesis, in silico, molecular and biochemical studies of tetrazole-derived organic
selenides with increased selectivity against hepatocellular carcinoma, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.06.005.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Combinatorial Synthesis, In Silico, Molecular and Biochemical Studies of

Tetrazole-Derived Organic Selenides with Increased Selectivity Against
Hepatocellular Carcinoma
PT
Saad Shaaban,a,e* Amr Negm,b Abeer M. Ashmawy,c Dalia M. Ahmedd and Ludger A.
Wessjohanne**
RI
a
Organic Chemistry Division, Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhorya
Street, 35516 Mansoura, Egypt.
b
Biochemistry Division, Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhorya Street,
SC
35516 Mansoura, Egypt.
c
Cancer Biology Department, National Cancer Institute, Cairo University, Egypt.
d
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Egypt.
e
U
Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, D-06120 Halle (Saale),
Germany.
AN
M
D
TE
C EP
AC
*
Corresponding author. Tel.: +2 01010 094 177; Fax: +20 502 397900; E-mail address: dr_saad_chem@mans.edu.eg
**
Corresponding author. Tel.: +49 345 5582 1301; fax: +49 345 5582 1309; E-mail address: wessjohann@ipb-halle.de
ACCEPTED MANUSCRIPT

PT
Wessjohanne**
RI
a
b
SC
c
d
e
U
Germany.
AN
ABSTRACT
M
Novel tetrazole-based diselenides and selenoquinones were synthesized via azido-Ugi and
sequential nucleophilic substitution (SN) strategy. Molecular docking study into mammalian
D
TrxR1 was used to predict the anticancer potential of the newly synthesized compounds. The
TE
cytotoxic activity of the compounds was evaluated using hepatocellular carcinoma (HepG2) and
breast adenocarcinoma (MCF-7) cancer cells and compared with their cytotoxicity in normal
fibroblast (WI-38) cells. The corresponding redox properties of the synthesized compounds were
EP
assessed employing 2,2-diphenyl-1-picrylhydrazyl (DPPH), glutathione peroxidase (GPx)-like

activity and bleomycin dependent DNA damage. In general, diselenides showed preferential
C
cytotoxicity to HepG2 compared to MCF-7 cells. These compounds exhibited also good GPx
AC
catalytic activity compared to ebselen (up to 5 fold). Selenoquinones 18, 21, 22 and 23 were
selected to monitor the expression levels of caspase-8, Bcl-2 and Ki-67 molecular biomarkers.
Interestingly, these compounds downregulated the Bcl-2 and Ki-67 expression levels and
activated the expression of caspase-8 in HepG2 cells compared to untreated cells. These results
indicate that some of the newly synthesized compounds possess anti-HepG2 activity.
*
**
1
ACCEPTED MANUSCRIPT
Keywords: organoselenium; diselenides; quinone; tetrazole; azido-Ugi reaction; redox

modulators; caspase-8, apoptosis, Ki-67, Bcl-2.
Abbreviations: Diversity-oriented synthesis (DOS), oxidative stress (OS); hepatocellular
PT
carcinoma (HCC); breast adenocarcinoma (MCF-7); nucleophilic substitution (SN); glutathione
peroxidase (GPx); reactive oxygen species (ROS); reactive nitrogen species (RNS); isocyanide-
RI
based multicomponent reactions (IMCRs); 2,2-diphenyl-1-picrylhydrazyl (DPPH).
1. Introduction
SC
Diversity-oriented synthesis (DOS) has emerged as a powerful tool in pharmaceutical
industry and now it is routinely used, for drug discovery, but mostly for improvement of desired
U
properties using focused libraries [1]. Lead candidates in this context can be identified and
AN
optimized via screening of skeletally diverse small molecules for specific biological target(s) [2].
Recent years have witnessed the developments of new DOS pathways; however,
multicomponent reactions (MCRs) are among the extensively investigated synthetic strategy for
M
the efficient generation of small chemical entities with increased molecular diversity and
complexity. Fortunately, isocyanide-based multicomponent reactions (IMCRs), the most
D
common subclass of MCR, offer a straightforward way to introduce structural diversity and
TE
molecular complexity in a single step [3]. Amongst all known IMCRs, Passerini and Ugi
reactions have emerged as a robust mean in the construction of peptide-like compounds by
giving access to depsipeptides [4,5], N-methyl peptides [6], and peptoids [7-9], including dimeric
EP
ones.
Despite the potential of the classical, basic form of Passerini and Ugi products, they are
C
limited by their nature; that is, ester and/ amide based structures. These compounds are therefore;
AC
prone to metabolism, i.e. in vivo they might be cleaved or modified to potentially more or less
active or desirable metabolites. The potential of Passerini and Ugi reaction was
accordingly extended to attain more constrained scaffolds i.e. heterocyclic moieties. This is
where Groebke and azido-Ugi reactions come to play [10]. These reactions give access to
bioactive imidazole- and tetrazole-based, constrained motifs [11,12].
Another alternative approach to attain further diversity and complexity is to tag the Ugi
reaction with subsequent post-condensation modifications, often via attaching orthogonal
2
ACCEPTED MANUSCRIPT
functional group(s) at the backbone of the initial Ugi product [12,13,14]. The incorporation of
bifunctional building block allows subsequent secondary transformations (e.g., nucleophilic
substitution (SN) reactions, cyclocondensation and cycladdition) and attains the second level of
diversity [15]. In view of the former, sequential coupling of Ugi reaction to other reactions have
been reported with different post-modification reactions such as Heck, Diels-Alder, Pictet–
PT
Spengler, Petasis, Mannich, Wittig and Click reactions for the efficient synthesis of a number of
therapeutically important heterocyclic scaffolds with dense structural features and functionalities
RI
[12,15-20]. This double-layered approach has gained vast importance, and now it is routinely
used for drug discovery.
SC
Recently, we employed a multicomponent strategy (e.g., Passerini, Ugi and Ugi/SN, Michael
reactions) for the synthesis of hybrid structures containing diverse organoselenium libraries
U
coupled to bioactive pharmacophores (e.g., quinones, naphthalene, cyclic imides) or
pharmacologically relevant heterocycles (e.g., thiazolidinone, pyrazole and thiazolopyrimidine)
AN
[21-31]. Some of these compounds exhibit cytotoxicity at sub-micromolar concentrations against
various types of cancer cells, such as hepatocellular carcinoma (HepG2), breast adenocarcinoma
M
(MCF-7), A-498 (human kidney carcinoma) and A-431 (human epidermoid carcinoma) cell
lines. It is worthwhile to mention that the toxicity was more pronounced in case of HepG2 and
D
MCF-7 cells. Furthermore, some of these compounds show lower cytotoxicity when tested
against normal cells such as HUVEC (human umbilical vein endothelial), WI-38 (human lung
TE
fibroblast) and HF (primary human fibroblast) cell lines. The underlying cytotoxicity and
selectivity mechanisms are quite interesting since these compounds can either function as
EP
antioxidant or pro-oxidants relying on their redox properties and the intracellular redox
environment in which they are placed [21,25,27].
C
In normal cells, organoselenium compounds act as antioxidants and thus protect cells form
oxidative damage. On the other hand, these compounds become pro-oxidants in oxidatively
AC
stressed cells (e.g., MCF-7 and HepG2) [30,32,33]. This bimodal function proposes
organoselenium compounds not only as chemopreventive agents, but also as selective
chemotherapeutics [34-36].
Although one can only speculate about the organoselenium possible mode(s) of action, their
cytotoxicity has been attributed primarily to caspase 3/7 activation and subsequent induction of
apoptosis. Additionally, different phenotypical changes were observed and these included
3
ACCEPTED MANUSCRIPT
endoplasmic reticulum, actin cytoskeleton and cellular morphology alterations as well as cell
cycle arrest and various biochemical changes (e.g., ROS and GSH levels) [21,22,26,27,29].
Interestingly, we found that selenium based quinones were among the most active compounds
exhibiting increased anticancer activity [22,29,31].
In continuation of our program directed towards the development of therapeutically
PT
promising organoselenium agents, we herein report the development of a facile route towards
symmetrical diselenide and selenoquinone based-tetrazoles via azido-Ugi and azido-Ugi/SN
RI
methodology. The respective mode-of-action(s) of the newly synthesized compounds are
assessed in twofold: a) addressing their corresponding cytotoxicity in cell assays using HepG2,
SC
MCF-7 and normal cells (WI-38) as well as estimating their corresponding effect on the
expression levels of caspase-8, Bcl-2 and Ki-67 molecular biomarkers; b) exploration of the
U
redox modulation activities of the synthesized compounds employing DPPH, GPx-like activity
and bleomycin dependent DNA damage assays. Furthermore, in silico molecular modeling
AN
studies, including field alignment and docking studies, will be applied as a preliminary
prediction tool to estimate the antioxidant and cytotoxic properties of the compounds.
M
2. Results and Discussions

2.1. Design and synthesis
D
Until recently, the synthesis of organic selenides was not an easy task and included the use of
TE
expensive/toxic starting materials. Recent years have witnessed significant progress in the
synthesis of different classes of organoselenium compounds such as selenaheterocyclic,
EP
selenocyanates, selenides and diselenides) [25, 37-42].

As a part of our project aimed towards the development of organoselenium-based
chemotherapeutic agents, we herein report the synthesis of tetrazole-based symmetrical
C
diselenide and selenoquinone compounds (6-22) synthesized via azido-Ugi and sequential SN
AC
strategy (Figure 1).

Figure 1.
Based on recent findings reported by our group, we envisioned that tetrazole-based

organoselenium scaffolds thus might be more efficient anticancer agents. Tetrazoles have
received considerable attention from the pharmaceutical market as they constitute the core
4
ACCEPTED MANUSCRIPT
scaffold of several bioactive compounds and many marketed drugs (e.g., pentylenetetrazol,
cilostazol, ceftezole, irbesartan, losartan) [43]. Furthermore, these compounds are of particular
interest because tetrazole is a bioisostere for carboxylic acid group (COOH) but yet with better
potency, favorable physicochemical properties, improved pharmacokinetic profiles and
metabolic stability compared. This is mainly due to the tetrazole larger size and superior
PT
lipophilicity (≈10 times more) which in turn leads to an increase of the substrate receptor
interaction [44,45].
RI
There are many methods for the construction of tetrazole ring system; however, the azido-
Ugi reaction is superior to classical methods in terms of automation, reaction time and overall
SC
yields [46]. In order to guarantee a second level of diversity, 4,4'-diselanediyldianiline (4) was
used as a bifunctional key synthon as it possesses an amino group convenient for the azido-Ugi
U
reaction and a readily liberated selenolate nucleophile (formed in situ via reduction of the
diselenide using NaBH4) suitable for subsequent SN reactions.
AN
With regard to library construction, six structurally diverse isonitriles (e.g., tert-
butylisocyanide (2a), cyclohexyl isocyanide (2b), 4-isocyanopermethylbutane-1,1,3-triol (IPB,
M
2c) [47], benzylisocyanide (2d), 4-methoxybenzylisocyanide (2e) and 2,4-

dimethoxybenzylisocyanide (2f) were included for library validation purposes, and partly there
D
potential for additional post-Ugi reactions. As oxo component, four different aldehydes were
used including both aliphatic (paraformaldehyde (1a), isobutyraldehyde (1b)) and aromatic (4-
TE
methylbenzaldehyde (1d) and furfuraldehyde (1c)) ones, whereas trimethylsilyl azide (TMSN3) 3
was used as the acid component.
EP
Figurer 2.
C
Tetrazole-based symmetric diselenides (6–19) were synthesized by the addition of two

AC
equivalents of aldehyde 1 to a methanolic solution of 4 (one equivalent), followed by the

subsequent addition of 2.5 equivalents of TMSN3 and isocyanide 2. After completion of the
reactions, reduction of diselenides (6–19) with NaBH4 afforded the corresponding sodium
selenolate which in turn reacts with 2-bromo-3-methyl-1,4-naphthoquinone (5) via a SN
mechanism (or addition/elimination sequence) to give tetrazole-based selenoquinones (20-22).
It is noteworthy that the reaction sequence can be also performed in a one pot (five
components) set up without the isolation of the initial azido-Ugi products. Overall, the syntheses
5
ACCEPTED MANUSCRIPT
of 6–22 proceeded smoothly and compounds were obtained in excellent yields (up to 96 %,
Figure 3).
Figure 3.
2.2. In silico prediction of biological activity
PT
Molecular modeling techniques have been shown to be of a high value in antineoplastic drug
development. Therefore, they were used in order to virtually assess the redox modulation and the
RI
potential anticancer properties of the newly synthesized compound prior to the biological
screening. Field alignment was applied for the 3D similarity study between the symmetrical
SC
diselenides of this work and two related reference compounds with known GPx-like activity.
Furthermore, a docking study was also performed at the Thioredoxin reductase (TrxR1) [48-50]
U
dimerization interface domain to explore the ability of these compounds to act as TrxR1
AN
inhibitors.
2.2.1. 3D similarity study:

M
3D similarity study is performed for a focused library of symmetrical diselenides to figure

out if these compounds possess similar electrostatic pattern, conformation and/or shape to
D
reference compounds with known GPx mimetic activity. Compounds (Z)-N-(4-

TE
methylbenzylidene)-1-(2-((2-(1-((E)-4-
methylbenzylideneamino)ethyl)phenyl)diselanyl)phenyl)ethanamine (24) [51] and 2,2'-
((diselanediylbis(4,1-phenylene))bis(acetylazanediyl))bis(N-(tert-butyl)-3-methylbutanamide)
EP
(25) [21] with recognized GPx mimetic activities were used as templates and for comparison
purposes. Field aligning technique is used to align molecules based on their molecular fields. The
C
molecules with similar field patterns to 24 and 25 are expected to possess similar patterns of
AC
biological activity, especially in redox reactions [52]. As molecular similarity approaches one,
the alignment and accordingly the electrostatic structural similarities are better.
Due to the lack of the availability of bioactive conformations of 24 and 25, a field template
was primary generated using the Field Templater Module using Forge 10.4. This was followed
by field alignment of selenol forms of our diselenides to that template employing Field Align
Module in Forge 10.4 [53].
6
ACCEPTED MANUSCRIPT
The selenol forms of compounds 24 and 25 were loaded as single 2D structures and then
passed to the conformation hunter to generate a set of diverse conformations. Field Templater
searches for common field patterns across the explored conformational space of a set of
molecules. The top-ranked field template was selected (scores: molecular similarities: 0.713,
field similarity: 0.656, raw field score: -67.014, shape similarity: 0.769 and raw shape score:
PT
154.4). The generated field template shows the negative field point (blue) localized around the
diselenide groups while multiple positive field points (red) surround the para substitution region.
RI
Conformers of compound 25 showed some additional negative field points (Figure 4).
Figure 4.
SC
Table 1.
U
After applying Field Alignment, the most probable conformer of each test molecules was
AN
selected on the basis of the pair-wise matching. The average molecular similarity is about 0.619-
0.713. The score values for the compounds after alignment over the template generated are
illustrated in Table 1. The essential field points are also present in our test compounds (Figure 5).
M
Accordingly, the field alignment technique demonstrated that our novel diselenides share the
same electrostatic pattern and – at least in the more stable conformations - shape with reference
D
compounds 24 and 25, suggesting that they would have similar GPx mimetic activity.
TE
Figure 5.
2.2.2. Docking study:

EP
Many organoselenium compounds were reported to inhibit Thioredoxin Reductase (TrxR) in

some cancer cells and therefore, activate an apoptosis pathway [54]. To predict the anticancer
C
activity and apoptotic ability of our compounds, docking into mammalian TrxR enzyme was
AC
performed. TrxR1 shows a homodimeric quaternary arrangement of its four monomers. Each
monomer has two main regions: FAD and NAD binding domains at the N-terminal and the
dimerization interface domain at the flexible C-terminal side [50,55-57].
Wang et al [54] reported the in silico simulation model of the proposed interaction
mechanism of the organoselenium compound ethaselen 26 with mammalian TrxR1. They
provided an evidence-based on explanation that ethaselen 26 targets the C-terminal active site of
7
ACCEPTED MANUSCRIPT
the mammalian TrxR1 at the dimerization interface domain forming two intermolecular
selenenyl sulfide (Se–S) and selenenyl selenide (Se-Se) covalent bonds with the Cys497 and
Sec498 amino acids of the protein, respectively; and thus it is acting as an irreversible inhibitor
for this enzyme [54].
Figure 6.
PT
Flexible docking was applied using Flexible Docking protocol. This protocol allows some
receptor flexibility during the docking of the flexible ligands [58]. The side-chains of the
RI
specified amino acids are allowed to move during docking. This allows the receptor to adapt to
the different ligands in an induced-fit model.
SC
Ethaselen 26 was successfully docked and the ideal pose was selected i.e. the pose which
orients the selenium atoms towards the interacting cystein residues 497 and 498 while pointing
U
the phenyl group of the other selenenylbenzene group towards His 472 (Figure 7). Such pose is
similar to that reported and proposed by the molecular dynamics simulation study [59].
AN
The test compounds were docked using Flexible Docking protocol, and the best pose for each
structure was selected. Compound 23 showed the best -CDOCKER energy (32.17 kcal/mole).
M
The carbonyl oxygen of the tertiary amide interacts with Cys 497 via intermolecular hydrogen
bonding (1.5 Å). Also, there is an additional hydrogen bond between Glu 477 and NH of the
D
secondary amide (1.6 Å). Such interactions orient the selenium atom close to the second
interacting Cys 498 (corresponding to human Sec 498 in human TrxR) within a distance of 5.9 Å
TE
(Figure 7 a and b).

Furthermore, compounds 21 and 22 exhibited also higher binding energies, 7.07 and 13.92,
EP
respectively, compared to ethaselen 26 with 0.75 kcal/mol. On the other hand, compound 18
manifested the poorest binding energy (see Supplementary material).
C
The calculated binding modes of structures 21 and 22 show π-π interactions between the
aromatic ring of His 472 or Phe 406 and the tetrazole ring (Figure 7 d and f). Morever, the 22
AC
docked pose shows additional interactions: σ-π interaction between Gln 494 and the quinone
ring, two hydrogen bonds between Ile 478 and the tetrazole ring nitrogens (N3 and N4) of 2.3 Å
each and a hydrogen bond between Ser 404 and the carbonyl oxygen of quinone (2.5 Å). These
interactions anchor the compounds 21 and 22 in the binding site in a such way that a selenium
atom gets closer to the cystein residue 498 with a distance of 6.8 Å and 5.8 Å, respectively.
Figure 7.
8
ACCEPTED MANUSCRIPT
2.3. Pharmacology, toxicity and antioxidant profiles

2.3.1. Cytotoxic activity of redox active compounds against human cell
lines
PT
In order to check if the chemistry developed here has led to potentially interesting candidates
in cancer therapy, our initial aim is directed towards checking if the newly synthesized
RI
compounds possess any anticancer activities as predicted by the in silico study. Cytotoxicity
screen was therefore performed against two mammalian cancer cell lines, HepG2 and MCF-7
SC
cells. These cell lines were chosen based on our previous broad-spectrum screening results,
which showed that the toxicity of organoselenium compounds are generally more pronounced in
case of MCF-7 and HepG2 cells. Furthermore, these cell lines are rather suitable models for drug
U
targeting. On the other hand, WI-38 normal lung fibroblast cells were also used to gain an initial
AN
insight into the selective cytotoxicity of these compounds and to narrow down the number of
most interesting compounds for further in-depth studies.
M
Cells were incubated with different concentrations of the test compounds for 48 h and the
viability was estimated by quantifying the number of metabolically active cells using the
D
standard colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

assay. 5-FU was used as positive control because it is commonly used in adjuvant and palliative
TE
cancer chemotherapy. Cytotoxicity was estimated from the respective dose response curves and
is expressed as the compounds’ concentration that reduces the absorbance of the treated cells by
EP
50% (IC50) i.e. 50% growth inhibition. The therapeutic index (TI) is defined as the ratio of the
concentration of compound that inhibits 50% of the normal WI-38cells viability to the
concentration that inhibits 50% of HepG2 cell viability (Table 2).
C
AC
Table 2.
Initial screening indicates that some of the compounds were able to reduce the viability of
MCF-7 and HepG2 with moderate IC50s. Furthermore, a distinct correlation was observed
between the chemical structures of test compounds and their corresponding cytotoxicity
9
ACCEPTED MANUSCRIPT
indicating that it is not selenium cytotoxicity that is observed. Contrary to our expectations,
tetrazol-based symmetrical diselenides were generally more cytotoxic than the analogous
tetrazol-based selenoquinones. Moreover, cytotoxicity was more pronounced in HepG2
compared to MCF-7 cells. Notably, phenotypic changes, including cell morphology and cell-cell
adhesion were also observed, in culture, after the treatment with organoselenium compounds.
PT
Additionally, cell blebs formation, shrinkage and detachment were also noticed in the culture
plate, and these can be considered as an early morphological sign of apoptosis.
RI
Interestingly, a clear difference in the toxicity zones was observed between the normal (WI-
38) and cancer HepG2 cells. Therefore, TI was calculated in correspondence to HepG2 cells
SC
(IC50WI-38/IC50HepG2). Compounds 9 and 18 were found to be the most selective ones
(selectivity index= 32 and 11, respectively). Moreover, compounds 6, 11, 14 and 20 have TI
U
values up to 3 which in turn were also higher than the standard benchmark compound 5-FU.
Of course, such initial studies are not directly transferable and therefore, require more
AN
investigations using a wider arsenal of normal and tumor cells, and eventually organismic
experiments.
M
2.3.2. Evaluation of caspase-8, Bcl-2 and Ki-67 molecular biomarkers in

D
Hep-G2 cells
TE
In order to further study the possibly addressed signaling pathways and obtain hints on the
mode(s) of action of the most promising compounds 18, 21, 22 and 23 at the molecular level, the
EP
expression levels of selected tumor proliferation, anti-apoptotic and apoptotic protein markers
were assessed in HepG2 cells. In view of the former, the Ki-67 proliferative marker was chosen
due to its strict association with cell proliferation, whereas pro-apoptotic caspase-8 and anti-
C
apoptotic Bcl-2 proteins were selected to monitor apoptosis induction.

AC
Figure 8.
As shown in Figure 8, compounds 18, 21, 22 and 23 were able to upregulate the expression
of caspase-8 and downregulate the expression of Ki-67 and Bcl-2 compared with untreated cells.
It is worthwhile to mention that a distinct correlation was observed between the chemical
structures of test compounds and their corresponding expression modulation activity showing
that it is not a general selenium cytotoxic effect. Not surprisingly, and in agreement with TrxR1
10
ACCEPTED MANUSCRIPT
docking study, the selenoquinones 21, 22 and 23 were more active compared to the symmetric
diselenides. Interestingly, 23 exhibited a superior activity within the quinoid analogues and this
worth further study. These results were in line with our previous studies, in which selenium
based quinones induced a chain of biochemical alterations among which is the cell caspases
activation and apoptosis induction [27,29]. These alterations seem to take place primarily in
PT
specific cells (e.g., HepG2) with a disturbed intracellular redox balance.
While it is premature to explain why compound 23 was among the most active agents in
RI
these assays, one may speculate that this compound may hit more than one specific cellular
target(s) and cause widespread modification of proteins and enzymes for the benefit of
SC
activation. Furthermore, it’s likely that 23 might also be taken up by cells and modified in vivo
into active metabolic intermediates. As these are unknown, it is too early to speculate over
U
details on its exact metabolism, pharmacokinetics in animals and enrichment in specific tissues
or degradation, although these issues are clearly important and will form part of our future
AN
studies. Ultimately, as the structure of these compounds provides considerable scope for
modifications, and the synthesis of derivatives is now straightforward, this will become a
M
promising starting point for future studies of structural variants.

2.3.3. Assessment of antioxidant activity
D
Organoselenium compounds have recently acquired much attention, particularly from the
TE
public health and pharmaceutical industry due to their chemotherapeutic potential as antitumor
agents. Accordingly, different modes of action have been proposed for such compounds.
EP
However, ROS-modulation has been accepted as a key hallmark mechanism for organoselenium
compounds [32,59].
Owing to the fact that organoselenium compounds are on occasion considered as redox-
C
modulators [59-62], the redox activities of the synthesized compound are further estimated
AC
employing different biochemical assays such as DPPH, GPx-like activity and bleomycin-
dependent DNA damage assays. The redox status can be crucial in fighting and differentiating
tumor cells from normal ones [63,64].
2.3.3.1. DPPH free radical scavenging assay
11
ACCEPTED MANUSCRIPT
There are many methods used for the evaluation of the antioxidant properties of organic
compounds; however, the in vitro DPPH chemical assay is frequently used to estimate the radical
scavenging activities of organoselenium compounds and nutritional products due to its simplicity
and rapidity, but with limited in vivo relevance. The antioxidant activity of a compound is
estimated by its ability to reduce DPPH. radical (purple color in methanol) to DPPHH (colorless)
PT
and the corresponding radical-scavenging activity is evaluated by the decrease in the absorbance
at 517 nm [65,66]. The standard water soluble antioxidant, vitamin C was used as the positive
RI
control in this assay.
Table 3.
SC
As depicted in Table 3, tetrazole-selenoquinones 20 and 21 were the most active compounds
in this assay showing a free-radical scavenging activity compared to ascorbic acid. This was in
U
excellent agreement with our previous report that quinones are good antioxidants at lower
concentrations [67].
AN
2.3.3.2. Bleomycin DNA damage Assay
M
The bleomycin-iron DNA damage assay was used to evaluate the pro-oxidant activity of the
synthesized compound. This assay has been established as an accurate and distinct method to test
D
potential of drugs and food antioxidants [68,69]. Bleomycin antibiotics exert their anticancer
TE
activity via ferrous (Fe2+) ions assisted damage of DNA. Bleomycin forms a complex with Fe2+,
which in turn catalyzes DNA degradation. In brief, pro-oxidant compounds reduce the
bleomycin-Fe+3 to bleomycin-Fe+2, under aerobic conditions, and thus induce DNA degradation,
EP
which can be followed spectrophotometrically by the increase in absorbance at 532 nm (Table

3). In this assay, vitamin C is used as a reducing agent and reduces bleomycin-Fe+3 complex to
C
induce DNA degradation.

AC
Using a modified assay [70,71], as absorbance increased, more bleomycin-Fe3+is converted

into bleomycin-Fe2+, thereby inducing the DNA degradation supporting the idea of the pro-
oxidant activity of these compounds. Compounds 6, 9, 10, 13 and 16 suppressed the reduction of
bleomycin-Fe+3, thus diminishing the bleomycin-Fe+2 chromogen formation and protecting the
DNA. On the other hand, compounds 14, 19 and 22 induced DNA degradation significantly
more than the other investigated compounds (Table 3).
12
ACCEPTED MANUSCRIPT
In accordance to the ROS-modulation theory, that explains the cytotoxic activity of the
organoselenium compounds. Compound 22 primarily is exerting its cytotoxic effect via its
pronounced pro-oxidant properties. On the other hand, there are likely other mechanisms for the
other compounds involved in their cytotoxic activity which need further investigation.
PT
2.3.3.3. Glutathione peroxidase-like activity assay
RI
Selenium trace element is a pivotal component of many enzymes, including GPx [72] and
thioredoxin reductases [73]. Most of these enzymes contain selenium cofactor as selenocysteine
SC
amino acid, which is responsible for their corresponding antioxidant properties. Since the
discovery of organoselenium compounds as good GPx mimics, and many of these agents have
been developed for the control of different illness related to oxidative stress, such as cancer,
Alzheimer and inflammatory diseases [74-75].
U
AN
The GPx-like activities of the novel compounds were evaluated using NADPH-reductase
coupled assay [76-81]. This assay is frequently used for the assessment of the GPx-like activity
of organoselenium compounds. Reduction of peroxides by GPx is followed by subsequent
M
conversion of the oxidized glutathione (GSSG) to its reduced form and oxidation of NADPH to
NADP+. Therefore, the activity of GPx is monitored spectrophotometrically by the decrease in
D
absorbance (340 nm) due to oxidation of NADPH to NADP+. The standard GPx-like seleno-
TE
organic ebselen was used as the positive control.

Figure 9.
EP
As shown in Figure 9, most of the compounds exhibited moderate to good GPx-like activity.
Symmetrical diselenides exhibited higher activity in this assay compared to their selenoquinone
analogues, as can be expected from theory. Furthermore, compounds 9, 12, 14, 19, and 21 were
C
even more active, up to 5 fold compared to the GPx mimetic ebselen.

AC
3. Conclusion
New symmetrical organodiselenides and selenium-based quinones containing tetrazol moiety

were synthesized, employing sequential azido-Ugi/SN reactions in one step and in good yields. In
silico prediction of the compounds’ antioxidant activity and 3D similarity studies revealed a
comparable electrostatic pattern and shape with reference compounds suggesting a similar GPx
mimetic activity which was further validated by biological screening. Furthermore, docking
13
ACCEPTED MANUSCRIPT
study performed into mammalian TrxR1 enzyme, showed that compounds 21, 22 and 23 have
promising binding energies and binding mode that orients the selenium atom towards Cys 498
for interaction; and thus they may act as TrxR inhibitors.
Moreover, compounds were evaluated for their cytotoxic activity against HepG2 and MCF-7
cell lines and compared with their cytotoxicity to normal WI-38 employing standard MTT
PT
assays. In general, diselenides were found to be more cytotoxic than selenoquinones, and this
was more pronounced in HepG2 compared to MCF-7 cells. Furthermore, a distinct selectivity
RI
was noticed by comparing the cytotoxicity in HepG2 with that of normal WI-38 cells.
Interestingly, compounds 9 and 18 exhibited a selectivity index of ca. 32 and 11, respectively.
SC
Moreover, compounds 6, 11, 14 and 20 showed a therapeutic index up to 3 for the model cell
lines, which in turn were all higher than that for the benchmark drug 5-FU. To this point, some
U
of the compounds exhibited a preferential cytotoxicity, and this was obvious in HepG2 compared
to MCF-7 cells. Furthermore, distinct selectivity patterns were observed by comparing the
AN
cytotoxicity in HepG2 with that of normal WI-38 cells.
Although it might appear that, compared to some standard drugs, these compounds do not
M
provide nanomolar activity, there is enough evidence of selectivity and the perspective of an
unknown (and thus potentially new) mode of action to warrant further studies. Within this
D
context, we are fully aware that a clear QSAR will require large and diverse sets of compounds
including sulfur- and for completion maybe even tellurium-analogues, to ascertain a specific role
TE
of organoselenium and to screen for compounds with improved activity and selectivity.
As expected, diselenides showed superior GPx catalytic activity and among the compounds,
EP
9, 12, 14, 19 and 21 were more active (up to 5 fold) than ebselen. Additionally, tetrazole-
selenoquinones 20 and 21 showed the highest radical scavenging activity in the DPPH assay
C
which was also in line with our previous reports, ascertaining that quinones are efficient
antioxidants at lower concentrations. Further the pro-oxidant activity of the synthesized
AC
compounds evaluated with the bleomycin DNA damage assay revealed that compounds 14, 19
and 22 were able to induced significant DNA degradation more than the other investigated
compounds (e.g., 6, 9, 10, 13 and 16).
Overall, the selenium-based quinones were the most promising candidates and therefore,
were selected for further in-depth studies of the underlying molecular mechanism. Treatment of
HepG2 cells with compounds 18, 21, 22 and 23 revealed a significant downregulation in the
14
ACCEPTED MANUSCRIPT
expression levels of Bcl-2 and Ki-67 and activation of caspase-8 compared to untreated cells.
Importantly, 23 showed the highest modulation activity the three biomarkers. These results
support the molecular docking study into mammalian TrxR1 that predicted the best binding
energies for compounds 23, 21 and 22 than 18. TrxR1 inhibition increases caspases level and
thus induces apoptosis.
PT
At this point, it should be noted that the results presented here are preliminary in nature and
need more in-depth studies in the future. In consequence, a more in-depth studies and additional
RI
experiments to investigate the exact mode(s) of action and to identify possible intracellular
targets (such as specific organelles, or membranes) are warranted. Furthermore, optimization of
SC
diaryl diselenides scaffold is required to improve their pharmcodynamic and pharmacokinetic
properties. This opens plenty of room for further, multi-disciplinary studies involving synthetic,
U
bioorganic and medicinal chemistry, cell biology, and pharmacology in order to develop a
strategy to treat cancer applying organoselenium compounds.
AN
4. Experimental protocols
4.1. Material and methods
M
All chemical reagents for the synthesis of the compounds were purchased from Sigma-
D
Aldrich-Fluka or Merck (AMD) and used without further purification unless stated otherwise.
Reactions in inert atmosphere were carried out under argon (4.6) using standard Schlenck
TE
techniques. Silica gel 60 (Macherey-Nagel, 50-200 µm) was used for column chromatography.
Unless noted otherwise, the dimensions of columns used were 2.5 cm (diameter) and 25-30 cm
EP
(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, 13
C NMR spectra at 100 MHz on a
Varian INOVA spectrometer. Chemical shifts are reported in δ (ppm), expressed relative to the
C
solvent signal at 7.26 ppm (CDCl3, 1H NMR) and at 77.16 ppm (CDCl3, 13C NMR), as well as
AC
3.31 ppm (1H NMR, CD3OD) and 49.00 ppm (13C NMR, CD3OD).
Coupling constants (J) are given in Hz. MS analysis: analyses were performed using a TSQ
quantum mass spectrometer equipped with an ESI source and a triple quadrupole mass detector
(Thermo Finnigan). High-resolution mass spectrometry (HRMS) 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 ≥ 99% MS-purity of the
15
ACCEPTED MANUSCRIPT
prepared compounds. DNA (Calf Thymus type1), bleomycin sulfate, thiobarbituric acid (TBA),
1,1-diphenyl-1-picrylhydrazyl, ethylenediaminetetraacetic acid (EDTA) and ascorbic acid were
obtained from Sigma. All other chemicals were of analytical grade. Compounds
were prepared and purified according to the experimental and purification procedures in the
Supplementary Materials. Further details on the spectral data and copies of 1H and 13
C NMR
PT
spectra for all synthesized compounds can also be found in Supplementary Materials.
4-(2-(4-aminophenyl)diselanyl)benzenamine (4) and 23 were synthesized according to a
RI
literature reported method [82,83].
4.2. Molecular Modeling
SC
4.2.1. Preparation of TrxR1:
The mammalian TrxR1 protein (PDB id: 1H6V) was retrieved from Protein Data Bank
U
(PDB) for docking. Only one monomer was used while others were deleted. All water molecules
AN
were removed. Protonation of the protein, completing missed residues, atom ordering corrections
if necessary and checking bonds and bond ordering and their correction if necessary were
M
performed by the use of the clean protein tool under protein report and utilities of Discovery
Studio 2.5. CHARMm force field was applied followed by energy minimization using Adopted
D
Basis NR algorithm with maximum steps of 2000 while other parameters maintained as default.
The binding site of TrxR1 was defined by: finding sites from receptor cavities, selecting the one
TE
present at the interface domain and definition of the docking sphere with dimensions X: 9 , Z:
10.36 and Y: 149.54 and a radius of 13Å.
EP
4.2.2. Ligands Preparation and Docking
Ligand test molecules were prepared using the prepare ligands protocol under general
C
purposes, whereby generation of all possible isomers have been considered to be generated.
AC
While generation of tautomers, application of Lipiniski's rule filtration and changing ionization
parameters were set to false. For the docking process, flexible docking protocol was applied. The
selected residues for generation of flexible side chain conformations were that of dimerization
interface domain (368-499). Generation of 10 protein conformations of the selected residues was
determined with a maximum number of 8 residues. Generation of ligand conformations was
enabled as fast method by generating 25 different conformers of each test compound with energy
threshold of 20 kcal.
16
ACCEPTED MANUSCRIPT
All other parameters of docking remained as default. After docking run finished, the highest
binding energy pose was selected to visualize the ligand protein interactions as reported above
(See supplementary material).
4.3. Biological assays
PT
4.3.1. Cytotoxicity assay
RI
The HepG2 human liver carcinoma, breast adenocarcinoma (MCF-7) and lung fibroblast (WI-
38) cell lines were purchased from American Type Culture Collection (HTB‑37; Rockville, MD,
SC
USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% (v/v) calf serum (Hyclone Laboratories, Ogden, UT), 60 mg/mL penicillin G and 100
mg/mL streptomycin sulfate maintained at 37 °C in a humidified atmosphere containing about
15% (v/v) CO2 in air.
U
AN
MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] (Sigma) was used to
measure the metabolic activity of cells which are capable of reducing it by dehydrogenases to a
M
violet colored formazan product. Briefly, 120 µL aliquots of a cell suspension (50,000 cells
mL-1) in 96-well microplates were incubated at 37 °C and 10% CO2 and allowed to grow for two
D
days. Then 60 µL of serial dilutions of the test compounds were added. After 48 h of incubation
at 37 oC and 10% CO2, 75 µL MTT in phosphate buffered saline (PBS) were added to a final
TE
concentration of 0.5 mg mL-1. After 2 h the precipitate of formazan crystals was centrifuged and
the supernatant discarded. The precipitate was washed three times with 100 µL PBS and
EP
dissolved in 100 µL DMSO. The resulting color was measured at 590 nm using an ELISA plate
reader. 5-FU was used as a positive control because it is commonly used in adjuvant and
palliative cancer chemotherapy. All investigations were carried out in two parallel experiments.
C
The IC50 values were determined as the concentrations of tested materials, which showed 50% of
AC
the absorbance of untreated control cells as estimated from the dose-response curves. The
therapeutic index (TI) is defined as the ratio of the concentration of compound that inhibits 50%
of the growth of the normal WI-38cells to the concentration that inhibits 50% of HepG2 cell
viability.
4.3.2. In vitro studies
4.3.2.1. DPPH free radical scavenging activity
17
ACCEPTED MANUSCRIPT
The hydrogen atom or electron donation ability of the corresponding compounds was measured
by the bleaching of the purple color of a methanolic solution of DPPH. This spectrophotometric
assay uses stable radical diphenylpicrylhydrazyl as a reagent. The test compounds were
dissolved in methanol to obtain a final concentration of 1µM. 200 µL of each sample were added
to 0.4 mL of 0.1 mM DPPH in methanol. After 30 min of incubation in the dark, the absorbance
PT
was read against a blank at 517 nm. Ascorbic acid (vitamin C) was used as standard antioxidant
(positive control). Blank sample was run without DPPH and using methanol instead of sample.
RI
Negative control sample was run with methanol instead of tested compound. The radical
scavenging activity was calculated from the following equation:
SC
I%= (Ablank-Asample)/(Ablank)x100
4.3.2.2. Glutathione peroxidase like activity
U
GPx kit (Biodiagnostic, Egypt) was used for the determination of GPx according to Paglia et
AN
al.[84]The reaction mixture contained 1 mL assay buffer (50 mM phosphate buffer containing
0.1 % triton X-100) and 0.1 mL NADPH reagent (24 µmol Glutathione, 12 unit Glutathione
M
reductase and 4.8 µmol NADPH) and 0.01 mL (41 µM) tested compounds and the reaction was
started by the addition of H2O2 (0.8 mM). The contents were mixed well and the absorbances
were recorded at 340 nm over a period of 3 min against deionized water. The change of
D
absorbance per minute (A340nm / min) was estimated using ebselen (41 µM) as the positive
TE
control. The values represented in Figure 9 are expressed after background correction for the
reaction with H2O2 and GSH. In case of colored compounds, their activities were estimated after
EP
subtracting their own absorbance at the used wave length.
4.3.2.3. Bleomycin-dependent DNA damage

C
The reaction mixture contained calf thymus DNA (0.5 mg/mL), bleomycin sulfate (0.05 mg/mL),
AC
MgCl2 (5 mM), FeCl3 (50 mM), samples to be tested (2 mM) and L-ascorbic acid was used as
positive control. The mixture was incubated at 37 oC for 1 hour. The activity of test compounds
was evaluated as malondialdehyde (MDA) equivalents. Thiobarbituric reactive substances which
arose from deoxyribose degradation of DNA were assessed. The reaction was terminated by
addition of 0.05 mL EDTA (0.1 M). The color was developed by adding 0.5 mL thiobarbituric
acid (1% w/v) and 0.5 mL HCl (25% v/v). The tube was capped with a screw cap and heated at
18
ACCEPTED MANUSCRIPT
80 oC for 30 min. After cooling in ice water, the mixture was then shaken and centrifuged and
the extent of DNA damage was measured by increase in absorbance at 532 nm.
4.3.2.4. Detection of caspase-8 protein expression levels
HepG2 cells were treated with the corresponding IC50 of each drug and incubated for 48 hours.
PT
The cells were detached by trypsin and lysed by freezing at liquid nitrogen and then thawing
with gentle mixing. Cell lysates incubated with Horseradish Peroxidase conjugated anti-CASP8
RI
antibodyfor 30 min at 37 oC. The reaction product was detected at 450 nm using enzyme-linked
immunosorbent assay (Platinum ELISA; Biospess).
SC
4.3.2.5. Detection of Bcl-2 protein expression levels
U
Bcl-2 levels were evaluated in HepG2 cells treated with the corresponding IC50 of each
AN
compound and incubated for 48 hours and compared with their levels in control untreated HepG2
cell line. The cells were harvested by applying trypsin and lysed by freezing with liquid nitrogen
and then thawing with gentle mixing. According to the instructions of the manufacturer of
M
(Platinum ELISA; eBioscience©), cell lysates were incubated with biotin-conjugate for 2 hours
25oC and then with streptavidin-HRP for 1h at 25oC. The reaction product was detected at
D
450 nm.
TE
4.3.2.6. Detection of Ki-67 protein expression levels
HepG2 cells were treated with the IC50 dose of each compound for 48 hours, then were detached
EP
by trypsin and lysed by ultrasonication for 4 times. The reaction product was detected at 450 nm
using enzyme-linked immunosorbent assay (Platinum ELISA; Biomatic) according to the
C
instructions of the manufacturer.

AC
4.3.2.7. Statistical analysis
Data analysis was performed using the statistical package for the social sciences software,
release 15.0 for Windows (SPSS version 15.0, Chicago: SPSS Inc). All results were expressed as
mean ± SD. A P-value <0.05 was considered statistically significant. Statistical analysis was
performed by analysis of variance (ANOVA) with LSD and Dunnett's test for PostHoc.
19
ACCEPTED MANUSCRIPT
4.4. Synthesis and characterization
General procedure I for the preparation of symmetrical diselenide based-tetrazoles derivatives

(6-19) by azido-Ugi reaction
Under argon, a mixture of 4 (1 mmol), aldehyde (2.2 mmol), TMSN3 (2.5 mmol) and isonitrile
PT
(2.5 mmol) in 1 mL methanol was stirred overnight at room temperature. Upon completion
(monitored by TLC), 10 mL dichloromethane were added to dissolve the sticky product, washed
RI
with water (3x50 mL). The organic layer was separated, dried over anhydrous Na2SO4 and
evaporated under reduced pressure. The residue was purified by chromatography on silica gel
SC
employing petroleum ether: ethyl acetate (4:3) to give yellow solid pure compound.
General procedure II for the preparation of selenoquinone based-tetrazoles (20-22)
U
General procedure IIA: two-step synthesis of selenoquinone based-tetrazoles (20-22) via
AN
sequential azido-Ugi/SN methodology
To a solution of the isolated azido-Ugi adduct (1 mmol), 2-methyl-3-bromo-l,4-naphthoquinone

M
(5) (552.2 mg, 2.2 mmol) in ethyl acetate (20 mL) and water (20 mL) heterogeneous solvent
system, was added tricaprylmethylammonium chloride (45 mg, 5 % mol). NaBH4 (189.15 mg, 5
D
mmol) was then added in small portions with caution and the mixture was stirred at room
TE
temperature for further 2 h. Upon completion (monitored by TLC), the organic layer was
separated and solvent was dried over anhydrous Na2SO4 and removed under reduced pressure
and the residue was purified by chromatography on silica gel employing petroleum ether and
EP
ethyl acetate as eluent to give reddish brown solid.

C
General procedure IIB: one-potsequential azido-Ugi/reduction/SN synthesis of selenoquinone

based-tetrazoles (20-22)
AC
Upon completion of the azido-Ugi reaction (monitored by TLC), 2-methyl-3-bromo-l,4-

naphthoquinone (5) (552.2 mg, 2.2 mmol) and tricaprylmethylammonium chloride (45 mg, 5 %
mol) was added to a heterogeneous solvent system of ethyl acetate (20 mL) and water (20 mL).
NaBH4 (189.15 mg, 5 mmol) was then added in small portions with caution and the mixture was
stirred at room temperature for further 2 h. Upon completion (monitored by TLC), the organic
layer was separated, dried over Na2SO4 and solvent was removed under reduced pressure and the
20
ACCEPTED MANUSCRIPT
residue was purified by chromatography on silica gel employing petroleum ether and ethyl
acetate as eluent to give reddish brown solid.
4,4'-Diselanediylbis(N-(1-(1-(tert-butyl)-1H-tetrazol-5-yl)-2-methylpropyl)aniline) (6)
Compound 6 was synthesized according to the general procedure I from 4,4'-diselanediyldianiline (4)
PT
(344 mg, 1 mmol), isobutyraldehyde (1b) (201 µL, 2.2 mmol), trimethylsilyl azide (3) (332 mg, 2.5
mmol) and tert-butyl isocyanide (2a) (283 µL, 2.5 mmol). Its formation was monitored by TLC petrol
RI
ether: ethyl acetate = 4:1, Rf = 0.36, purified by column chromatography on silica gel with petrol ether:
ethyl acetate= 3:1. Yield: 620 mg (88 %). 1H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 8.6 Hz, 4H, Ar-H),
6.56 – 6.46 (m, 4H, Ar-H), 4.91 (s, 2H, 2NH), 4.38 (d, J = 7.4 Hz, 2H, 2CH), 2.43 – 2.30 (m, 2H, 2CH),
SC
1.76 (s, 18H, 6CH3), 1.13 (J = 6.6 Hz, 6H, 2CH3), 1.02 (d, J = 6.7 Hz, 6H, 2CH3).13C NMR (101 MHz,
CDCl3) δ 155.74 (C-tetrazol), 147.30 (C-Ar), 136.05 (CH-Ar), 135.98 (CH-Ar), 119.36 (C-Ar), 114.41
U
(CH-Ar), 70.79 (C-t-butyl), 61.53 (CH), 55.53 (CH), 34.53 (CH-isopropyl), 30.23 (CH3-t-butyl), 19.99
(CH3), 18.04 (CH3). MS (ESI): m/z = found 727.6 [M++Na]; calcd. 704.21[M++Na]; HRMS calcd. for
AN
C30H44N10Se2 [M++Na]: 727.1998, found 727.1973 [M++Na].
M
4,4'-Diselanediylbis(N-((1-(tert-butyl)-1H-tetrazol-5-yl)methyl)aniline) (7)
D
(344 mg, 1 mmol), paraformaldehyde (1a) (66 mg, 2.2 mmol), trimethylsilyl azide (3) (332 mg, 2.5
TE
ethyl acetate= 3:1. Yield: 533 mg (86 %). 1H NMR (400 MHz, CDCl3) δ 7.50 – 7.38 (m, 4H, Ar-H), 6.67
EP
– 6.55 (m, 4H, Ar-H), 4.86 (s, 2H, 2NH), 4.66 (s, 4H, 2CH2), 1.80 (s, 18H, 6CH3).13C NMR (101 MHz,
CDCl3) δ 151.67 (C-tetrazol), 147.12 (C-Ar), 136.05 (CH-Ar), 119.78 (C-Ar), 113.80 (CH-Ar), 61.45 (C-
t-butyl), 39.98 (CH2), 29.59 (CH3-t-butyl). MS (ESI): m/z = found 643.4 [M++Na]; calcd. 643.1 [M++Na];
C
HRMS calcd. for C24H32N10Se2 [M++Na]: 643.0998, found 643.10396 [M++Na].

AC
4,4'-Diselanediylbis(N-((1-(tert-butyl)-1H-tetrazol-5-yl)(p-tolyl)methyl)aniline) (8)
(344 mg, 1 mmol), 4-methylbenzaldehyde (1d) (264 mg, 2.2 mmol), trimethylsilyl azide (3) (332 mg, 2.5
21
ACCEPTED MANUSCRIPT
(d, J = 8.2 Hz, 4H, Ar-H), 7.16 (d, J = 8.1 Hz, 4H, Ar-H), 6.52 (dd, J = 12.2, 6.0 Hz, 4H, Ar-H), 6.09 (s,
2H, 2CH), 5.01 (s, 2H, 2NH), 2.32 (s, 6H, 2CH3), 1.68 (s, 18H, 6CH3).13C NMR (101 MHz, CDCl3) δ
155.04 (C-tetrazol), 146.22 (C-Ar), 138.73 (C-Ar), 135.89 (CH-Ar), 134.73 (C-Ar), 129.85 (CH-Ar),
127.64 (CH-Ar), 119.66 (Ar-C), 114.40 (Ar-CH), 61.78 (C-t-butyl), 53.99 (CH-), 30.05 (CH3-t-butyl),
21.09 (CH3). MS (ESI): m/z = found 825.21 [M++2+Na]; calcd. 823.21 [M++Na]; HRMS calcd. for
PT
C38H44N10Se2 [M++Na]: 823.1998, found 823.19729 [M++Na].
4,4'-Diselanediylbis(N-(1-(1-(2,4-dimethoxybenzyl)-1H-tetrazol-5-yl)-2-methylpropyl)aniline) (9)
RI
SC
mmol) and 1-(isocyanomethyl)-2,4-dimethoxybenzene) (2f) (443 mg, 2.5 mmol). Its formation was
monitored by TLC petrol ether: ethyl acetate = 4:1, Rf = 0.31, purified by column chromatography on
U
silica gel with petrol ether: ethyl acetate= 3:1. Yield: 732 mg (82 %). 1H NMR (400 MHz, CDCl3) δ 7.19
AN
(dd, J = 23.9, 7.3 Hz, 4H, Ar-H), 7.05 (dd, J = 8.4, 4.1 Hz, 2H, Ar-H), 6.49 – 6.42 (m, 4H, Ar-H), 6.30 (d,
J = 7.7 Hz, 4H, Ar-H), 5.58 – 5.44 (s, 4H, 2CH2), 4.61 (dd, J = 9.2, 7.2 Hz, 2H, 2CH), 4.35 (d, J = 9.4 Hz,
2H, 2CH), 3.79 (s, 12H, 4CH3), 2.09 (dd, J = 13.5, 6.7 Hz, 2H, 2CH), 1.65 (s, 2H, 2NH), 1.03 (d, J = 6.8
M
Hz, 6H, 2CH3), 0.85 (d, J = 6.0 Hz, 6H, 2CH3).13C NMR (101 MHz, CDCl3) δ 161.72 (O-C-Ar), 157.67
(O-C-Ar), 155.47 (C-tetrazol), 147.02 (C-Ar), 135.87 (CH-Ar), 131.01 (CH-Ar), 119.24 (C-Ar), 113.99
D
(CH-Ar), 105.00 (CH-Ar), 98.80 (CH-Ar), 55.49 (H3C-O), 54.12 (H3C-O), 45.70 (CH2), 33.08 (CH-
isopropyl), 19.14 (CH3), 18.61 (CH3). MS (ESI): m/z = found 915.5 [M++Na]; calcd. 915.22[M++Na];
TE
HRMS calcd. for C40H48N10O4Se2 [M++Na]: 915.2098, found 915.20992[M++Na].

EP
4,4'-Diselanediylbis(N-(1-(1-(4-methoxyphenyl)-1H-tetrazol-5-yl)-2-methylpropyl)aniline) (10)
C
mmol) and 1-(isocyanomethyl)-4-methoxybenzene (2e) (368 mg, 2.5 mmol). Its formation was monitored
AC
by TLC petrol ether: ethyl acetate = 4:1, Rf = 0.34, purified by column chromatography on silica gel with
petrol ether: ethyl acetate= 3:1. Yield: 675 mg (84 %). 1H NMR (400 MHz, CDCl3) δ 7.32 – 7.25 (m, 4H,
Ar-H), 7.22 – 7.12 (m, 4H, Ar-H), 7.04 (ddd, J = 17.1, 7.8, 2.1 Hz, 4H, Ar-H), 6.37 (dd, J = 18.2, 5.9 Hz,
4H, Ar-H), 4.49 (dd, J = 9.6, 8.4 Hz, 2H, 2CH), 4.20 – 4.07 (m, 2H, 2CH), 3.91 (s, J = 8.1 Hz, 6H,
2CH3),2.26 – 2.13 (m, 2H, 2CH), 1.65 (s, 2H, 2NH), 1.04 (d, J = 9.3 Hz, 6H, 2CH3), 0.92 (d, J = 7.8 Hz,
6H, 2CH3).13C NMR (101 MHz, CDCl3) δ 161.29 (O-C-Ar), 156.37 (C-tetrazol), 146.58, 135.85, 127.32,
125.97, 119.64 (C-Ar), 114.89 (CH-Ar), 114.34 (CH-Ar), 55.75 (H3C-O), 54.57 (H3C-O), 33.35 (CH-
22
ACCEPTED MANUSCRIPT
+ +
isopropyl), 19.17 (CH3-isopropyl). MS (ESI): m/z = found 827.5 [M +Na]; calcd. 827.17[M +Na];
4,4'-Diselanediylbis(N-(2-methyl-1-(1-(2,4,4-trimethoxybutyl)-1H-tetrazol-5-yl)propyl)aniline) (11)
PT
(344 mg, 1 mmol), isobutyraldehyde (1b) (201 µL, 2.2 mmol), trimethylsilyl azide 3 (332 mg, 2.5 mmol)
and IPB (2c) (433 mg, 2.5 mmol). Its formation was monitored by TLC petrol ether: ethyl acetate = 4:1,
RI
Rf = 0.32, purified by column chromatography on silica gel with petrol ether: ethyl acetate= 3:1. Yield:
796 mg (90 %). 1H NMR (400 MHz, CDCl3) δ 7.39 – 7.25 (m, 4H, Ar-H), 6.61 – 6.49 (m, 4H, Ar-H),
SC
4.73 (dd, J = 16.4, 8.7 Hz, 2H, 2CH), 4.62 – 4.52 (m, 2H, 2CH), 4.48 – 4.29 (m, 4H, 2CH2), 3.85 – 3.78
(m, 2H, 2CH), 3.37 (s, 6H, 2CH3), 3.35 (s, 3H, CH3), 3.34 (s, 3H, CH3), 3.13-3.11 (s, 3H, CH3), 3.01 (s,
3H, CH3), 2.42 – 2.25 (m, 2H, 2CH), 1.95 – 1.84 (m, 4H, 2CH2), 1.09 (d, J = 6.7 Hz, 6H, 2CH3), 1.02 –
U
0.94 (d, J = 6.7 Hz, 6H, 2CH3).13C NMR (101 MHz, CDCl3) δ 156.35 (C-tetrazol), 147.08 (C-Ar), 135.89
AN
(CH-Ar), 119.18 (C-Ar), 113.99 (CH-Ar), 101.51 (CH-Ar), 54.45 (CH), 53.98 (CH3-O), 53.44 (CH3-O),
51.33 (CH2), 34.47 (CH2), 33.18 (CH-isopropyl), 32.73 (CH), 19.20 (CH3), 18.63 (CH3). MS (ESI): m/z =
found 907.6 [M++Na]; calcd. 907.2[M++Na]; HRMS calcd. for C36H56N10O6Se2 [M++Na]: 907.2598,
M
found 907.26106 [M++Na].

D
4,4'-Diselanediylbis(N-((1-(tert-butyl)-1H-tetrazol-5-yl)(furan-2-yl)methyl)aniline) (12)
TE
(344 mg, 1 mmol), 4- furan-2-carbaldehyde (1c) (182 mg, 2.2 mmol), trimethylsilyl azide (3) (332 mg,
2.5 mmol) and tert-butyl isocyanide (2a) (283 µL, 2.5 mmol). Its formation was monitored by TLC petrol
EP
C
(d, J = 8.4 Hz, 4H, Ar-H), 6.38 – 6.31 (s, 2H, 2NH), 6.23 (dd, J = 9.8, 4.7 Hz, 4H, Ar-H), 5.11 (s, 2H,
2CH), 1.73 (s, 18H, 6CH3).13C NMR (101 MHz, CDCl3) δ 153.32 (C-tetrazol), 150.60 (C-furan), 145.80
AC
(C-Ar), 142.94 (CH-Ar), 135.76 (CH-furan), 135.57 (CH-Ar), 133.56 (CH-Ar), 121.69 (CH-Ar), 120.30
(C-Ar), 114.66 (CH-Ar), 110.92 (CH-furan), 109.05 (CH-Ar), 62.03 (C-t-butyl), 48.51 (CH), 29.96 (CH3-
t-butyl). MS (ESI): m/z = found 777.4 [M++2+Na]; calcd. 775.14 [M++Na]; HRMS calcd. for
C32H36N10O2Se2 [M++Na]: 775.1298, found 775.12466[M++Na].
4,4'-Diselanediylbis(N-((1-(2,4-dimethoxybenzyl)-1H-tetrazol-5-yl)(furan-2-yl)methyl)aniline) (13)
23
ACCEPTED MANUSCRIPT
2.5 mmol) and 1-(isocyanomethyl)-2,4-dimethoxybenzene) (2f) (443 µL, 2.5 mmol). Its formation was
silica gel with petrol ether: ethyl acetate= 3:1. Yield: 809 mg (86 %). 1H NMR (400 MHz, CDCl3) δ 7.36
PT
(t, J = 3.7 Hz, 2H, Ar-H), 7.30 (t, J = 7.5 Hz, 2H, Ar-H), 7.09 (dd, J = 8.1, 3.7 Hz, 2H, Ar-H), 6.48 – 6.38
(m, 8H, Ar-H), 6.32 (dd, J = 3.3, 1.9 Hz, 2H, Ar-H), 6.22 (d, J = 2.4 Hz, 2H, Ar-H), 6.09 (dd, J = 10.1,
6.2 Hz, 2H, Ar-H),5.55 (s, 4H, 2CH2), 5.01 (s, 2H, 2CH), 3.80 (s, 6H, 2CH3), 3.73 (s, 6H, 2CH3).13C
RI
NMR (101 MHz, CDCl3) δ 161.81 (O-C-Ar), 157.91 (O-C-Ar), 153.25 (C-tetrazol), 149.53 (C-furan),
145.80 (C-Ar), 143.16 (CH-Ar), 135.70 (CH-Ar), 135.68 (CH-Ar), 131.24 (CH-Ar), 119.95 (C-Ar),
SC
114.18 (CH-Ar), 113.40 (C-Ar), 110.79 (CH-Ar), 108.68 (CH-Ar), 104.85 (CH-Ar), 98.74 (CH-Ar),
55.47 (CH), 47.07 (H3CO), 46.32 (CH2). MS (ESI): m/z = found 963.4 [M++Na]; calcd. 963.1[M++Na];
U
AN
4,4'-Diselanediylbis(N-((1-cyclohexyl-1H-tetrazol-5-yl)(furan-2-yl)methyl)aniline) (14)
M
2.5 mmol) and isocyanocyclohexane (2b) (310 µL, 2.5 mmol). Its formation was monitored by TLC
D
petrol ether: ethyl acetate = 4:1, Rf = 0.31, purified by column chromatography on silica gel with petrol
ether: ethyl acetate= 3:1. Yield: 715 mg (89 %). 1H NMR (400 MHz, CDCl3) δ 7.65 – 7.46 (m, 2H, Ar-
TE
H), 7.43 – 7.37 (m, 2H, Ar-H), 7.37 – 7.28 (m, 2H, Ar-H), 7.20 – 7.10 (m, 1H, Ar-H), 7.06 – 6.88 (s, 1H,
NH), 6.72 – 6.48 (m, 3H, Ar-H), 6.43 – 6.29 (m, 3H, Ar-H), 6.19 – 5.98 (m, 2H, Ar-H), 5.13 (s, 1H, NH),
EP
4.47-4.45 (s, 2H, 2CH), 2.11 – 1.85 (m, 8H, 4CH2), 1.59 – 1.55 (m, 6H, 3CH2), 1.46 – 1.25 (m, 6H,
3CH2).13C NMR (101 MHz, CDCl3) δ 152.38 (C-tetrazol), 149.40 (C-furan), 145.75 (C-Ar), 143.18 (CH-
Ar), 136.12 (CH-Ar), 133.60 (CH-Ar), 121.70 (CH-Ar), 120.39 (C-Ar), 115.49 (CH-Ar), 114.29 (CH-
C
Ar), 114.20 (CH-Ar), 111.16 (CH-Ar), 108.99 (CH-Ar), 58.74 (CH), 48.01 (CH-cyclohexyl), 32.79 (CH2-
cyclohexyl), 25.37 (CH2-cyclohexyl), 24.76 (CH2-cyclohexyl). MS (ESI): m/z = found 827.5 [M++Na];
AC
calcd. 827.17[M++Na]; HRMS calcd. for C36H40N10O2Se2 [M++Na]: 827.1598, found 827.15663[M++Na].
4,4'-Diselanediylbis(N-((1-cyclohexyl-1H-tetrazol-5-yl)methyl)aniline) (15)
(344 mg, 1 mmol), paraformaldehyde (1a) (66 mg, 2.2 mmol), trimethylsilyl azide (3) (332 mg, 2.5
mmol) and isocyanocyclohexane (2b) (310 µL, 2.5 mmol). Its formation was monitored by TLC petrol
24
ACCEPTED MANUSCRIPT
ethyl acetate= 3:1. Yield: 598 mg (89 %). 1H NMR (400 MHz, DMSO) δ 7.31 (dd, J = 36.9, 11.3 Hz, 4H,
Ar-H), 6.61 (d, J = 8.3 Hz, 4H, Ar-H), 4.66 (s, 4H, 2CH2), 4.54 (s, 2H, 2NH), 2.11-210 (m, 4H), 1.98 –
1.74 (m, 10H), 1.37 (dt, J = 25.2, 11.7 Hz, 6H).13C NMR (101 MHz, DMSO) δ 151.90 (C-tetrazol),
147.83 (C-Ar), 135.35 (CH-Ar), 116.80 (C-Ar), 112.71 (CH-Ar), 56.74 (CH), 35.88 (CH2-cyclohexyl),
PT
32.32 (CH2-cyclohexyl), 30.40 (CH-cyclohexyl), 24.54 (CH2-cyclohexyl), 24.40 (CH2-cyclohexyl). MS
(ESI): m/z = found 695.5 [M++Na]; calcd. 695.15[M++Na]; HRMS calcd. for C28H36N10Se2 [M++Na]:
695.1398, found 695.13601[M++Na].
RI
4,4'-Diselanediylbis(N-((1-cyclohexyl-1H-tetrazol-5-yl)(p-tolyl)methyl)aniline) (16)
SC
U
AN
– 7.13 (m, 4H, Ar-H), 6.54 (ddd, J = 14.4, 8.6, 6.6 Hz, 4H, Ar-H), 5.88 (s, 2H, 2CH), 5.20 (dd, J = 14.6,
M
6.3 Hz, 2H, CH2), 4.33 (s, 2H, 2NH), 2.33 (s, 6H, 2CH3), 2.11 – 1.89 (m, 6H, 3CH2), 1.82 – 1.68 (m, 6H,
13
3CH2), 1.42 – 1.16 (m, 8H, 4CH2). C NMR (101 MHz, CDCl3) δ 154.23 (C-tetrazol), 146.09 (C-
D
Ar), 139.00 (C-Ar), 136.17 (CH-Ar), 135.97 (CH-Ar), 134.40 (C-Ar), 129.99 (CH-Ar), 127.19
TE
(CH-Ar), 119.65 (C-Ar), 114.09 (CH-Ar), 58.38 (CH), 53.33 (CH), 32.64 (CH2-cyclohexyl),
25.33 (CH2-cyclohexyl), 25.21 (CH2-cyclohexyl), 21.12 (CH3-Ar). MS (ESI): m/z = found 875.5
[M++Na]; calcd. 875.24[M++Na]; HRMS calcd. for C42H48N10Se2 [M++Na]: 875.2298, found
EP
875.22903[M++Na].
C
4,4'-Diselanediylbis(N-(1-(1-cyclohexyl-1H-tetrazol-5-yl)-2-methylpropyl)aniline) (17)
AC
ethyl acetate= 3:1. Yield: 605 mg (80 %). 1H NMR (400 MHz, CDCl3) δ 7.24 (ddd, J = 29.9, 9.9, 4.1 Hz,
4H, Ar-H), 6.61 – 6.36 (m, 4H, Ar-H), 4.73-4.72 (m, 2H), 4.62 – 4.52 (m, 2H), 4.43 – 4.35 (m, 2H), 2.29
(ddd, J = 27.6, 14.0, 7.2 Hz, 2H, CH2), 2.08 – 1.89 (m, 8H, 4CH2), 1.79 (dd, J = 21.4, 10.6 Hz, 4H,
25
ACCEPTED MANUSCRIPT
2CH2), 1.40 (d, J = 6.7 Hz, 6H, 2CH3), 1.15 (d, J = 6.7 Hz, 6H, 2CH3), 0.94 (d, J = 6.7 Hz, 4H, 2CH2),
0.87 (d, J = 6.8 Hz, 2H, CH2).13C NMR (101 MHz, CDCl3) δ 154.49 (C-tetrazol), 147.00 (C-Ar), 136.15
(CH-Ar), 119.72 (C-Ar), 114.08 (CH-Ar), 70.64 (CH), 58.35 (CH), 55.26 (CH), 33.39 (CH2-cyclohexyl),
33.07 (CH2-cyclohexyl), 25.33 (CH2-cyclohexyl), 24.72 (CH2-cyclohexyl), 19.34 (CH3), 18.77 (CH3). MS
(ESI): m/z = found 755.0 [M+-1]; calcd. 756.24[M+]; HRMS calcd. for C34H48N10Se2 [M++Na]: 779.2298,
PT
found 779.22847[M++Na].
4,4'-Diselanediylbis(N-(1-(1-benzyl-1H-tetrazol-5-yl)-2-methylpropyl)aniline) (18)
RI
SC
mmol) and (isocyanomethyl)benzene (2d) (304 µL, 2.5 mmol). Its formation was monitored by TLC
petrol ether: ethyl acetate = 4:1, Rf = 0.31, purified by column chromatography on silica gel with petrol
U
ether: ethyl acetate= 3:1. Yield: 633 mg (82 %). 1H NMR (400 MHz, CDCl3) δ 7.40 – 7.29 (m, 6H, Ar-
AN
H), 7.28 – 7.09 (m, 8H, Ar-H), 6.27 (dd, J = 8.5, 3.2 Hz, 4H, Ar-H), 5.74 – 5.47 (dd, J = 8.5, 3.2 Hz,4H,
2CH2),4.60 – 4.46 (m, 2H, 2CH), 4.30 (d, J = 8.4 Hz, 2H, 2CH), 2.11 (s, 2H, 2NH), 1.12 (d, J = 6.7 Hz,
6H, 2CH3), 0.93 (d, J = 6.7 Hz, 6H, 2CH3).13C NMR (101 MHz, CDCl3) δ 155.64 (C-tetrazol), 146.70 (C-
M
Ar), 135.89 (CH-Ar), 133.29 (C-Ar), 129.21 (CH-Ar), 129.00 (CH-Ar), 127.49 (CH-Ar), 119.55 (C-Ar),
113.99 (CH-Ar), 70.67 (CH), 54.87 (CH), 51.38 (CH2), 32.93 (CH-isopropyl), 19.25 (CH3), 18.72 (CH3).
D
MS (ESI): m/z = found 795.3 [M++Na]; calcd. 795.18[M++Na].

TE
4,4'-Diselanediylbis(N-((1-(2,4-dimethoxybenzyl)-1H-tetrazol-5-yl)(p-tolyl)methyl)aniline) (19)
EP
mmol) and 1-(isocyanomethyl)-2,4-dimethoxybenzene) (2f) (443 mg, 2.5 mmol). Its formation was
C
1
silica gel with petrol ether: ethyl acetate= 3:1. Yield: 870 mg (88 %). H NMR (400 MHz, CDCl3) δ
AC
7.27 – 7.21 (m, 4H, Ar-H), 7.14 (dq, J = 16.8, 8.4 Hz, 8H, Ar-H), 7.06 – 7.00 (m, 2H, Ar-H),
6.47 – 6.40 (m, 4H, Ar-H), 6.38 – 6.29 (m, 4H, Ar-H), 5.87 (s, 2H, 2CH), 5.42 (s, 4H, 2CH2),
4.99 (s, 2H, 2NH), 3.80 (s, 6H, 2CH3), 3.75 (s, 6H, 2CH3), 2.31 (s, 6H, 2CH3).13C NMR (101
MHz, CDCl3) δ 161.78 (O-C-Ar), 157.77 (O-C-Ar), 155.04 (C-tetrazol), 146.22 (C-Ar), 138.73
(C-Ar), 135.79 (CH-Ar), 133.70 (C-Ar), 131.09 (CH-Ar), 129.82 (CH-Ar), 127.25 (CH-Ar),
121.54 (CH-Ar), 119.32 (C-Ar), 113.91 (CH-Ar), 113.45 (C-Ar), 104.89 (CH-Ar), 98.72 (CH-
26
ACCEPTED MANUSCRIPT
Ar), 55.46 (CH), 55.46 (H3C-O), 52.34 (H3C-O), 45.93 (CH2), 21.08 (CH3-Ar). MS (ESI): m/z =
found 989.8 [M++1]; calcd. 888.22[M+]; HRMS calcd. for C48H48N10O4Se2 [M++Na]:
1011.2098, found 1011.20776[M++Na].
PT
2-((4-(((1-(tert-Butyl)-1H-tetrazol-5-yl)methyl)amino)phenyl)selanyl)-3-methylnaphthalene-1,4-dione
(20)
RI
Compound 20 was synthesized according to the general procedure II from2-methyl-3-bromo-l,4-
naphthoquinone (5) (552.2 mg, 2.2 mmol), compound 7 ( 620 mg, 1 mmol), NaBH4 (189.15 mg, 5 mmol)
SC
and tricaprylmethylammonium chloride (45 mg, 5 % mol). Its formation was monitored by TLC petrol
U
– 7.63 (m, 2H, Ar-H), 7.49 – 7.43 (m, 2H, Ar-H), 6.68 – 6.59 (m, 2H, Ar-H), 4.93 (s, 1H, NH), 4.62 (s,
2H, CH2), 2.16 (s, 3H, CH3), 1.72 (s, 9H, 3CH3).13C NMR (101 MHz, CDCl3) δ 182.46 (C=O, quinone),
AN
181.96 (C=O, quinone), 151.55 (C-tetrazol), 148.45 (C-Ar), 147.74 (C-quinone), 146.86 (C-quinone),
136.29 (CH-Ar), 133.35 (CH-Ar), 132.25 (C-quinone), 132.02 (C-quinone), 126.92 (CH-Ar), 126.63
M
(CH-Ar), 116.86 (C-Ar), 114.04 (CH-Ar), 61.43 (C-t-butyl), 39.95 (CH2), 29.74 (CH3-t-butyl), 17.23
(CH3-quinone). MS (ESI): m/z = found 504.3 [M++Na]; calcd. 504.10[M++Na]; HRMS calcd. for
C23H23N5O2Se [M++Na]: 504.0898, found 504.0909[M++Na].
D
TE
2-((4-(((1-(tert-Butyl)-1H-tetrazol-5-yl)(p-tolyl)methyl)amino)phenyl)selanyl)-3-methylnaphthalene-1,4-
dione (21)
EP

naphthoquinone (5) (552.2 mg, 2.2 mmol), compound 8 ( 800 mg, 1 mmol), NaBH4 (189.15 mg, 5mmol)
C
AC
(ddd, J = 12.2, 5.7, 3.7 Hz, 2H, Ar-H), 7.38 (d, J = 8.6 Hz, 2H, Ar-H), 7.17 (q, J = 8.2 Hz, 4H, Ar-H),
6.54 (d, J = 8.6 Hz, 2H, Ar-H), 6.05 (s, 1H, CH), 2.32 (s, 3H, CH3), 2.12 (s, 3H, CH3), 1.68 (s, 9H).13C
NMR (101 MHz, CDCl3) δ 182.51 (C=O), 181.95 (C=O), 154.79 (C-tetrazol), 148.58 (C-Ar), 148.16 (C-
quinone), 147.69 (C-Ar), 146.02 (C-quinone), 136.24 (C-Ar), 133.53 (C-Ar), 133.56 (CH-Ar), 133.36
(CH-Ar), 132.25 (C-quinone), 132.05 (C-quinone), 129.88 (CH-Ar), 129.22 (CH-Ar), 127.63 (CH-Ar),
27
ACCEPTED MANUSCRIPT
127.09 (CH-Ar), 114.72 (CH-Ar), 65.29 (C-t-butyl), 54.05 (CH), 39.90 (CH3-t-butyl), 21.12 (CH3-Ar),
17.26 (CH3-quinone).
2-((4-(((1-(tert-Butyl)-1H-tetrazol-5-yl)(furan-2-yl)methyl)amino)phenyl)selanyl)-3-methylnaphthalene-
1,4-dione (22)
PT
naphthoquinone (5) (552.2 mg, 2.2 mmol), compound 12 (752 mg, 1 mmol), NaBH4 (189.15 mg, 5 mmol)
RI
SC
– 7.63 (m, 2H, Ar-H), 7.47 – 7.30 (m, 4H, Ar-H), 6.63 (dd, J = 11.9, 8.7 Hz, 2H, Ar-H), 6.33 (s, 1H, CH),
6.22 (dd, J = 7.5, 4.2 Hz, 2H, Ar-H), 2.17 (s, 3H, CH3), 1.76 (s, 9H, 3CH3).13C NMR (101 MHz, CDCl3)
U
δ 182.51 (C=O, quinone), 181.93 (C=O, quinone), 153.26 (C-tetrazol), 150.58 (C-furan), 147.53 (C-Ar),
AN
146.11 (C-quinone), 145.61 (C-Ar), 142.94 (C-quinone), 136.21 (CH-Ar), 133.60 (CH-Ar), 133.39 (C-
quinone), 132.04 (C-quinone), 127.36 (CH-Ar), 126.96 (CH-Ar), 126.60 (CH-Ar), 114.94 (CH-Ar),
110.95 (CH-Ar), 109.05 (CH-Ar), 62.35 (C-t-butyl), 48.52 (CH), 30.92 (CH3-t-butyl), 17.29 (CH3-
M
quinone). MS (ESI): m/z = found 570.2 [M++Na]; calcd. 570.11[M++Na]; HRMS calcd. for C27H25N5O3Se
[M++Na]: 570.0998, found 570.1015[M++Na].
D
Acknowledgements
TE
The authors thank the Egyptian Ministry of Higher Education, Deutscher Akademischer
Austauschdienst (DAAD), Leibniz Institute of Plant Biochemistry and Mansoura University for
EP
financial support.
Reference List
C
[1] S. Dandapani, A.R. Germain, I. Jewett, S. le Quement, J.C. Marie, G. Muncipinto, J.R.
AC
Duvall, L.C. Carmody, J.R. Perez, J.C. Engel, J. Gut, D. Kellar, J.L. Siqueira-Neto, J.H.
McKerrow, M. Kaiser, A. Rodriguez, M.A. Palmer, M. Foley, S.L. Schreiber, B. Munoz,
Diversity-oriented synthesis yields a new drug lead for treatment of Chagas disease, ACS Med.
Chem. Lett. 5 (2013) 149-153.
[2] S. Dandapani, E. Comer, J.R. Duvall, B. Munoz, Hits, leads and drugs against malaria
through diversity-oriented synthesis, Future Med. Chem. 4 (2012) 2279-2294.
28
ACCEPTED MANUSCRIPT
[3] I. Akritopoulou-Zanze, Isocyanide-based multicomponent reactions in drug discovery, Curr.

Opin. Chem. Biol. 12 (2008) 324-331.
[4] A. Domling, Recent developments in isocyanide based multicomponent reactions in applied

chemistry, Chem. Rev. 106 (2006) 17-89.
[5] F. Barreto Ade, O.E. Vercillo, L.A. Wessjohann, C.K. Andrade, Consecutive isocyanide-
PT
based multicomponent reactions: synthesis of cyclic pentadepsipeptoids, Beilstein J. Org. Chem.
10 (2014) 1017-1022.
RI
[6] R.A. Neves Filho, S. Stark, B. Westermann, L.A. Wessjohann, The multicomponent
approach to N-methyl peptides: total synthesis of antibacterial (-)-viridic acid and analogues,
Beilstein J. Org. Chem. 8 (2012) 2085-2090.
SC
[7] F. Barreto Ade, O.E. Vercillo, M.A. Birkett, J.C. Caulfield, L.A. Wessjohann, C.K. Andrade,
Fast and efficient microwave-assisted synthesis of functionalized peptoids via Ugi reactions,
Org. Biomol. Chem. 9 (2011) 5024-5027.
U
[8] W. Brandt, T. Herberg, L. Wessjohann, Systematic conformational investigations of peptoids
AN
and peptoid-peptide chimeras, Biopolymers. 96 (2011) 651-668.
[9] M.C. Brauer, R.A. Neves Filho, B. Westermann, R. Heinke, L.A. Wessjohann, Synthesis of
M
antibacterial 1,3-diyne-linked peptoids from an Ugi-4CR/Glaser coupling approach, Beilstein J.

Org. Chem. 11 (2015) 25-30.
[10] S. Shaaban, B.F. Abdel-Wahab, Groebke-Blackburn-Bienayme multicomponent reaction:

D
emerging chemistry for drug discovery, Mol. Divers. 20 (2015) 233-54.

TE
[11] C. Kalinski, M. Umkehrer, S. Gonnard, N. Jäger, G. Ross, W. Hiller, A new and versatile
Ugi/SNAr synthesis of fused 4,5-dihydrotetrazolo[1,5-a]quinoxalines, Tetrahedron Lett. 47
(2006) 2041-2044.
EP
[12] S.J. Welsch, M. Umkehrer, C. Kalinski, G. Ross, C. Burdack, J. Kolb, M. Wild, A. Ehrlich,
L.A. Wessjohann, Synthesis of substituted imidazolines by an Ugi/Staudinger/aza-Wittig
sequence, Tetrahedron Lett. 56 (2015) 1025-1029.
C
[13] F. Leon, D.G. Rivera, L.A. Wessjohann, Multiple multicomponent macrocyclizations

AC
including bifunctional building blocks (MiBs) based on Staudinger and Passerini three-
component reactions, J. Org. Chem. 73 (2008) 1762-1767.
[14] L.A. Wessjohann, D.G. Rivera, F. Coll, Synthesis of steroid-biaryl ether hybrid macrocycles
with high skeletal and side chain variability by multiple multicomponent macrocyclization
including bifunctional building blocks, J. Org. Chem. 71 (2006) 7521-7526.
[15] S.J. Welsch, C. Kalinski, M. Umkehrer, G. Ross, J. Kolb, C. Burdack, L.A. Wessjohann,
Palladium and copper catalyzed cyclizations of hydrazine derived Ugi products: facile synthesis
29
ACCEPTED MANUSCRIPT
of substituted indazolones and hydroxytriazafluorendiones, Tetrahedron Lett. 53 (2012) 2298-

2301.
[16] P. Pramitha, D. Bahulayan, Stereoselective synthesis of bio-hybrid amphiphiles of coumarin

derivatives by Ugi–Mannich triazole randomization using copper catalyzed alkyne azide click
chemistry, Bioorg. Med. Chem. Lett. 22 (2012) 2598-2603.
PT
[17] M. Oikawa, M. Ikoma, M. Sasaki, Parallel synthesis of tandem Ugi/Diels–Alder reaction
products on a soluble polymer support directed toward split-pool realization of a small molecule
library, Tetrahedron Lett. 46 (2005) 415-418.
RI
[18] M.K. Sinha, K. Khoury, E. Herdtweck, A. Dömling, Tricycles by a New Ugi Variation and
Pictet-Spengler Reaction in One Pot, Chem. Eur. J. 19 (2013) 8048-8052.
SC
[19] Z. Xu, M. Ayaz, A.A. Cappelli, C. Hulme, General one-pot, two-step protocol accessing a
range of novel polycyclic heterocycles with high skeletal diversity, ACS Comb. Sci. 14 (2012)
460-464.
U
[20] C.R. Rhoden, D.G. Rivera, O. Kreye, A.K. Bauer, B. Westermann, L.A. Wessjohann, Rapid
AN
access to N-substituted diketopiperazines by one-pot Ugi-
4CR/deprotection+activation/cyclization (UDAC), J. Comb. Chem. 11 (2009) 1078-1082.
M
[21] N. A. Abbas, S. Shaaban, H. E. Gaffer, E. Abdel-latif, Synthesis and antitumor activity of

some new symmetrical diselenide derivatives, Research Journal of Pharmaceutical, Biological
and Chemical Sciences. 6 (2015) 1655-1664.
D
[22] S. Shabaan, L.A. Ba, M. Abbas, T. Burkholz, A. Denkert, A. Gohr, L.A. Wessjohann, F.
Sasse, W. Weber, C. Jacob, Multicomponent reactions for the synthesis of multifunctional agents
TE
with activity against cancer cells, Chem. Commun. (Camb). 31 (2009) 4702-4704.
[23] S. Shaaban, E.H. Gaffer, M. Alshahd, S.S. Elmorsy, Cytotoxic symmetrical

EP
thiazolediselenides with increased selectivity against MCF-7 breast cancer cells, International
Journal of Research and Development in Pharmacy & Life Sciences. 4 (2015) 1654-1668.
[24] S. Shaaban, E.H. Gaffer, Y. Jabar, S.S. Elmorsy, Cytotoxic naphthalene based-symmetrical
C
diselenides with increased selectivity against MCF-7 breast cancer cells, International Journal of
Pharmacy. 5 (2015) 738-746.
AC
[25] S. Shaaban, A. Negm, M.A. Sobh, L.A. Wessjohann, Organoselenocyanates and

symmetrical diselenides redox modulators: Design, synthesis and biological evaluation, Eur. J.
Med. Chem. 97 (2015) 190-201.
[26] B. F. Abdel-Wahab, S.Shaaban, Thiazolothiadiazoles and Oxazolothiadiazoles: Synthesis

and Biological Applications. Synthesis. 46 (2014) 1709-1716.
30
ACCEPTED MANUSCRIPT
[27] S. Shaaban, F. Sasse, T. Burkholz, C. Jacob, Sulfur, selenium and tellurium pseudopeptides:
Synthesis and biological evaluation, Bioorg. Med. Chem. 22 (2014) 3610-3619.
[28] S. Shaaban, M.A. Arafat, H.E. Gaffer, W.S. Hamama, Synthesis and anti-tumor evaluation
of novel organoselenocyanates and symmetrical diselenides dyestuffs, Der Pharma Chemica. 6
(2014) 186-193.
PT
[29] S. Shaaban, R. Diestel, B. Hinkelmann, Y. Muthukumar, R.P. Verma, F. Sasse, C. Jacob,
Novel peptidomimetic compounds containing redox active chalcogens and quinones as potential
anticancer agents, Eur. J. Med. Chem. 58 (2012) 192-205.
RI
[30] S. Shaaban, Synthesis and biological activity of multifunctional sensor/effector catalysts,
(2011), PhD Dissertation, http://scidok.sulb.uni-saarland.de/frontdoor.php.
SC
[31] S. Mecklenburg, S. Shaaban, L.A. Ba, T. Burkholz, T. Schneider, B. Diesel, A.K. Kiemer,
A. Roseler, K. Becker, J. Reichrath, A. Stark, W. Tilgen, M. Abbas, L.A. Wessjohann, F. Sasse,
C. Jacob, Exploring synthetic avenues for the effective synthesis of selenium- and tellurium-
U
containing multifunctional redox agents, Org. Biomol. Chem. 7 (2009) 4753-4762.
AN
[32] F.H. Fry, C. Jacob, Sensor/effector drug design with potential relevance to cancer, Curr.
Pharm. Des. 12 (2006) 4479-4499.
M
[33] V. Jamier, L.A. Ba, C. Jacob, Selenium- and tellurium-containing multifunctional redox
agents as biochemical redox modulators with selective cytotoxicity, Chemistry. 16 (2010)
10920-10928.
D
[34] M. Abbas, L.A. Wessjohann, Direct synthesis of sensitive selenocysteine peptides by the
Ugi reaction, Org. Biomol. Chem. 10 (2012) 9330-9333.
TE
[35] L.A. Wessjohann, A. Schneider, G.N. Kaluderovic, W. Brandt, Solid-phase synthesis of

reduced selenocysteine tetrapeptides and their oxidized analogs containing selenenylsulfide
EP
eight-membered rings, Mol. Divers. 17 (2013) 537-545.
[36] M. Abbas, J. Bethke, L.A. Wessjohann, One pot synthesis of selenocysteine containing
peptoid libraries by Ugi multicomponent reactions in water, Chem. Commun. (Camb). 5 (2006)
C
541-543.
AC
[37] L.A. Wessjohann, U. Sinks, Benzeneselenenyl Reagents in Organic Synthesis, J. Prak.

Chem-Chem-Ztg. 340 (1998) 189-203.
[38] B. Boualy, S. El Houssame, L. Sancineto, C. Santi, M.A. Ali, H. Stoeckli-Evans, L. El

Firdoussi, A mild and efficient method for the synthesis of a new optically active diallyl selenide
and its catalytic activity in the allylic chlorination of natural terpenes, New J. Chem. 40 (2016)
3395-3399.
31
ACCEPTED MANUSCRIPT
[39] L. Savegnago, M. do Sacramento, L.M. Brod, M.G. Fronza, N. Seus, E.J. Lenardão, M.W.
Paixão, D. Alves, Phenylselanyl-1 H-1, 2, 3-triazole-4-carbonitriles: synthesis, antioxidant
properties and use as precursors to highly functionalized tetrazoles, RSC Advances. 6 (2016)
8021-8031.
[40] L. Wolf, N. Quoos, J.C. Mayer, D. de Souza, A.C. Sauer, L. Meichtry, V. Bortolotto, M.
Prigol, O.E. Rodrigues, L. Dornelles, Synthesis and free radical scavenging activity of 2-
PT
alkyl/arylchalcogenyl-N-(4-aryl-1, 3-thiazol-2-yl) acetamides compounds. Tetrahedron Lett. 57
(2016) 1031-1034.
RI
[41] I. Yavari, S. Mosaferi, Synthesis of 1, 3, 5-Triazepineselone Derivatives from Acyl
Isoselenocyanates and Benzene-1, 2-diamine, Helv. Chim. Acta. 99 (2016) 130-132.
SC
[42] J.H. Lee, C. Kim, M.S. Park, Selenium-containing Bis (alkylselanyl) pyridazines: Synthesis,
and Evaluation of Antiproliferative Activities, Bull. Korean Chem. Soc. 37 (2016) 234-237.
[43] L.V. Myznikov, A. Hrabalek, G.I. Koldobskii, Drugs in the tetrazole series. (Review),
U
Chemistry of Heterocyclic Compounds. 43 (2007) 1-9.
AN
[44] W.H. Song, M.M. Liu, D.W. Zhong, Y.L. Zhu, M. Bosscher, L. Zhou, D.Y. Ye, Z.H. Yuan,
Tetrazole and triazole as bioisosteres of carboxylic acid: discovery of diketo tetrazoles and
diketo triazoles as anti-HCV agents, Bioorg. Med. Chem. Lett. 23 (2013) 4528-4531.
M
[45] V.A. Ostrovskii, R.E. Trifonov, E.A. Popova, Medicinal chemistry of tetrazoles, Russian
Chemical Bulletin. 61 (2013) 768-780.
D
[46] O.I. Shmatova, V.G. Nenajdenko, Synthesis of tetrazole-derived organocatalysts via azido-
Ugi reaction with cyclic ketimines, J. Org. Chem. 78 (2013) 9214-9222.
TE
[47] R.A.W. Neves Filho, S. Stark, M.C. Morejon, B. Westermann, L.A. Wessjohann, 4-
Isocyanopermethylbutane-1,1,3-triol (IPB): a convertible isonitrile for multicomponent reactions,
EP
Tetrahedron Lett. 53 (2012) 5360-5363.
[48] W. Brandt, L.A. Wessjohann, The functional role of selenocysteine (Sec) in the catalysis
mechanism of large thioredoxin reductases: proposition of a swapping catalytic triad including a
C
Sec-His-Glu state, Chembiochem. 6 (2005) 386-394.

AC
[49] C. Jakupoglu, G.K. Przemeck, M. Schneider, S.G. Moreno, N. Mayr, A.K. Hatzopoulos,
M.H. de Angelis, W. Wurst, G.W. Bornkamm, M. Brielmeier, M. Conrad, Cytoplasmic
thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development,
Mol. Cell. Biol. 25 (2005) 1980-1988.
[50] K. Fritz-Wolf, S. Kehr, M. Stumpf, S. Rahlfs, K. Becker, Crystal structure of the human
thioredoxin reductase-thioredoxin complex, Nat. Commun. 2 (2011) 383.
32
ACCEPTED MANUSCRIPT
[51] M. Ibrahim, N. Muhammad, M. Naeem, A.M. Deobald, J.P. Kamdem, J.B. Rocha, In vitro
evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of an
organoselenium compound, Toxicol. In. Vitro. 29 (2015) 947-952.
[52] T. Cheeseright, M. Mackey, S. Rose, A. Vinter, Molecular field extrema as descriptors of

biological activity: definition and validation, J. Chem. Inf. Model. 46 (2006) 665-676.
PT
[53] Forge 10.4.2, http://www.cresset-group.com/forge.
[54] L. Wang, Z. Yang, J. Fu, H. Yin, K. Xiong, Q. Tan, H. Jin, J. Li, T. Wang, W. Tang, J. Yin,
RI
G. Cai, M. Liu, S. Kehr, K. Becker, H. Zeng, Ethaselen: a potent mammalian thioredoxin
reductase 1 inhibitor and novel organoselenium anticancer agent, Free Radical Biology and
Medicine. 52 (2012) 898-908.
SC
[55] S. Gromer, L.A. Wessjohann, J. Eubel, W. Brandt, Mutational studies confirm the catalytic
triad in the human selenoenzyme thioredoxin reductase predicted by molecular modeling,
Chembiochem. 7 (2006) 1649-1652.
U
[56] W. Brandt, L.A. Wessjohann, The functional role of selenocysteine (Sec) in the catalysis
AN
mechanism of large thioredoxin reductases: proposition of a swapping catalytic triad including a
Sec-His-Glu state, Chembiochem. 6 (2005) 386-394.
M
[57] T. Sandalova, L. Zhong, Y. Lindqvist, A. Holmgren, G. Schneider, Three-dimensional

structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a
selenocysteine-dependent enzyme, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 9533-9538.
D
[58] J. Koska, V.Z. Spassov, A.J. Maynard, L. Yan, N. Austin, P.K. Flook, C.M. Venkatachalam,
Fully automated molecular mechanics based induced fit protein-ligand docking method, J. Chem.
TE
Inf. Model. 48 (2008) 1965-1973.
[59] V. Jamier, L.A. Ba, C. Jacob, Selenium- and tellurium-containing multifunctional redox
EP
agents as biochemical redox modulators with selective cytotoxicity, Chem. Eur. J. 16 (2010)
10920-10928.
[60] A. Pircher, M. Medinger, J. Drevs, Liver cancer: Targeted future options, World J. Hepatol.
C
3 (2011) 38-44.
AC
[61] D. Trachootham, J. Alexandre, P. Huang, Targeting cancer cells by ROS-mediated

mechanisms: a radical therapeutic approach. Nat. Rev. Drug Discov. 8 (2009) 579-591.
[62] N.M. Giles, G.I. Giles, J.E. Holley, N.J. Gutowski, C. Jacob, Targeting oxidative stress-
related diseases: organochalcogen catalysts as redox sensitizers, Biochem. Pharmacol. 66 (2003)
2021-2028.
33
ACCEPTED MANUSCRIPT
[63] C. Lennicke, J. Rahn, N. Heimer, R. Lichtenfels, L.A. Wessjohann, B. Seliger, Redox

proteomics: Methods for the identification and enrichment of redox-modified proteins and their
applications, Proteomics. 16 (2016) 197-213.
[64] C. Lennicke, J. Rahn, R. Lichtenfels, L.A. Wessjohann, B. Seliger, Hydrogen peroxide -

production, fate and role in redox signaling of tumor cells, Cell. Commun. Signal. 13 (2015) 39.
doi: 10.1186/s12964-015-0118-6
PT
[65] D. Meriane, G. Genta-Jouve, M. Kaabeche, S. Michel, S. Boutefnouchet, Rapid
Identification of Antioxidant Compounds of Genista saharae Coss. & Dur. by Combination of
RI
DPPH Scavenging Assay and HPTLC-MS, Molecules. 19 (2014) 4369-4379.
[66] M. Ibrahim, W. Hassan, J. Anwar, A.M. Deobald, J.P. Kamdem, D.O. Souza, J.B. Rocha, 1-
SC
(2-(2-(2-(1-Aminoethyl)phenyl)diselanyl)phenyl)ethanamine: An amino organoselenium
compound with interesting antioxidant profile, Toxicol. In. Vitro. 28 (2014) 524-530.
[67] S. Shaaban, A. Negm, M.A. Sobh, L.A. Wessjohann, Expeditious Entry to Functionalized
U
Pseudo-peptidic Organoselenide Redox Modulators via Sequential Ugi/SN Methodology,
Anticancer Agents Med. Chem. 16 (2016) 621-632.
AN
[68] A. Mira, E.M. Gimenez, A.D. Bolzan, M.S. Bianchi, D.M. Lopez-Larraza, Effect of thiol
compounds on bleomycin-induced DNA and chromosome damage in human cells, Arch.
M
Environ. Occup. Health. 68 (2013) 107-116.
[69] Q. Wang, K. Cui, O. Espin-Garcia, D. Cheng, X. Qiu, Z. Chen, M. Moore, R.G. Bristow,
W. Xu, S. Der, G. Liu, Resistance to bleomycin in cancer cell lines is characterized by prolonged
D
doubling time, reduced DNA damage and evasion of G2/M arrest and apoptosis, PLoS One. 8
(2013) e82363.
TE
[70] O.I. Aruoma, A. Murcia, J. Butler, B. Halliwell, Evaluation of the antioxidant and
prooxidant actions of gallic acid and its derivatives, J. Agric. Food Chem. 41 (1993) 1880-1885.
EP
[71] P.J. Evans, B. Halliwell, Measurement of iron and copper in biological systems: bleomycin
and copper-phenanthroline assays, Methods Enzymol. 233 (1994) 82-92.
C
[72] S. Krehl, M. Loewinger, S. Florian, A.P. Kipp, A. Banning, L.A. Wessjohann, M.N. Brauer,
R. Iori, R.S. Esworthy, F.F. Chu, R. Brigelius-Flohe, Glutathione peroxidase-2 and selenium
AC
decreased inflammation and tumors in a mouse model of inflammation-associated carcinogenesis

whereas sulforaphane effects differed with selenium supply, Carcinogenesis. 33 (2012) 620-628.
[73] L.A. Wessjohann, A. Schneider, M. Abbas, W. Brandt, Selenium in chemistry and

biochemistry in comparison to sulfur, Biol. Chem. 388 (2007) 997-1006.
[74] K.J. Haselton, R. David, K. Fell, E. Schulte, M. Dybas, K.W. Olsen, S.M. Kanzok,
Molecular cloning, characterization and expression profile of a glutathione peroxidase-like
34
ACCEPTED MANUSCRIPT
thioredoxin peroxidase (TPx) of the rodent malaria parasite Plasmodium berghei, Parasitol. Int.
64 (2015) 282-289.
[75] A. Wendel, M. Fausel, H. Safayhi, G. Tiegs, R. Otter, A novel biologically active seleno-
organic compound--II. Activity of PZ 51 in relation to glutathione peroxidase, Biochem.
Pharmacol. 33 (1984) 3241-3245.
PT
[76] H. Safayhi, G. Tiegs, A. Wendel, A novel biologically active seleno-organic compound--V.
Inhibition by ebselen (PZ 51) of rat peritoneal neutrophil lipoxygenase, Biochem. Pharmacol. 34
(1985) 2691-2694.
RI
[77] A.S. Hodage, P.P. Phadnis, A. Wadawale, K.I. Priyadarsini, V.K. Jain, Synthesis,
characterization and structures of 2-(3,5-dimethylpyrazol-1-yl)ethylseleno derivatives and their
SC
probable glutathione peroxidase (GPx) like activity, Org. Biomol. Chem. 9 (2011) 2992-2998.
[78] B.K. Sarma, D. Manna, M. Minoura, G. Mugesh, Synthesis, structure, spirocyclization

mechanism, and glutathione peroxidase-like antioxidant activity of stable spirodiazaselenurane
U
and spirodiazatellurane, J. Am. Chem. Soc. 132 (2010) 5364-5374.
AN
[79] P. Prabhu, B.G. Singh, M. Noguchi, P.P. Phadnis, V.K. Jain, M. Iwaoka, K.I. Priyadarsini,
Stable selones in glutathione-peroxidase-like catalytic cycle of selenonicotinamide derivative,
Org. Biomol. Chem. 12 (2014) 2404-2412.
M
[80] F. Kumakura, B. Mishra, K.I. Priyadarsini, M. Iwaoka, A Water-Soluble Cyclic Selenide

with Enhanced Glutathione Peroxidase-Like Catalytic Activities, Eur. J. Org. Chem. 2010 (2010)
440-445.
D
[81] V. Nascimento, E.E. Alberto, D.W. Tondo, D. Dambrowski, M.R. Detty, F. Nome, A.L.
TE
Braga, GPx-Like activity of selenides and selenoxides: experimental evidence for the
involvement of hydroxy perhydroxy selenane as the active species, J. Am. Chem. Soc. 134
(2012) 138-141.
EP
[82] V.A. Kachanov, Y.O. Slabko, V.O. Baranova, V.E. Shilova, A.V. Kaminskii, Triselenium
dicyanide from malononitrile and selenium
dioxide. One-pot synthesis of selenocyanates, Tetrahedron letters. 45 (2004) 4461-4463.
C
[83] D. Plano, Y. Baquedano, D. Moreno-Mateos, M. Font, A. Jiménez-Ruiz, J.A. Palop, C.

AC
Sanmartín, Selenocyanates and diselenides: A new class of potent antileishmanial agents, Eur. J.
Med. Chem. 46 (2011) 3315-3323.
[84] D.E. Paglia, W.N. Valentine, Studies on the quantitative and qualitative characterization of
erythrocyte glutathione peroxidase, J. Lab. Clin. Med. 70 (1967) 158-169.
35
ACCEPTED MANUSCRIPT
Figures captions
Figure 1. Synthesis of functionalized diseleno- and selenium-based quinone compounds via
sequential Ugi/SN and herein emphasizes azido-Ugi/SN reactions.
Figurer 2. Aldehydes, isonitriles, azide (“acid”), amine, and quinone used in the azido-Ugi and
subsequent reduction/SN reactions.
PT
Figure 3.. Structures of tetrazole/napthoquinone-based organoselenium redox modulators (6-22).
RI
Figure 4. Field alignment of diselenide intermediates of template molecules, yellow and green
24 and purple and pink 25 in lowest energy conformations; with the positive (red) andnegative
(blue), Van der Waals (yellow), and hydrophobic (orange) field points are represented as balls or
SC
cubes or polygons, while black arrows indicate the essential field points
Figure 5. Field alignment of diselenide forms of the test molecules (silver) on the right over
reference templates 24, 25 on the left in a separate display showing the presence of the same
U
essential field points.
AN
Figure 6. Docked pose of ethaselen 26 at the dimerization interface domain as predicted by
Flexible Docking by Discovery studio 2.5 showing the orientation of selenium atom approaching
Cys 497 and 498 and the other selenylphenyl moiety towards His 472.
M
Figure 7. Docked pose and 3D binding mode of structures 23 (a, b), 21 (c, d) and 23 (e, f)
showing H bonds in green dotted lines and σ-π and π-π interactions as orange lines.
D
Figure 8. Expression levels of Ki-67, Bcl-2, and caspase-8 in Hep-G2 cells after 48 hrs
incubation with 18, 21, 22 and 23 at their respective IC50s compared to untreated cells.
TE
Figure 9. Glutathione peroxidase-like activity assay in µM. min-1 of the investigated compounds.
The reaction was monitored to completion and the reaction rate was linear throughout the entire
EP
time course.
C
AC
36
ACCEPTED MANUSCRIPT
List of Figures
PT
RI
U SC
Figure 1. Synthesis of functionalized diseleno- and selenium-based quinone compounds via
AN
sequential Ugi/SN and herein emphasizes azido-Ugi/SN reactions.
M
D
TE
C EP
AC
Figurer 2. Aldehydes, isonitriles, azide (“acid”), amine, and quinone used in the azido-Ugi and
subsequent reduction/SN reactions.
37
ACCEPTED MANUSCRIPT
PT
RI
U SC
AN
M
D
TE
C EP
AC
Figure 3.. Structures of tetrazole/napthoquinone-based organoselenium redox modulators (6-22).
38
ACCEPTED MANUSCRIPT
N Se
Se N
24
PT
HN
O O
Se N
N Se
O O
RI
NH
25
Figure 4. Field alignment of diselenide intermediates of template molecules, yellow and green
SC
24 and purple and pink 25 in lowest energy conformations; with the positive (red) andnegative
(blue), Van der Waals (yellow), and hydrophobic (orange) field points are represented as balls or
cubes or polygons, while black arrows indicate the essential field points
U
AN
M
D
TE
Figure 5. Field alignment of diselenide forms of the test molecules (silver) on the right over
EP
reference templates 24, 25 on the left in a separate display showing the presence of the same
essential field points.
C
AC
39
ACCEPTED MANUSCRIPT
PT
RI
Figure 6. Docked pose of ethaselen 26 at the dimerization interface domain as predicted by
SC
Flexible Docking by Discovery studio 2.5 showing the orientation of selenium atom approaching
Cys 497 and 498 and the other selenylphenyl moiety towards His 472.
U
AN
M
D
TE
C EP
AC
40
ACCEPTED MANUSCRIPT
(a) (b)
PT
RI
SC
(c) (d)
U
AN
M
D
(e) (f)
TE
C EP
AC
Figure 7. Docked pose and 3D binding mode of structures 23 (a, b), 21 (c, d) and 23 (e, f)
showing H bonds in green dotted lines and σ-π and π-π interactions as orange lines.
41
ACCEPTED MANUSCRIPT
PT
RI
U SC
AN
Figure 8. Expression levels of Ki-67, Bcl-2, and caspase-8 in Hep-G2 cells after 48 hrs
M
incubation with 18, 21, 22 and 23 at their respective IC50s compared to untreated cells.
D
0.05
TE
0.04
Rate (µm. min-1)
0.03
EP
0.02
C
0.01
AC
Figure 9. Glutathione peroxidase-like activity assay in µM. min-1 of the investigated compounds.
The reaction was monitored to completion and the reaction rate was linear throughout the entire
time course.
42
ACCEPTED MANUSCRIPT
List of Tables
Table 2. Scores values for diselenides (7- 19) after alignment over field template reference
compounds 24 and 25.
Compound No of Conformers Molecular similarity Field similarity Shape similarity
PT
7 3 0.709 0.660 0.758
8 28 0.648 0.630 0.665
9 546 0.634 0.577 0.691
RI
10 9 0.694 0.644 0.744
11 829 0.619 0.562 0.677
SC
12 45 0.671 0.645 0.696
13 615 0.633 0.597 0.669
14 132 0.682 0.674 0.689
U
15 1 0.713 0.666 0.761
16 1 0.629 0.581 0.676
AN
17 48 0.700 0.642 0.759
18 149 0.678 0.621 0.734
19 414 0.641 0.610 0.673
M
D
TE
C EP
AC
43
ACCEPTED MANUSCRIPT
Table 2. GIC50 values* in µM of the compounds (6-23) from MTT viability assays of HepG2,
MCF-7 (both cancer) and WI-38 (fibroblast) cell lines. TI = therapeutic index*
Compd. No. MCF-7 HepG2 WI-38 TI
5-FU 3 ± 0.13 8 ± 1.07 4 ± 0.63 0.5
6 a 37 ± 2.31 a >3
PT
7 28 ± 2.07 44 ± 4.58 21 ± 1.34 -
8 62 ± 2.34 46 ± 3.15 41 ± 3.47 -
9 70 ± 4.51 2 ± 0.34 64 ± 2.33 32
RI
10 a a a -
11 a 47 ± 1.98 a >2
SC
12 14 ± 1.35 51 ± 1.03 11 ± 0.97 -
13 a a 88 ± 2.36 -
14 a 36 ± 2.32 83 ± 2.21 2
U
15 a a 68 ± 2.58 -
AN
16 78 ± 1.52 a 56 ± 3.67 -
17 52 ± 1.27 a 43 ± 2.82 -
18 a 10 ± 0.86 a >10
M
19 a a a -
20 42 ± 3.24 33 ± 2.08 48 ± 3.25 1.5
D
21 a 47 ± 3.14 36 ± 2.98 0.8

22 a 9 ± 0.64 5 ± 0.38 0.6
TE
23 34 ± 2.11 53 ± 3.67 29 ± 1.54 >0.5

*
The metabolic activity of the cells was measured after 48h of incubation with different concentrations of the
investigated compounds by means of MTT assay. The IC50 (µM) was determined from the dose-response curves as
EP
the mean of two parallel experiments; 5-Fluorouracil was used as a positive control; aGrowth inhibition ≥ 100 µM
(inactive). TI is the therapeutic index and calculated as follow: IC50WI-38/IC50HepG2.
C
AC
44
ACCEPTED MANUSCRIPT
Table 3. The redox modulation activity of the compounds.

Redox modulation activity
Bleomycin-dependent DNA
Compd. No. DPPH assay
damage assay
PT
Antioxidant
Fold Absorbance
activity %
vitamin Ca 96.5 ± 1.2 1 310 ± 3.8
RI
6 38.6 ± 1.6 0.4 67 ± 0.43
7 44.3 ± 0.9 0.5 93 ± 0.89
SC
8 30.4 ± 1.5 0.3 98 ± 0.93
9 31.8 ± 1.8 0.3 81 ± 0.81
10 30.5 ± 1.1 0.3 77 ± 0.56
U
11 26.3 ± 1.3 0.3 94 ± 0.72
12 24.0± 1.1 0.3 108 ± 1.5
AN
13 18.8 ± 1.5 0.2 81 ± 0.69
14 49.5 ± 1.9 0.5 222 ± 3.9
15 19.4 ± 1.9 0.2 96 ± 1.9
M
16 22.9 ± 1.2 0.3 79 ± 0.98

17 22.9 ± 2.1 0.2 99 ± 1.3
D
18 20.5 ± 1.1 0.2 92 ± 1.7

19 22.1 ± 1.9 0.2 130 ± 2.1
TE
20 69 ± 1.7 0.8 103 ± 1.6

21 93 ± 1.4 1.0 90 ± 1.8
22 95.3 ± 1.6 1.0 138 ± 2.7
EP
a
Ascorbic acid is used as standard for antioxidant in case of DPPH assay while it is used as a reducing
agent in bleomycin-dependent DNA damage assay; Values are means of 3 replicates
C
AC
45
ACCEPTED MANUSCRIPT

PT
Wessjohanne**
RI
a
b
SC
c
d
e
U
Germany.
AN
Highlights
•
M
A series of tetrazole-based diselenides and selenoquinones were synthesized.

• Most of the compounds were selectively more cytotoxic to HepG2 cells.
D
• Some compounds showed good free radical scavenging activity in the DPPH assay.
• Compounds 9, 12, 14, 19 and 21 manifested good GPx catalytic activity.
TE
• Compounds 18, 21, 22 and 23 modulated the levels of caspase-8, Bcl-2 and Ki-67.
C EP
AC
*
**

2016 Eur J Med Chem

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

2016 Eur J Med Chem

Uploaded by

Copyright:

Available Formats

Accepted Manuscript

Combinatorial synthesis, in silico, molecular and biochemical studies of tetrazole-

Saad Shaaban, Amr Negm, Abeer M. Ashmawy, Dalia M. Ahmed, Ludger A.

To appear in: European Journal of Medicinal Chemistry

Received Date: 2 March 2016

Combinatorial Synthesis, In Silico, Molecular and Biochemical Studies of

Combinatorial Synthesis, In Silico, Molecular and Biochemical Studies of

assessed employing 2,2-diphenyl-1-picrylhydrazyl (DPPH), glutathione peroxidase (GPx)-like

Keywords: organoselenium; diselenides; quinone; tetrazole; azido-Ugi reaction; redox

Abbreviations: Diversity-oriented synthesis (DOS), oxidative stress (OS); hepatocellular

2. Results and Discussions

selenocyanates, selenides and diselenides) [25, 37-42].

strategy (Figure 1).

Based on recent findings reported by our group, we envisioned that tetrazole-based

2c) [47], benzylisocyanide (2d), 4-methoxybenzylisocyanide (2e) and 2,4-

Tetrazole-based symmetric diselenides (6–19) were synthesized by the addition of two

equivalents of aldehyde 1 to a methanolic solution of 4 (one equivalent), followed by the

2.2. In silico prediction of biological activity

2.2.1. 3D similarity study:

3D similarity study is performed for a focused library of symmetrical diselenides to figure

reference compounds with known GPx mimetic activity. Compounds (Z)-N-(4-

2.2.2. Docking study:

Many organoselenium compounds were reported to inhibit Thioredoxin Reductase (TrxR) in

(Figure 7 a and b).

2.3. Pharmacology, toxicity and antioxidant profiles

standard colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

2.3.2. Evaluation of caspase-8, Bcl-2 and Ki-67 molecular biomarkers in

apoptotic Bcl-2 proteins were selected to monitor apoptosis induction.

promising starting point for future studies of structural variants.

2.3.3.1. DPPH free radical scavenging assay

which can be followed spectrophotometrically by the increase in absorbance at 532 nm (Table

induce DNA degradation.

Using a modified assay [70,71], as absorbance increased, more bleomycin-Fe3+is converted

organic ebselen was used as the positive control.

even more active, up to 5 fold compared to the GPx mimetic ebselen.

New symmetrical organodiselenides and selenium-based quinones containing tetrazol moiety

4.2.2. Ligands Preparation and Docking

4.3. Biological assays

4.3.2.2. Glutathione peroxidase like activity

subtracting their own absorbance at the used wave length.

4.3.2.3. Bleomycin-dependent DNA damage

4.3.2.4. Detection of caspase-8 protein expression levels

4.3.2.6. Detection of Ki-67 protein expression levels

instructions of the manufacturer.

4.3.2.7. Statistical analysis

4.4. Synthesis and characterization

General procedure I for the preparation of symmetrical diselenide based-tetrazoles derivatives

General procedure II for the preparation of selenoquinone based-tetrazoles (20-22)

To a solution of the isolated azido-Ugi adduct (1 mmol), 2-methyl-3-bromo-l,4-naphthoquinone

ethyl acetate as eluent to give reddish brown solid.

General procedure IIB: one-potsequential azido-Ugi/reduction/SN synthesis of selenoquinone

Upon completion of the azido-Ugi reaction (monitored by TLC), 2-methyl-3-bromo-l,4-

HRMS calcd. for C24H32N10Se2 [M++Na]: 643.0998, found 643.10396 [M++Na].

HRMS calcd. for C40H48N10O4Se2 [M++Na]: 915.2098, found 915.20992[M++Na].

found 907.26106 [M++Na].

MS (ESI): m/z = found 795.3 [M++Na]; calcd. 795.18[M++Na].

Compound 21 was synthesized according to the general procedure II from2-methyl-3-bromo-l,4-

[3] I. Akritopoulou-Zanze, Isocyanide-based multicomponent reactions in drug discovery, Curr.

[4] A. Domling, Recent developments in isocyanide based multicomponent reactions in applied

antibacterial 1,3-diyne-linked peptoids from an Ugi-4CR/Glaser coupling approach, Beilstein J.

[10] S. Shaaban, B.F. Abdel-Wahab, Groebke-Blackburn-Bienayme multicomponent reaction:

emerging chemistry for drug discovery, Mol. Divers. 20 (2015) 233-54.

[13] F. Leon, D.G. Rivera, L.A. Wessjohann, Multiple multicomponent macrocyclizations

of substituted indazolones and hydroxytriazafluorendiones, Tetrahedron Lett. 53 (2012) 2298-