Cytoprotective and antioxidant properties of organic selenides for the myelin-forming cells, oligodendrocytes

Article abstract of DOI:10.1016/j.bioorg.2018.05.019

Here a new series of twenty-one organoselenides, of potential protective activity, were synthesized and tested for their intrinsic cytotoxicity, anti-apoptotic and antioxidant capacities in oligodendrocytes. Most of the organoselenides were able to decrease the ROS levels, revealing antioxidant properties. Compounds 5b and 7b showed a high glutathione peroxidase (GPx)-like activities, which were 1.5 folds more active than ebselen. Remarkably, compound 5a diminished the formation of the oligodendrocytes SubG1 peak in a concentration-dependent manner, indicating its anti-apoptotic properties. Furthermore, based on the SwissADME web interface, we performed an in-silico structure-activity relationship to explore the drug-likeness of these organoselenides, predicting the pharmacokinetic parameters for compounds of interest that could cross the blood-brain barrier. Collectively, we present new organoselenide compounds with cytoprotective and antioxidant properties that can be considered as promising drug candidates for myelin diseases.

Full text of DOI:10.1016/j.bioorg.2018.05.019

Accepted Manuscript
Cytoprotective and antioxidant properties of organic selenides for the myelin-
forming cells, oligodendrocytes
Saad Shaaban, Dominique Vervandier-Fasseur, Pierre Andreoletti, Amira
Zarrouk, Philippe Richard, Amr Negm, Georg Manolikakes, Claus Jacob,
Mustapha Cherkaoui-Malki
PII:
S0045-2068(18)30335-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.05.019
YBIOO 2372
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
31 March 2018
16 May 2018
20 May 2018
Please cite this article as: S. Shaaban, D. Vervandier-Fasseur, P. Andreoletti, A. Zarrouk, P. Richard, A. Negm, G.
Manolikakes, C. Jacob, M. Cherkaoui-Malki, Cytoprotective and antioxidant properties of organic selenides for the
myelin-forming cells, oligodendrocytes, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2
018.05.019
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Cytoprotective and antioxidant properties of organic selenides for the myelin-
forming cells, oligodendrocytes
a,b,c,f *
b
b
a
a,d
Saad Shaaban,
Dominique Vervandier-Fasseur, Pierre Andreoletti, Amira Zarrouk ,
e
f
g
Philippe Richard, Amr Negm, Georg Manolikakes, Claus Jacob and Mustapha Cherkaoui-
a**
Malki
a
Univ. Bourgogne-Franche Comté, Laboratoire BioPeroxIL (Biochimie du Peroxysome, Inflammation et
Métabolisme Lipidique) EA 7270, 21000 Dijon, France.
b
Institut de Chimie Moléculaire de l'Université de Bourgogne, UMR6302, CNRS, Université Bourgogne Franche-
Comté, F-21000 Dijon, France.
c
Organic Chemistry Division, Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhorya
Street, 35516 Mansoura, Egypt.
d
Faculté de Médecine, Laboratoire de Nutrition-Aliments Fonctionnels et Santé Vasculaire (LR12ES05), Monastir
&
Faculté de Médecine, Université de Monastir, Sousse, Tunisia.
e
Biochemistry Division, Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhorya Street,
3
5516 Mansoura, Egypt.
f
Institute of Organic Chemistry and Chemical Biology, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438,
Frankfurt/Main, Germany.
g
Division of Bioorganic Chemistry, School of Pharmacy, Saarland University, Campus B2 1, D-66123 Saarbuecken,
Germany.
ABSTRACT
Here a new series of twenty-one organoselenides, of potential protective activity, were
synthesized and tested for their intrinsic cytotoxicity, anti-apoptotic and antioxidant capacities in
oligodendrocytes. Most of the organoselenides were able to decrease the ROS levels, revealing
*
Corresponding author: Saad Shaaban Tel.: +201010094177; Fax: +20502397900; E-mail address:
dr_saad_chem@mans.edu.eg
*
*
Corresponding author: Mustapha Cherkaoui-Malki, Laboratoire BioPeroxIL EA7270, Univ. Bourgogne-Franche Comté,
Faculté des Sciences Gabriel, 6 Bd Gabriel, 21000 Dijon, France. Tel.: 33380396205; malki@u-bourgogne.fr
1

antioxidant properties. Compounds 5b and 7b showed a high glutathione peroxidase (GPx)-like
activities, which were 1.5 folds more active than ebselen. Remarkably, compound 5a diminished
the formation of the oligodendrocytes SubG1 peak in a concentration-dependent manner,
indicating its anti-apoptotic properties. Furthermore, based on the SwissADME web interface,
we performed an in-silico structure-activity relationship to explore the drug-likeness of these
organoselenides, predicting the pharmacokinetic parameters for compounds of interest that could
cross the blood-brain barrier. Collectively, we present new organoselenide compounds with
cytoprotective and antioxidant properties that can be considered as promising drug candidates for
myelin diseases.
Keywords: Organic selenides; apoptosis; neurodegeneration; oligodendrocytes; multicomponent
reactions; cytoprotective.
Abbreviations: Central nervous system (CNS), 3-(4,5- dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), 7-ketocholesterol (7kc), dichlorofluorescein (DCF),
dihydroethidium (DHE), glutathione peroxidase (GPx); reactive oxygen species (ROS); 2,2-
diphenyl-1-picrylhydrazyl (DPPH); 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid
(
ABTS).
1. Introduction
In the brain, the myelin sheath plays a crucial role for neuron signal transmission and their
1
survival. Such a sheath is built by a multilayered spiral extension of oligodendrocyte cell
2
membranes to wrap the neuron’s axons. Chronic erosion of this myelin sheath leads to
3
demyelination, and disrupting the neuron integrity and the normal axonal function .
Demyelination is largely associated with numerous neurodegenerative diseases (e.g., Parkinson’
and Alzheimer’s diseases) as well as multiple sclerosis and peroxisomal leukodystrophies rare
4
,5
diseases. Thus for the myelination process, oligodendrocyte integrity is central and their
2

dysfunction was also detected in several psychiatric disorders, including autism, schizophrenia,
6
and depression. Oligodendrocytes support the long-term neurons integrity by myelinating their
7
axons. One of the hallmarks of oligodendrocyte dysfunction and apoptosis is their high
susceptibility to oxidative stress (OS), which results from the excess production of reactive
oxygen species (ROS; e.g., hydrogen peroxide, superoxide, and hydroxyl radicals), by cellular
8
aerobic metabolism, particularly from mitochondrial respiration. Oxidative damage, caused in a
variety of neurodegenerative diseases, is mediated by excessive production of ROS, which can
9
be linked to cell lysis or oxidative burst associated to the immune response. This overproduction
occurs when the generation of reactive oxygen species (ROS) exceeds the antioxidant cellular
capacity, including enzymatic (catalase, glutathione peroxidase and superoxide dismutase) and
1
0
non-enzymatic defense systems (glutathione and ascorbic acid). The selenoprotein family,
including glutathione peroxidases, represents one of the first-line defenses against ROS.
Selenoproteins play also a crucial role for neuronal function, as demonstrated by deactivation of
1
1
the tRNA [Ser] (Sec) gene in mice, resulting in extensive neurodegeneration. The second line
of defense is represented by cellular antioxidants, such as glutathione (GSH) and selenium (Se),
the latter for its implication in the active center of several glutathione peroxidases and in the
1
2
catalytic activity of thioredoxin reductase. Depleted states of the reduced form of GSH and
1
3-15
selenium were associated with the progression of neurodegenerative diseases.
This underlines
the overarching importance of an essential dietary supply of Se for mammals. Interestingly, there
seems to be a connection between Se and other antioxidants on the one side, and the role of ROS
16-18
in the pathogenesis of neurodegenerative diseases on the other.
Yeo et al., reported that Se
1
9
antioxidant activity prevented cell apoptosis in an experimental rat spinal cord injury.
Numerous data revealed that Se levels decline in humans with age and that Se might play
3

2
0,21
different roles in the evolution of Alzheimer’s disease.
Se deficiency leads to inhibition of
the normal upregulation of myelin genes in differentiating oligodendrocytes and the progression
2
2
from immature to mature oligodendrocytes as well. Curiously, experiments using animal
models reveal that organoselenium has a better bioavailability and biological activity than
2
3
inorganic Se. Therefore, during the last decade or two, the biological chemistry of selenium
2
4
derivatives has often focused on potential antioxidant and neuroprotective activities, and here
2
5, 26
primarily on organic rather than inorganic selenium agents.
During our research on selenium
derivatives with possible antioxidant and neuroprotective activities, we recently developed
2
7-32
organoselenium compounds with enhanced antioxidant and GPx-like activities.
The
mechanism by which these compounds exert their antioxidant effect is related to the modulation
of the ROS and GSH levels in cancer cells. Interestingly, some of these compounds showed
2
7,
better antioxidant and GPx-like activities than classical antioxidants (e.g., vitamin C, ebselen).
3
0, 32-36
Based on these results, we decided to further direct our research to obtain evidences
regarding the structural requirements underlying the potential cytoprotective/neuroprotective
activities of organic selenides taking the myelin-forming cells, oligodendrocytes as a model of
study.
Different reports on the synthesis of organoselenium compounds have already been
published; however, they generally require the handling of hazard reagents (e.g., KSeCN), harsh
3
7,38
reaction conditions (e.g., strongly acidic or basic), and mostly multistep procedures
.
Therefore, we decided to avoid rather sophisticated synthetic procedures and employ simple,
straight forward and facile routes towards the synthesis of our preliminary organoselenium-based
library.
4

Here, we present the results obtained during extensive in vitro evaluations of the
selenium derivatives in oligodendrocytes. Besides a rational design of synthetic organoselenium
compounds, we have aimed at gaining a deeper insight into the potential use of these compounds
as neuroprotectors in neurodegenerative diseases through the protective effect on
oligodendrocytes, the myelinating cells. For the chemical synthesis, we adopted both multistep
and one-pot synthesis as part of a diversity-oriented approach to achieve the synthesis of the
twenty-one organoselenides. The in vitro evaluations were conducted using immortalized murine
3
9
oligodendrocyte cell lines. Flow cytometry was used to estimate the ROS levels employing the
H2-DCFDA and the DHE assays. Furthermore, the antioxidant potential of these compounds was
further investigated employing different chemical and biochemical assays and the apoptotic/anti-
apoptotic properties of the compounds were detected by flow cytometry follow-up of the sub-G1
peak. Finally, to assess the structure-activity relationship of the synthesized organoselenium
compounds, we determined in silico their physicochemical and pharmacokinetic parameters, and
their drug-likeness as well through the free web tools in SwissADME.
2. Results and Discussions
2.1. Design and syntheses
The last decade has witnessed significant advance in the synthesis of diverse classes of organic
selenides e.g., diselenides, selenocyanates, and seleno-heterocyclic compounds. Although, these
compounds have a pivotal role in human health and disease prevention; they have limited
application in biological systems due to their often toxicity especially if higher concentrations
were administrated. Combining selenium with better physico-chemical properties bioactive
pharmacophores will interactively not only stimulate their corresponding cytotoxicity but will
also foster their bioavailability. Within this context, our design strategy relies on the synthesis of
5

agents in which selenium is combined with different functionalities such as quinone, cyclic
imides, N-substituted maleanilic and succinanilic acids, formamide, acetanilide, acetaldehyde
diethyl acetal, and/or benzyl. Most of these functionalities are commonly found in bioactive
compounds, thus enabling the chemical diversity and increasing polarity.
2
8-35
Based on our recent reports on organoselenium agents
, we envisioned the synthesis of novel
selenium-based primary aromatic amines 2a-d. The amine functionality in turn can give an
access to a large group of agents either via isocyanide-based multicomponent reactions (IMCR)
(
e.g., Passerini, azido-Ugi Passerini, Ugi, and Groebke-Blackburn-Bienaymé reactions) or via the
classical amines reactions (e.g., formylation, acetylation, … etc.).
The key synthons selenoamines 2a-d were synthesized in good yields (up to 91%) via
reduction of the diselenide (1) with sodium tetrahydridoborate (NaBH
4
) followed by subsequent
nucleophilic substitution (S ) reaction with appropriate organic halides (Scheme 1).
N
Chalcogen-bearing isonitriles are not commercially available and usually challenging to
synthesize. In 2009, we reported the synthesis of three chalcogen-based isonitriles, including the
32, 34
tellurium isonitrile for the first time.
Therefore, our preliminary target was to convert the
amines 2a, 2b, 2c, and 2d into the corresponding isonitriles, suitable for further applications in
2
9, 40
IMCR.
employing acetic formic anhydride followed by subsequent dehydration using phosphoryl
chloride (POCl ). In accordance with our aim, we decided to apply the same methodology for the
The synthesis of isocyanides is usually performed in two steps i.e. amine formylation
3
transformation of organoselenium-based amines 2a-d into the corresponding isocyanides.
Unexpectedly, acetanilides 4a-c were invariably obtained instead of the corresponding
formamides. This promoted us to use only formic acid in place of the acetic formic anhydride.
6

Under neat conditions, the desired corresponding formamides were obtained in good yields (up
to 88%).
Scheme 1
The dehydration of formamides was thereafter investigated using POCl
3
. A non-polar
product (eluted with petroleum ether) with the characteristic isonitrile odor was detected by
1
3
TLC; however, the triplet CNMR peak (≈ 156 ppm) corresponding to the isocyanide carbon did
not appear, suggesting that the isocyanide may be unstable. Therefore, we designed a one-pot
synthetic approach based on the use of the in situ formed isocyanide in subsequent isocyanide-
4
0
based MCRs (IMCRs) such as Groebke-Blackburn-Bienaymé and Ugi reactions.
Unfortunately, very poor conversion (˂5%) was constantly observed with subsequent
disappearance of the isocyanide from the reaction mixture (monitored by TLC). Hence, we
concluded, that this result is related to the high reactivity and low stability of the in situ formed
4
1
42
isocyanides. Different dehydrating agents such as cyanuric chloride , triphenylphosphine , 4-
4
ꢀ
44
4ꢁ
toluenesulfonyl chloride , DABCO and methyl N-(triethylammoniumsulfonyl)carbamate
were investigated; however, these reagents turned out to be either low yielding or ineffective
with our seleno-formamides.
Further efforts were then directed to evaluate the reactivity of the novel selenoamines 2a-d
via their reactions with various anhydrides aiming to synthesize selenocyclic imides which we
recently proved to possess enhanced antioxidant activities (e.g., GPx-mimicking and free radical-
8
2-31
scavenging activity).
Amines 2b-c reacted with maleic and succinic anhydrides in dry toluene
at ambient temperature afforded the corresponding N-substituted maleanilic 5a-b and N-
substituted succinanilic 6a-b derivatives in quantitative yields (Scheme 2). In contrast, 2a failed
to react under these conditions and a tar material was formed. This may be attributed to the side
7

reaction(s) such as hydrolysis and sequential condensation and/or polymerization which might
take place at the acetal residue. Furthermore, the resulting N-substituted monoamidic acids 5a-b
and 6a were subjected to acid-catalyzed esterification and the corresponding methyl esters 7a-b
and 8a were obtained in good yields (up to 87%). The esterification of 6b was also performed
under the same conditions as of 6a. Unfortunately, the expected quinone-based methylester was
not obtained; instead we observed a decomposition of 6b. We attributed this behaviour to the
formation of the corresponding hydroquinone (under acidic conditions) and subsequent cleavage
of the amidic bond.
As expected, selenocyclic imides 10a-b were obtained via ring-closure of the corresponding
o
N-substituted succinanilic acids 6a-b with acetic anhydride at 55 C in the presence of sodium
acetate (Scheme 2).
Scheme 2
Unexpectedly, kinetically controlled isomaleimides 9a-b were obtained in good yields (up to
7
7%) instead of the thermodynamically, more stable, maleimides. This was confirmed via
spectral data and X-ray diffraction. A perspective view and atom numbering are given in Figure
and crystal structure determination and refinement parameters are shown in the Experimental
section.
The X-ray single crystal structure of 9a shows a triclinic system with a space group of P-1.
1
The phenyl ring is directly bonded to the imine moiety (-N=). There are three different types of
C-O bonds: C(14)-O(2) single bond with a bond distance of 1.389 Å, C(17)-O(2) single bond
with a bond distance of 1.405 Å, and C(17)-O(2) double bond with a bond distance of 1.195 Å.
Such efficient and clean pathway to isomaleimides synthesis is highly useful and worth further
investigations using a wider arsenal of N-substituted selenomonoamidic acids.
8

Figure 1
2.2. Cytoprotective and antioxidant profiles
2.2.1. Organoselenides effect on the viability of the oligodendrocytes
1
58N and 158JP
Oligodendrocytes play a crucial in the long-term neurons integrity by myelinating their axons
2
, 3
and thus securing the fast signal conduction. Oligodendrocytes are the most vulnerable cells of
the CNS to ROS and there are only very few reports on the cytoprotective/cytotoxic role of
organoselenides on these myelinating cells. It was therefore of interest to investigate the
protective activity of the synthesized organoselenides on two immortalized murine
oligodendrocytes: 158N and 158JP cell lines. The jimpy (158JP) cell line is mutated for the
proteolipid protein PLP/DM20, a predominant myelin protein that may have a role in the
ꢀ
3
maintenance of myelin sheaths. To determine the half maximal inhibitory concentration IC50 of
the organoselenides, a range of concentrations was evaluated on both oligodendrocytes cell lines.
The low cytotoxic concentration will be further selected to evaluate the compounds
corresponding antioxidant and protective properties. Accordingly, MTT assay was used to
evaluate the basal oligodendrocytes cytotoxicity of the twenty-one organic selenides on 158N
4
ꢂ
and 158JP oligodendrocytes using 7-ketocholesterol (7kc) as known cytotoxic positive control.
MTT is metabolized by the mitochondrial enzyme succinate dehydrogenase giving the blue
formazan product. Hence, this assay provides information on viability (and/or cell proliferation)
and mitochondrial metabolism. The concentration producing 50% growth inhibition (IC50, Table
1
) and the maximum concentration without toxicity were estimated for each of the compounds
from the respective dose-response curves.
Table 1
9

As shown in Table 1, most of the compounds did not exhibit any effect on the viability (IC50
≥
100 µM) of 158N and 158JP at the concentration range used, except for the quinoid-based
compounds 6b and 10b and N-substituted maleanilic ester 7a, they showed IC50 values down to 9
and 3µM in 158N cells and 158JP, respectively. Surprisingly, the cytotoxicity of acetanilides 4a
and 4c was more pronounced in the case of 158JP cells compared to 158N cells (IC50 = 0.02 and
1
4 µM, respectively). These compounds may act as pro-oxidants and their toxicity was
significant in 158JP owing to the fact that these cells have higher ROS levels (10-fold) compared
4
7
to 158N, due to the known higher mitochondrial activity and redox status in 158JP. A possible
explanation of acetanilides derivatives cytotoxicity, is may be related to the accumulation of
paracetamol, a well-known cytotoxin, produced during the intracellular xenobiotic metabolism of
4
2
acetanilides.
2.2.2. Assessment of antioxidant profiles of the selenide compounds
Organoselenium compounds are generally good nucleophiles and this fact led to the rational
design of several synthetic organoselenides as antioxidants, chemopreventive, and
36-38, 49,50
neuroprotective agents.
To the best of our knowledge, the underlying mechanism(s) of
action is (are) not fully understood. Organoselenium compounds can either act as pro-oxidants or
antioxidants depending on the redox status of their environment. In normal cells, organic
selenides behave as antioxidants and protect cells from ROS. In contrast, these compounds
5
1-54
switch into pro-oxidants in cells with elevated ROS levels.
Taking into account that the CNS is prone to oxidative damage and in the light of our search
for new neuroprotective agents, our aim is to further investigate the antioxidant potential of the
novel organic selenides employing different biochemical and chemical assays.
10

2
.2.2.1. Effect of organoselenides on oligodendrocytes ROS
production
ROS levels were estimated by flow cytometry in 158N cells treated with different
concentrations (0, 10, 20 and 50 µM) of the organoselenium compounds employing the
commonly used H2-DCFDA and DHE assays for the detection of ROS and of superoxide anions
•−
55,56
(
O
2
), respectively.
The commercial α-Tocopherol (vitamin E) was used as a standard
antioxidant (positive control).
In the H2-DCFDA assay, most of the compounds were able to decrease the ROS levels in
1
58N cells. Interestingly, compounds 2b, 2c, 3b, 4a, 4b, 4c, 5a, 5b, 6b, and 9a showed a
significant decrease in the H2-DCF fluorescence intensity, thus lower ROS production at 10 and
0 µM compared to vitamin E (Table 2). On the other hand, compounds 7a, 8a and 10a and most
2
of the quinone-based organoselenium 7b, 9b, and 10b were able to mediate significant
overproduction of the intracellular ROS levels at all the tested concentrations proposing a pro-
oxidant activity of these compounds (Table 2).
Table 2
Interestingly, the pro-oxidant activity was more pronounced in the case of the quinoid
compounds 2d which showed the highest H2-DCF fluorescence intensity. This is hardly
surprising, as quinones in general and selenium-containing naphthoquinones, in particular, are
well known to generate ROS in a catalytic fashion - and via the reduction of dioxygen in the
presence of a suitable reducing agent.
11

2
.2.2.2. Effect of organoselenium compounds on superoxide radical
.
-
anion (O ) production by oligodendrocytes
2
In living cells and under aerobic conditions, quinones undergo redox cycling with triplet
•
−
oxygen to generate superoxide radical anion (O ). The later causes oxidative DNA damage,
2
5
5
apoptosis and subsequent cell death. This biochemical feature is responsible for most of the
quinone-based drug’s activity. From this perspective, all the selenoquinones were selected for
•−
further estimation of their respective ability to produce O
2
by the DHE assay. The latter is
•−
believed to be a selective “gold standard” O
2
detector. DHE is a lipophilic cell-permeable probe,
•−
55
which is rapidly oxidized by O
2
, to give the fluorescent compound 2-hydroxyethidium. As
shown in Table 3, and in agreement with the literature and our hypothesis of quinones as ROS-
•−
generating substances, all quinoid-based compounds were able to increase the O
2
levels,
compared to vitamin E. Interestingly, compound 2d was among the most active compounds and
•−
was able to induce a dose-dependent increase of O
2
in 158N cells.
Table 3
2.2.3. Anti-apoptotic effect of selenide compounds on the
oligodendrocytes
Apoptosis was estimated by staining fixed 158N cells with PI followed employing flow
cytometry quantification of the sub-G1 peak in the cell cycle. The sub-G1 is indicator of
apoptotic cells and can be identified by a nuclear fragmentation and/or condensation associated
5
7, 58
with an internucleosomal fragmentation of the DNA.
7Kc is known to induce oxidative stress
and trigger apoptosis in 158N murine oligodendrocytes and therefore was used as apoptosis
5
9
promoter and the positive control.
12

Figure 2
To detect apoptosis, cell cycle analysis was performed after treatment of 158N cells with
different concentrations (50, 10, 5 and 1 μM) of organoselenium compounds treated with or
without 7kc. Interestingly, compound 5a was able to diminish the formation of the SubG1 peak
in a concentration-dependent manner, which is an indicator of its anti-apoptotic properties
(
Figure 2). The remaining compounds did not show any effect on the development of the SubG1
peak (data not shown).
Compound 5a is reducing ROS levels, which in essence is an anti-oxidative and hence often
also an anti-apoptotic event. Admittedly, some of the other compounds (e.g., 5b, 6b and 9a) are a
lot better in removing ROS, yet, they may not be as good in crossing membranes. This may be
due to that 5a is probably amphiphilic and hence a good molecule to enter cells, whilst others are
not. Compound 5a is also an antioxidant (albeit a weak one) and may act via its unsaturated
bond. There is another possible mode of action: is an attack of 5a on cysteine proteins via its
unsaturated double bond, and this may result in an upregulation of antioxidant pathways and
hence anti-apoptotic action as well (for instance via Nrf-2). All this is, of course, speculation,
and these results worth further in-depth studies to substantiate the anti-apoptotic effect of 5a.
Induction of anti-apoptotic effect in oligodendrocytes by 5a is a key target for chemotherapeutic
and chemopreventive applications of such a compound, opening the way for further
pharmacological studies.
2.2.4. Glutathione peroxidase-like activity assay
Many organoselenium compounds have long been known to mimic the antioxidant activity of
the GPx selenoenzyme. Among these compounds, the non-toxic seleno-organic drug, ebselen has
been extensively studied because of its superior GPx mimetic activity. Recently, we have
13

reported different selenium-based organic compounds that possess higher catalytic GPx-like
activity than ebselen. In view of the former, the novel organic selenides were evaluated for their
3
0,60
GPx mimicking activity.
The GPx catalyzes the reduction of ROOR, in presence of glutathione, thus protecting cells
ꢂ1-ꢂ3
from oxidative damage by H
2
O
2
.
The reaction takes place at the enzyme catalytic site, which
contains selenium redox center as a selenocysteine amino acid. It is worthwhile to mention that
selenium is an essential cofactor in all the selenocysteine-based GPx family members for the
ꢂ4
manifestation of their corresponding antioxidant activities.
The catalytic GPx mimic activities of the newly synthesized organic-selenides were
5
ꢂ-ꢂ2
evaluated employing the NADPH-reductase coupled assays.
The latter is repeatedly used for
the evaluation of the GPx-like activity of organochalcogens. The assay principle depends on the
reduction of H
2
O
2
by GPx followed by oxidation of glutathione disulfide (GSSG) and NADPH
+
to glutathione (GSH, reduced form) and NADP , respectively. The latter can be
spectrophotometrically detected by a decrease in the absorbance at 340 nm. The known, standard
GPx mimic compound, ebselen is used as the positive control. The background reaction of GSH
with H
2
2
O was measured and the activity of the GPx was calculated after correction. The reaction
was followed till the end and the rate of the reaction was constantly linear (Figure 3).
Figure 3
Interestingly, as shown in Figure 3, the quinoid-based N-substituted maleanilic 5b acid and
its corresponding methyl esters 7b were more active (≈1.5-fold) than ebselen. The rest of the
compounds showed moderate GPx activity, particularly: 2d, 3b, 8a and 9b compounds.
14

2
.2.5. Assessment of antioxidant/pro-oxidant activity: DPPH, ABTS radical
scavenging, and Bleomycin DNA damage assays
Among different assays used for the accounting of the antioxidant activities of organic
compounds, the 2,2-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azinobis-3-
ethylbenzothioazoline-6-sulphonate (ABTS) chemical assays are usually used to evaluate the
radical scavenging activities of natural products, foods (e.g., vegetables, fruits and beverages)
8
ꢃ, ꢀꢄ, 69, 70
and several organoselenium compounds.
Furthermore, the bleomycin-induced DNA
damage assay isconsidered as an accurate method to evaluate the pro-oxidant activity of many
69, 70
food antioxidants and antineoplastic agents.
As presented in Figure S1 and Table S1 (Supporting Information), most of the compounds
showed good-moderate scavenging activity compared to vitamine C. On the other hand,
selenoquinones 2d, 3c, 4c, 5b, 6b, 7b, 9b and 10b showed between 40-60% pro-oxidant
activities, when compared to vitamin C (bleomycin-reducing activity considered as 100%).
These results were in agreement with that obtained from DCF and DHE assays that
selenoquinones have more pro-oxidant properties compared to the rest of organoselenium
compounds. Although the DPPH and the ABTS assays are widely used, they are only in vitro
models and do not necessarily translated into in vivo action, in particular, the ADME factors are
not covered here either.
2.3. In silico structure-activity relationship
Based on the molecular structure, we explored in silico the synthesized organoselenides
drugability by performing a computer-aided structure-activity prediction through the free web
tools in SwissADME, which is a user-friendly interface website (http://www.swissadme.ch.) to
7
1
evaluate several physicochemical and pharmacokinetic parameters. Among these parameters
15

showed in Figure 4: the topological polar surface area (TPSA) based on the summation of
tabulated surface contributions of polar fragments in the compound; the lipophilicity represented
by the n-octanol/water partition coefficient (log Po/w), which is a key physicochemical parameter
in drug discovery; the pharmacokinetic (i.e. gastrointestinal (GI) absorption, blood-brain barrier
(
BBB) permeability), and the synthetic accessibility (SA) score, which ranges from 1 (very easy
synthesis) to 10 (very difficult synthesis). This computation of SA score was implemented by
7
2
Daina et al. in SwissADME interface to assist the medicinal chemist for the identification of
potentially problematic fragments and eventual lead optimization.
Figure 4
In parallel, to compare the structure-activity relationship of the synthesized organoselenide
derivatives, we summarized the obtained experimental results as a heatmap of these compounds
taking into account their chemical phylogeny (Figure 4): where the scalable red color indicates
the negative effect, while the scalable green color reveals the positive effect of the tested
compounds. The heatmap of our experimental results (Figure 4) shows that selenoamines 2a
(
diethoxyethyl selanyl aniline) and 2d (selanyl dihydronaphthalene, dione), derived from
diselinide as a precursor, exhibited a low or a mild cytotoxicity respectively, while selenylaniline
derivatives 2b and 2c revealed a protective effect on oligodendrocytes. It’s noteworthy, that
selenoamines, as selenocystine, induce ROS production and exert their pro-oxidant action
7
3-75
through depletion of the cellular antioxidant glutathione.
Interestingly, compound 2d, which
show a higher cytotoxicity of the 2-serie compounds, presents also strong pro-oxidant and DNA
damage activities and a lower antioxidant property and all 2d derivatives, containing the 2-
methyl-3-selanylnaphthalene-1,4-dione moiety, conserved a significant pro-oxidant and DNA
damage activities. However, among 2d derivatives, three compounds (5b, 7b and 9b) exhibited a
16

protective effect on oligodendrocytes and showed a GPx-like activity, but only compound 5b
revealed antioxidant and anti-apoptotic properties (Figure 4). Several studies reviewed by
7
6
Bhabak and Mugesh described the mechanism of GPx activity of Ebselen and related
…
organoselenium compounds, for which the strength of Se O non-bonded interactions in the
selenenyl sulfides fine-tuned the balance between the antioxidant scavenging and the GPx
activity. Additionally, 7b showed a negative predicted pharmacokinetic to cross the blood-brain
barrier. Structurally, compound 7b is an esterified derivative of the N-substituted monoamidic
acid 5b. Nonetheless, a strong correlation exists between the absorbed fraction and the hydrogen
77
bond descriptors. Here the physicochemical analysis in SwissADME revealed that compound
7
b has additional 4 rotatable bonds and one H-bond donor, which may account for its
78
unfavorable molecular dynamic. Furthermore, TPSA was defined by Ertl et al. as the sum of
77
the surface areas of polar fragments in a molecule. As documented by Hitchcook et al. ,
2
compounds with PSA˂ 90 Å are considered easily absorbable with higher capacity in
2
penetrating cellular membranes, while the preferred range is compounds with PSA˂ 70 Å .
Moreover, substances with high TPSA are more prone to better interact with hydrophobic
environment of their transporter proteins, as shown for the Multidrug resistance-associated
7
9
protein 1.
In line to this, compounds 5b, 6b, and 7b are either equally or above the PSA limit (72.80
2
2
2
Å , 100.54 Å , and 89.54 Å , respectively). Interestingly, compounds 2c, 5a and 7a showed
2
2
2
lower polar surface than the recommended PSA limit (26.02 Å , 66.40 Å , and 55.40 Å
2
respectively). Within this context, the 7a esterified derivative with a lower TPSA (55.40Å ) and
a poor solubility, showed a strong cytotoxicity and a significant GPx activity (Figure 4). While
2
compound 5a which derived from 2c, with a TPSA of 66.40 Å , showed protective and highest
17

anti-apoptotic effects on oligodendrocytes as well as a good antioxidant property, but has a low
GPx activity. Thus, compound 5a presents interesting protective properties for oligodendrocytes
and could become a lead compound in the development of new drug. The pioneer work on drug-
8
0
likeness was setup by Lipinski et al. as a rule-of-five to predict poor bioavailability for a drug
candidate when it contains more than 5 H-bond donors, 10 H-bond acceptors, a molecular weight
superior to 500 and a calculated log Po/w higher than 5. Remarkably, all the substituted
organoselenides that we synthesized show favorable Lipinski rules (with zero violation)
indicative of their likelihood drug-likeness to become an oral drug with respect to bioavailability.
Pharmacokinetically, except compounds 6b and 7b, all the other synthesized organoselenides
would potentially cross the blood-brain barrier. The predicted synthetic accessibility scores range
from 2.6 to 3.8, revealing that these compounds are easily synthesizable. Such prediction is in
agreement with the obtained good experimental synthesis yields ranging from 77% to 91%.
Collectively, the in silico and in cellulo collected data are in favor of additional investigations as
the organotypic cerebellar slice model to study the effects of lead compound on axon
demyelination.
3. Conclusion
In the present work, we synthesized, in good yields, twenty-one novel organoselenium
compounds and evaluated their potential antioxidant and protective activities in silico and in
cellulo on oligodendrocytes. It’s noteworthy that most of the organic selenides demonstrated a
cytoprotective effect in two different oligodendrocyte cell lines, indicating that these compounds
may represent chemoprotective activity. Therefore, the potential antioxidant properties of the
compounds were evaluated employing different well-established biological and chemical assays,
including H2-DCFDA, DHE, DPPH, ABTS, bleomycin DNA damage and GPx-like assays. Our
18

data demonstrated that most of the selenium-based quinones manifested a pro-oxidant activity,
whereas, the rest of the compounds demonstrated antioxidant activity. Within this context,
organoselenium compounds 2c, 2b, 3b, 4a, 4b, 4c, 5a, 5b, 6b, and 9a were able to decrease the
intracellular ROS levels, as measured by H2-DCFDA assay, in oligodendrocytes compared to
vitamin E. On the other hand, selenoquinones 2d, 3c, 7b, 9b and 10b increased the ROS levels in
oligodendrocytes. Furthermore, the apoptotic/anti-apoptotic properties of the compounds were
detected by flow cytometric estimation of the sub-G1 peak in oligodendrocytes treated with the
7
kc apoptosis inducer. Interestingly, a significant anti-apoptotic effect was observed in the case
of compound 5a suggesting the potential protective activity of this compound. Moreover,
compounds 5b and 7b displayed good GPx-like (≈1.5-fold more active than ebselen) which in
turn may also account for their protective actions. These results were further confirmed by the
ABTS and DPPH scavenging activity and the bleomycin dependent DNA damage assay, where
most of the compounds showed good scavenging activity with respect to ascorbic acid and
protected DNA from damage; whereas, selenium-based quinones exhibited low scavenging
activity and caused DNA damage.
Together, the present results point to antioxidant properties of some, non-cytotoxic, organic
selenides resulting from their capacity to reduce ROS generation. These compounds are
considered promising candidates and might provide a new avenue for innovative antioxidant
research into prototype drugs for neurodegenerative disorders.
4. Experimental Section
4.1. Material and methods
19

All chemicals were obtained from Sigma. Solvents were dried prior to use. The spectroscopic
studies were measured at the “Pôle Chimie Moléculaire” de l'Université de Bourgogne
(
PACSMUB)”. The mass of the organic selenides was obtained on an Electro-Spray Ionization
1
Mass Spectrometry (Thermo LTQ Orbitrap XL ESI–MS) and high-resolution mass spectra. H
1
3
(
300.13 MHz) and C (75.5 MHz) NMR spectra were analyzed on Bruker 300 Avance III,
spectrometers. The values of the chemical shifts (δ) are presented in parts per million relative to
tetramethylsilane, using deuterated solvent as an internal standard. All chemicals were purchased
from Sigma e.g., 7-ketocholesterol, and ascorbic acid and vitamin E. More details on the
spectroscopic data can be found in the Supporting Informations. 2-methyl-3-bromo-1,4-
8
1
naphthaoquinone was synthesized according the reported literature method. Compound 1 was
8
2,83, 84
synthesized according to a literature reported method.
4.2.Biological assays
4.2.1. Cytotoxicity assay
Murine oligodendrocytes (158N and 158JP) were seeded at 240,000 cells per 24-well plates, and
were grown using the Dulbecco’s Modified Eagle Medium supplemented with penicillin
antibiotics (1% v/v, streptomycin) and fetal bovine serum (5% v/v) and kept at 37 °C in
5
7-59
atmosphere containing 5% CO
2
. The concentration of 7kc
and vitamin E, and the time of
8
5
treatment were chosen based on data obtained on 7kc treated 158N cells. Organoselenium
compounds were prepared in DMSO and then diluted with DMEM up to 1 mM.
4.2.2. MTT assay
The MTT assay was carried out on 158N and 158JP cells after one day of incubation with 7kc
50µM) different concentrations (0, 1, 10, 20, 50 and 100 µM) of the organic selenides. In live
cells, the MTT is reduced to formazan by the succinate dehydrogenase mitochondrial enzyme.
(
20

The microplate reader Tecan Sunrise (Tecan, Lyon, France) was used for the reading of the
plates at 570 nm. The IC50 values and the maximum concentrations without toxicity were
estimated for each of the compounds from the respective dose-response curves.
4.2.3. H2-DCFDA and DHE assays
•−
ROS overproduction and O
2
and were detected with DCF and DHE, respectively. Cultured
6
cells were treated with trypsin re-suspended in PBS (10 cells/mL) and kept in the dark for half
°
hour at 37 C with H2-DCFDA or DHE at 10 μM and 2 μM and cells were analyzed by flow
5
5,56
cytometry.
The green fluorescence of 2’,7’- dichlorofluorescein resulting from the oxidation
of H2-DCFDA was analyzed by flow cytometry and collected through a 520/10-nm band pass
•−
filter. DHE, a non-fluorescent compound, rapidly oxidized in ethidium under the action of O
2
to give 2-hydroxyethidium that intercalates with DNA giving red fluorescence of ethidium
collected through a 590/10-nm band pass filter using an excitation of 488 nm and an emission
5
5, 56
wavelength of 575 nm.
the data analysis.
Flomax (Partec) or FlowJo (Tree Star Inc.) software were used for
4.2.4. Apoptosis status and detection of Sub-G1 cells
The analysis of the cell cycle can reveal a Sub-G1 peak, which is representative of apoptotic
cells. Cell cycle analysis was detected on 158N cells stained with Propidium Iodide (PI).
Flow cytometric analyses were measured on a Galaxy flow cytometer. Cells were prepared by
re-suspension in cold ethanol (80%, −20 °C) for 2 h. Cells were then washed with phosphate
buffer, and re-suspended in phosphate buffer (300 μL) containing PI (80 μg/mL) and RNase (200
μg/mL) and kept for 1 h at 37°C.PBS 2 mL was then added, and Fluorescence of propidium
iodide was recorded using a 590 ± 10 nm band filter. The percentage of cells in different cell
cycle phase was manually estimated.
21

4.2.5. GPx -like activity
The GPx-kit was purchased from Biodiagnostic (Egypt) and used according to the reported
8
6
method. The sample was prepared by adding assay buffer and NADPH reagent (0.1 ml; 24
µmol Glutathione, 4.8 µmol NADPH and 12 units Glutathione reductase), and organic selenides
(
0.01 ml, 41 µM). H
2
2
O (0.8 mM) was then added and the absorbance was kinetically recorded
every 1 min at wavelength 340 nm over three successive minutes. A340nm/min was calculated.
Ebselen (41 µM) was used as the positive control. For colored organoselenium compounds, their
individual absorbance was subtracted at the used wavelength.
4.2.6. Synthesis and characterization
8
1,84
4
.2.6.1. Synthesis of 2-methyl-3-bromo-l,4-naphthoquinone
2
-Methyl-1,4-naphthoquinone (11 mg, 63 mmol), anhydrous sodium acetate (1.1 mg, 34 mmol)
were mixed together in glacial acetic acid (50 ml). Bromine (2 ml) was then added and the
mixture was allowed to settle in the dark for three days. A yellow crystalline mass formed, which
was removed. The supernatant liquid was poured on to 500 ml of distilled water. The formed
precipitate was filtered off and recrystallized from ethanol.
General procedure Ia: Reduction of 1 and subsequent modification of selenium based-amines
(2a-d)
Compound 1 (1 mmol) and appropriate halo derivative (2.2 mmol) were dissolved in EtOAc (20
ml). Water (20 ml) and N-Methyl-N,N,N-trioctylammonium chloride (Aliquat 336) (45 mg, 5%
mol) were then added. Sodium tetrahydridoborate (189.15 mg, 5 mmol) was added portion wise
over 1h. Then the reaction was stirred for additional 3 h. The organic layer was dried and
evaporated under vacuum. The residue was purified by silica gel chromatography.
22

General procedure Ib: Reduction of 1 and subsequent modification of selenium based-amines
2a-d)
(
Compound 1 (1 mmol) and appropriate halo derivative (2.2 mmol) were dissolved in EtOH (20
ml). Sodium tetrahydridoborate (189.15 mg, 5 mmol) was added portion wise over 0.5 h. Then
the reaction was reflux for additional 3 h. The organic layer was dried and evaporated under
vacuum. The residue was purified by silica gel chromatography.
General procedure IIa: Preparation of N-acetyl derivatives (4a-c)
A mixture of formic acid (1.0-1.2 equivalents) and acetic anhydride (1.5-2.0 equivalents) was
°
heated at 55 C for 2h and then one equivalent of the appropriate amine (2a-d) was added.
Heating was continued for another 2h. After complete consumption of the starting material (as
judged by TLC analysis), the reaction was cooled and poured onto water. The formed
precipitated was collected and purified by silica gel chromatography.
General procedure IIb: Preparation of N-acetyl derivatives (4a-c)
Acetic anhydride (2.0 equivalents) was added to one equivalent of the appropriate amine (2a-d)
°
and the reaction was heated for two hours at 60 C. The reaction was cooled and then poured
onto ice. The formed precipitated was purified by silica gel chromatography.
General procedure III: Preparation of N-formyl derivatives (3a-c)
A solution of the amine (1.0 mmol) and 85% aqueous formic acid (1.2 mmol) was heated at 60
°
C for 2h. After the disappearance of the amine, the reaction was poured on to water and the
crude N-formyl compound formed essentially as a solid product was purified by silica gel
chromatography.
General procedure IV: Preparation of amide-acids (5a-b and 6a-b)
23

To a solution of amine (1 mmol) in dry toluene (5 mL), the appropriate anhydride (1 mmol) was
added and the mixture was stirred at for 3h. The amide-acid precipitate was collected and further
washed with hot-toluene and dried under vacuum. The purity of the amide-acids was satisfactory
for further use. If necessary, further purification using silica gel chromatography was performed.
General procedure V: preparation of cyclic imides (10a-b) and/or isomaleimides (9a-b)
To an appropriate amide-acid (one equivalent) and 100 mg of sodium acetate, acetic anhydride
°
(
3 mL) was added and the mixture was gently heated for 2h at 50-60 C. Ice was added and the
resulting mixture was extracted with CH
2
Cl
2
(200 ml), dried with Mg
2
SO
4
. CH
2
Cl was
2
evaporated and the residue was purified by silica gel chromatography.
General procedure VI: preparation of methyl ester (7a-b and 8a)
An appropriate amide-acid (1 mmol) was suspended in methanol (10 ml) and then conc.H
2
SO
4
(
200 µl) was added and the reaction was stirred at ambient temperature for 8 h. The mixture was
poured onto ice and the formed precipitate was purified by silica gel chromatography.
Crystallization and X-ray data collection of 9a
Colorless single crystals of C17
H
12NO
2
SeBr were obtained by the slow evaporation method. A
3
suitable crystal with dimensions of 0.37 × 0.37 × 0.12 mm was selected, mounted on glass fibers
and diffraction data were collected on a Bruker D8 diffractometer using graphite
monochromated MoK/a radiation (λ = 0.71073 Å). The crystal was kept at 100 K during data
8
7
collection. APEX2 was used for the unit cell determination and intensity data collection. Using
8
8
89
Olex2 , the structure was solved with the ShelXT and structure solution program using Direct
8
9
Methods and refined with the XL refinement package using Least Squares minimisation. For
more details regarding the crystal parameters and data collection statistics for 9a, please refer to
the supporting information.
24

4
.2.6.2. 4-((2,2-Diethoxyethyl)selanyl)aniline (2a)
Compound 2a was prepared following procedure Ib from 1 (344 mg, 1 mmol),
bromoacetaldehyde diethyl acetal (331 μL, 2.2 mmol), Aliquat 336 (45 mg, 5% mol) and sodium
tetrahydridoborate (189.15 mg, 5 mmol). The progress of the product formation was followed by
TLC petroleum ether: EtOAc= 6:1, R
f
= 0.39, purified by column silica gel chromatography with
1
petroleum ether: EtOAc= 6:1.5. Colorless oil; Yield: 258.1 mg (89%). H NMR (300 MHz,
CDCl
3
) δ 7.42 – 7.34 (d, J = 7.37 HZ, 2H, Ar-H), 6.62 – 6.55 (d, J = 6.58, 2H, Ar-H), 4.66 (t, J =
), 3.03 (d, J = 5.7 Hz, 2H, SeCH ), 1.19 (t, J = 7.1
) δ 146.17, 135.90, 117.11, 115.70, 102.30, 61.70,
5
.7 Hz, 1H, CH), 3.67 – 3.46 (m, 4H, 2CH
2
2
1
3
Hz, 6H, 2CH
3
); C NMR (75 MHz, CDCl
3
+
+
3
2.23, 15.23; MS (ESI): m/z = found 312.04 [M +Na]; calcd. 289.06 [M ]; HRMS calcd. for
+
+
C
12
H19NO
2
Se [M +Na]: 312.0498, found 312.04597 [M +Na].
.2.6.3. 4-(Benzylselanyl)aniline (2b)
Compound 2b was prepared following procedure Ib from 1 (344 mg, 1 mmol), benzyl chloride
253 μL, 2.2 mmol), Aliquat 336 (45 mg, 5% mol) and sodium tetrahydridoborate (189.15 mg, 5
mmol). The progress of the product formation was followed by TLC petroleum ether: EtOAc=
4
(
6
6
:1, R
f
= 0.36, purified by column silica gel chromatography with petroleum ether: EtOAc =
1
:1.5. Colorless oil; Yield: 223.55 mg (85%). H NMR (300 MHz, CDCl
3
) δ 7.28 – 7.18 (m, 2H,
Ar-H), 7.14 – 7.08 (m, 2H, Ar-H), 6.94 – 6.81 (m, 2H, Ar-H), 6.50 – 6.39 (m, 2H, Ar-H), 3.85 (s,
1
3
2
1
H, SeCH
2
), 3.62 (s, 2H, NH
2
); C NMR (126 MHz, CDCl
3
) δ 146.68, 138.65, 137.03, 131.32,
+
30.48, 120.33, 116.48, 115.63, 32.51; MS (ESI): m/z = found 263.85 [M +1]; calcd. 263.02
+
+
+
[
M ]; HRMS calcd. for C13
H13NSe [M +1]: 264.02860, found 264.02748 [M +1].
4
.2.6.4. 4-((4-Bromobenzyl)selanyl)aniline (2c)
25

Compound 2c was prepared following procedure Ib from 1 (344 mg, 1 mmol), 4-bromobenzyl
bromide (550 mg, 2.2 mmol), Aliquat 336 (45 mg, 5% mol) and sodium tetrahydridoborate
(
189.15 mg, 5 mmol). The progress of the product formation was followed by TLC petroleum
ether: EtOAc= 6:1, R = 0.36, purified by column silica gel chromatography with petroleum
f
o
1
ether: EtOAc = 6:1.5. White solid; Yield: 310.31 mg (91%); mp 156-158 C. H NMR (300
MHz, DMSO) δ 7.37 – 7.33 (m, 2H, Ar-H), 7.24 – 7.20 (m, 2H, Ar-H), 7.01 – 6.94 (m, 2H, Ar-
1
3
H), 6.59 – 6.55 (m, 2H, Ar-H), 3.90 (s, 2H, SeCH
2
); C NMR (75 MHz, DMSO) δ 146.65,
1
38.63, 137.04, 131.31, 130.45, 120.32, 116.49, 115.61, 32.50; MS (ESI): m/z = found 364.84
+
+
+
[
M +Na]; calcd. 340.93 [M ]; HRMS calcd. for C13H12BrNSe [M +1]: 341.93911, found
+
3
41.93760 [M +1].
4
.2.6.5. 2-((4-Aminophenyl)selanyl)-3-methylnaphthalene-1,4-dione (2d)
Compound 2d was prepared following procedure Ia from 1 (344 mg, 1 mmol), 2-methyl-3-
bromo-l,4-naphthoquinone (550 mg, 2.2 mmol), Aliquat 336 (45 mg, 5% mol) and sodium
tetrahydridoborate (189.15 mg, 5 mmol). The progress of the product formation was followed by
TLC petroleum ether: EtOAc= 4:1.5, R = 0.32, purified by column silica gel chromatography
f
o
1
with petroleum ether: EtOAc= 3:1. Brown solid; Yield: 305.27 mg (89%); mp 162-164 C. H
NMR (300 MHz, DMSO) δ 7.97 (ddt, J = 11.8, 6.9, 3.4 Hz, 2H, Ar-H), 7.87 – 7.77 (m, 2H, Ar-
H), 7.28 – 7.19 (m, 2H, Ar-H), 6.55 – 6.44 (m, 2H, Ar-H), 5.39 (s, 2H, NH
2
), 1.98 (s, 3H, CH );
3
1
3
C NMR (75 MHz, CDCl
3
) δ 181.84, 181.73, 149.10, 147.31, 147.24, 135.69, 134.08, 133.77,
+
1
31.69, 131.65, 126.34, 126.27, 114.65, 111.92, 16.50; MS (ESI): m/z = found 344.95 [M +1];
+
+
calcd. 343.01 [M ]; HRMS calcd. for C17
H
13NO Se [M +Na]: 366.00037, found 365.99930
2
+
[
M +Na].
4
.2.6.6. N-(4-((2,2-Diethoxyethyl)selanyl)phenyl)formamide (3a)
26

Compound 3a was prepared following procedure III from 2a (289 mg, 1 mmol) and 85%
aqueous formic acid (1.2 mmol). The progress of the product formation was followed by TLC
petroleum ether: EtOAc = 8:1, R
f
= 0.40, purified by column silica gel chromatography with
o
1
petroleum ether: EtOAc= 8:1.5. Brown solid; Yield: 259.94 mg (82%); mp 175-177 C. H NMR
(
300 MHz, CDCl
3
) δ 8.66 (s, 1H, CHO), 8.28 (s, 1H, NH), 7.50 – 7.34 (m, 3H, Ar-H), 6.96 –
), 3.00 (d, J =
); C NMR (75 MHz, CDCl ) δ 162.52,
6
5
1
1
C
.88 (m, 1H, Ar-H), 4.62 (td, J = 5.6, 3.0 Hz, 1H, CH), 3.66 – 3.40 (m, 4H, 2CH
2
1
3
.6 Hz, 2H, SeCH
2
), 1.12 (t, J = 7.1 Hz, 6H, 2CH
3
3
59.18, 136.16, 135.92, 134.47, 133.84, 126.57, 125.76, 120.55, 119.28, 102.28, 62.13, 31.64,
+
+
5.22; MS (ESI): m/z = found 339.95 [M +Na]; calcd. 317.05 [M ]; HRMS calcd. for
+
+
13
H19NO
3
Se [M ]: 317.05123, found 199.96062 [M -CH
2
CH(OC
2
H
5
)
2
].
4
.2.6.7. N-(4-((4-Bromobenzyl)selanyl)phenyl)formamide (3b)
Compound 3b was prepared following procedure III from 2c (341 mg, 1 mmol) and 85%
aqueous formic acid (1.2 mmol). The progress of the product formation was followed by TLC
petroleum ether: EtOAc = 8:1, R
f
= 0.36, purified by column silica gel chromatography with
o
1
petroleum ether: EtOAc= 6:1. White solid; Yield: 324.72 mg (88%); mp 158-160 C. H NMR
(
300 MHz, MeOD) δ 8.47 (s, 1H, CHO), 7.56 – 7.38 (m, 5H, Ar-H), 7.13 – 6.97 (m, 3H, Ar-H),
13
4
.11 (s, 2H, SeCH
2
); C NMR (75 MHz, MeOD) δ 167.77, 132.13, 131.46, 127.58, 127.54,
+
+
1
26.51, 116.38, 115.17, 28.02; MS (ESI): m/z = found 391.86 [M +Na]; calcd. 368.93 [M ];
+
+
HRMS calcd. for C14H12BrNOSe [M +Na]: 391.91597, found 391.91555 [M +Na].
4
.2.6.8. N-(4-((3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)selanyl)phenyl)formamide (3c)
Compound 3c was prepared following procedure III from 2d (343 mg, 1 mmol) and 85%
aqueous formic acid (1.2 mmol). The progress of the product formation was followed by TLC
petroleum ether: EtOAc = 8:1, R = 0.36, purified by column silica gel chromatography with
f
27

o
1
petroleum ether: EtOAc= 8:3. Brown solid; Yield: 300.51 mg (81%); mp 168-170 C. H NMR
(
300 MHz, DMSO) δ 10.26 (s, 1H, CHO), 8.27 (d, J = 1.9 Hz, 1H, CHO), 8.04 – 7.97 (m, 2H,
13
Ar-H), 7.85 – 7.80 (m, 2H, Ar-H), 7.52 (t, J = 2.5 Hz, 4H, Ar-H), 2.09 (s, 3H, CH ); C NMR
3
(
75 MHz, DMSO) δ 182.33, 181.52, 160.17, 149.86, 138.32, 134.57, 134.03, 132.25, 126.98,
+
+
1
26.78, 123.89, 120.52, 18.06; MS (ESI): m/z = found394.01 [M +Na]; calcd. 371.01 [M ];
+
+
HRMS calcd. for C18
H
13NO Se [M +Na]: 393.99529, found 393.99469 [M +Na].
3
4
.2.6.9. N-(4-((2,2-diethoxyethyl)selanyl)phenyl)acetamide (4a)
Compound 4a was prepared following procedure IIa from 2a (289 mg, 1 mmol) and acetic
anhydride (2.0 equivalents). The progress of the product formation was followed by TLC
petroleum ether: EtOAc = 4:3, R
petroleum ether: EtOAc= 1:1. Yellow solid; Yield: 294.84 mg (89%); mp 165-167 C. H NMR
300 MHz, CDCl ) δ 8.37 (s, 1H, NH), 7.40 – 7.32 (m, 4H, Ar-H), 4.60 (t, J = 5.6 Hz, 1H, CH),
.63 – 3.37 (m, 4H, 2CH ), 3.01 – 2.93 (m, 2H, CH ), 2.06 (s, 3H, COCH ), 1.10 (t, J = 7.1 Hz,
); C NMR (75 MHz, CDCl ) δ 169.13 (s), 137.42 (s), 133.75 (s), 124.93 (s), 120.70
f
= 0.30, purified by column silica gel chromatography with
o
1
(
3
3
6
2
2
3
1
3
H, 2CH
3
3
(
[
s), 102.29 (s), 62.14 (s), 31.68 (s), 24.37 (s), 15.23 (s); MS (ESI): m/z = found 376.06
+
+
+
M +2Na]; calcd. 331.07 [M ]; HRMS calcd. for C14
H
21NO Se [M ]: 331.0687, found
3
+
3
31.06812 [M ].
4
.2.6.10. N-(4-(Benzylselanyl)phenyl)acetamide (4b)
Compound 4b was prepared following procedure II from 4-(benzylselanyl)aniline 2b (263 mg, 1
mmol) and acetic anhydride (2.0 equivalents). The progress of the product formation was
followed by TLC petroleum ether: EtOAc= 8:1, R
f
= 0.42, purified by column silica gel
chromatography with petroleum ether: EtOAc= 8:1.5. White solid; Yield: 280.6 mg (92%); mp
o
1
1
80-182 C. H NMR (300 MHz, DMSOd ) δ 6.83 – 6.55 (m, 4H, Ar-H), 6.49 – 6.41 (m, 4H,
6
28

13
Ar-H), 4.11 (s, 2H, SeCH
2
), 1.39 (s, 3H, CH
3
); C NMR (75 MHz, MeOD) δ 170.32, 139.06,
1
3
38.17, 134.54, 128.50, 127.87, 126.31, 124.32, 120.06, 31.70, 22.42; MS (ESI): m/z = found
+
+
+
27.94 [M +Na]; calcd. 305.03 [M ]; HRMS calcd. for C15
H
15NOSe [M + Na]: 328.02111,
+
found 328.01998 [M +Na].
4
.2.6.11. N-(4-((3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)selanyl)phenyl)acetamide (4c)
Compound 4c was prepared following procedure II from 2-((4-aminophenyl)selanyl)-3-
methylnaphthalene-1,4-dione 2d (344 mg, 1 mmol) and acetic anhydride (2.0 equivalents). The
progress of the product formation was followed by TLC petroleum ether: EtOAc= 8:1, R
f
= 0.43,
purified by column silica gel chromatography with petroleum ether: EtOAc= 8:3. Brown solid;
o
1
Yield: 288.75 mg (75%); mp 125-127 C. H NMR (300 MHz, DMSO) δ 8.04 – 7.92 (m, 2H,
Ar-H), 7.89 – 7.78 (m, 2H, Ar-H), 7.49 (qd, J = 6.7, 3.4 Hz, 4H, Ar-H), 2.07 (s, 3H, CH ), 2.03
); C NMR (75 MHz, DMSO) δ 182.33, 181.60, 168.89, 149.50, 146.11, 139.51,
34.57, 134.01, 132.16, 126.96, 122.88, 120.29, 24.47, 17.96; MS (ESI): m/z = found 384.83
3
1
3
(
s, 3H, CH
3
1
+
+
+
[
M ]; calcd. 385.02 [M ]; HRMS calcd. for C19
H
15NO Se [M +Na]: 408.01094, found
3
+
4
08,00933 [M +Na].
4
.2.6.12. (E)-4-((4-((4-Bromobenzyl)selanyl)phenyl)amino)-4-oxobut-2-enoic
acid (5a)
Compound 5a was prepared following procedure IV from 4-((4-bromobenzyl)selanyl)aniline 2c
344 mg, 1 mmol) and maleic anhydride (98 mg, 1mmol). Its formation was monitored by TLC
chloroform: methanol = 8:1, R = 0.36, purified by column silica gel chromatography with
chloroform: methanol = 6:1. Yellow solid; Yield: 412.66 mg (94%); mp 215-217 C. H NMR
300 MHz, DMSO) δ 10.45 (s, 1H, COOH), 7.55 (d, J = 8.6 Hz, 2H, Ar-H), 7.46 – 7.37 (m, 4H,
Ar-H), 7.17 (d, J = 8.4 Hz, 2H, Ar-H), 6.45 (d, J = 12.1 Hz, 1H, =CH), 6.30 (d, J = 12.0 Hz, 1H,
(
f
o
1
(
29