Organoselenocyanates and symmetrical diselenides redox modulators: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2015.05.002

Abstract Oxidative stress (OS) and disturbed intracellular redox balance have been predominantly observed in different types of cancer, including hepatocellular carcinoma (HCC). Agents which can stop OS multi-stressor events and modulate the intracellular redox state are becoming a major focus in HCC prevention. Among them, compounds with glutathione peroxidase (GPx)-like activity are of particularly concern. We herein report the synthesis of novel series of organoselenocyanates and symmetrical diselenide antioxidants, inspired by the natural redox enzyme, GPx and the synthetic organoselenium ebselen antioxidants. Their cytotoxic activity was evaluated against Hep G2 cells and their antimicrobial activities were evaluated against Candida albicans (C. albicans) fungus as well as against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), gram-negative and gram-positive bacteria, respectively. These compounds were also tested for their antioxidant activities using 2,2-diphenyl-1-picrylhydrazyl (DPPH), GPx-like activity and bleomycin dependent DNA damage assays and a basic structure-activity relationship was subsequently established. The physicochemical parameters and drug-likeness were computed employing the Molinspiration online property calculation toolkit and MolSoft software. Interestingly, some compounds proved to be more cytotoxic than ebselen and the known anticancer drug 5-Fu and in the same time they showed similar, sometime even more, antifungal activity than the reference antifungal drugs. Among these compounds, compound 16 was considered to be the most interesting with free radical-scavenging activity comparable to ascorbic acid and a GPx-like activity similar to ebselen. As most of these compounds comply with Lipinski's Rule of Five, they promise good bioavailability, which needs to be studied as part of future investigations.

Full text of DOI:10.1016/j.ejmech.2015.05.002

Accepted Manuscript
Organoselenocyanates and symmetrical diselenides redox modulators: Design,
synthesis and biological evaluation
Saad Shaaban, Amr Negm, Mohamed A. Sobh, Ludger A. Wessjohann
PII:
S0223-5234(15)30029-5
10.1016/j.ejmech.2015.05.002
EJMECH 7881
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 November 2014
Revised Date: 25 April 2015
Accepted Date: 2 May 2015
Please cite this article as: S. Shaaban, A. Negm, M.A. Sobh, L.A. Wessjohann, Organoselenocyanates
and symmetrical diselenides redox modulators: Design, synthesis and biological evaluation, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.05.002.
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ACCEPTED MANUSCRIPT
Organoselenocyanates and Symmetrical Diselenides Redox Modulators: Design,
Synthesis and Biological Evaluation
a,d*
b
c
d**
Saad Shaaban, Amr Negm, Mohamed A. Sobh and Ludger A. Wessjohann
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, 35516
Mansoura, Egypt.
c
Experimental Biology, Urology & Nephrology Center, Faculty of Medicine, Mansoura University, El-Gomhorya Street, 35516
Mansoura, Egypt.
d
Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, D-06120 Halle (Saale), Germany
Graphical abstract
OH
HO
O
O
O
Se Se
O
O
N
O
HO
NH
O
O
OH
HN
Se
H
2
N
Se
Se
O
Se
Se
NH
2
N
SOCl
2
Se
Se
O
Se
O
8
1
6
1
1
1
4
2
0
8
6
4
2
0
GPx-like activity
*
Corresponding author. Tel.: +2 1010 094 177; Fax: +20 502 397900.
E-mail address: dr_saad_chem@mans.edu.eg; dr.saad.chem@gmail.com
**
Corresponding author. Tel.: +49 345 5582 1301; fax: +49 345 5582 1309.
E-mail address: wessjohann@ipb-halle.de

ACCEPTED MANUSCRIPT
Organoselenocyanates and Symmetrical Diselenides Redox Modulators:
Design, Synthesis and Biological Evaluation
a,d*
b
c
d**
Saad Shaaban, Amr Negm, Mohamed A. Sobh and Ludger A. Wessjohann
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,
3
5516 Mansoura, Egypt.
c
Experimental Biology, Urology & Nephrology Center, Faculty of Medicine, Mansoura University, El-Gomhorya
Street, 35516 Mansoura, Egypt.
d
Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, D-06120 Halle (Saale),
Germany
ABSTRACT
Oxidative stress (OS) and disturbed intracellular redox balance have been predominantly
observed in different types of cancer, including hepatocellular carcinoma (HCC). Agents which
can stop OS multi-stressor events and modulate the intracellular redox state are becoming a
major focus in HCC prevention. Among them, compounds with glutathione peroxidase (GPx)-
like activity are of particularly concern. We herein report the synthesis of novel series of
organoselenocyanates and symmetrical diselenide antioxidants, inspired by the natural redox
enzyme, GPx and the synthetic organoselenium ebselen antioxidants. Their cytotoxic activity
was evaluated against Hep G2 cells and their antimicrobial activities were evaluated against
Candida albicans (C. albicans) fungus as well as against Escherichia coli (E. coli) and
Staphylococcus aureus (S. aureus), gram-negative and gram-positive bacteria, respectively.
These compounds were also tested for their antioxidant activities using 2,2-diphenyl-1-
picrylhydrazyl (DPPH), GPx-like activity and bleomycin dependent DNA damage assays and a
basic structure-activity relationship was subsequently established. The physicochemical
parameters and drug-likeness were computed employing the Molinspiration online property
calculation toolkit and MolSoft software. Interestingly, some compounds proved to be more
cytotoxic than ebselen and the known anticancer drug 5-Fu and in the same time they showed
*
Corresponding author. Tel.: +2 1010 094 177; Fax: +20 502 397900; E-mail address: dr_saad_chem@mans.edu.eg;
dr.saad.chem@gmail.com
**
Corresponding author. Tel.: +49 345 5582 1301; fax: +49 345 5582 1309; E-mail address: wessjohann@ipb-halle.de
1

ACCEPTED MANUSCRIPT
similar, sometime even more, antifungal activity than the reference antifungal drugs. Among
these compounds, compound 16 was considered to be the most interesting with free radical-
scavenging activity comparable to ascorbic acid and a GPx-like activity similar to ebselen. As
most of these compounds comply with Lipinski’s Rule of Five, they promise good
bioavailability, which needs to be studied as part of future investigations.
Keywords:
ebselen; organoselenocyanates; diselenides; GPx mimics; multicomponent
reactions.
Abbreviations: Oxidative stress (OS); hepatocellular carcinoma (HCC); glutathione peroxidase
GPx); reactive oxygen species (ROS); reactive nitrogen species (RNS); isocyanide based
multicomponent reactions (IMCRs).
(
1
. Introduction
Overproduction of reactive oxygen (ROS) and nitrogen species (RNS) and diminished
antioxidant capacity have been observed in wide range of diseases ranging from
neurodegenerative diseases and inflammation to cancer. The role of antioxidant therapeutics is to
scavenge free radicals, recover the cellular “redox homeostasis” and therefore they are widely
believed to reduce the risk of such diseases. This is where organoselenium compounds come into
play. These compounds attract the continuous interest of chemists and medicinal biologists due
to their largely untapped pharmacological applications. Selenium compounds are being used as
antioxidants, neuroprotectives, nootropics, anxiolytics, antinociceptives and antidepressants.[1,2]
It is noteworthy that not only the amount but also the nature of the selenium containing
compounds is essential for its pharmacological potency, or toxicity. In this context,
organoselenium compounds have been found to be less toxic than some inorganic selenium
compounds. Therefore, there has been a growing interest in the synthesis of organoselenium
compounds.
Symmetrical diorganyl diselenides have become a hot spot in selenium chemistry since they
are stable, easy to prepare and depending on the substituents can have reduced toxicity.[3] Some
compounds play a leading role in reducing the risk of cancer incidence and mortality in many
tumors.[4,5] In this context, various diselenides have shown promising hepatoprotective
properties as they were able to reduce hepatotoxicity induced by heavy metals.[4,6] Considering
2

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the fact that the difference between prevention and treatment are strictly dose dependent,
diselenides in this context can be model for cancer therapy and particularly for HCC.[7]
Recently, we have reported the synthesis of selenium-containing peptoids and peptide-like
compounds employing isocyanide based multicomponent reactions (IMCRs).[8-10] Some of
these compounds were able to induce cell cycle delay, reduction of glutathione levels and
apoptosis via dose-dependent activation of caspase 3 and 7 in melanoma (A-431) and breast
cancer (MCF-7) cells.[7,10-15] Based on these result and in continuation of our previous work
on organoselenium compounds and HCC, we would like to develop further sophisticated
diorganyl diselenides and evaluate their antitumor activity on HCC cells.
Twenty years ago, it was found that ebselen behaved against OS in a similar manner as the
common enzyme GPx (Figure 1).[16-18][19,20] More recently, it was reported that diphenyl
diselenide was able to increase both the cellular GPx expression and showed activity that was
two times higher than that of ebselen.[21] Another important factor is the relatively low toxicity
of diphenyl diselenide which is attributed to its in vivo stability. Recent evidence suggested that
diphenyl diselenide is a good substrate of hepatic and cerebral thioredoxin reductase (TrxR)
proposing another rational for its potent antioxidant action (Figure 1).[22-24] These studies
stimulated the quest for new organic diselenides that would be better soluble, more efficient and
less toxic.
<Figure 1 >
The objective of this study was to synthesize novel series of organoselenocyanates and
symmetrical diselenides with the aim to find out more efficient GPx mimics with a potent anti-
HCC activity.
2
. Results and Discussions
.1. Chemistry
2
Diverse trials were made to enhance ebselen catalytic activity.[25,26] These were only
focused to modulate the substituent effects which in turn have been marred with limited success.
Keeping this in mind and taking in considerations that diphenyl diselenide potentially is more
efficient mimetic of native GPx than ebselen and can be re-reduced by thioredoxin reductases,
our primary objective was to devise a rapid synthesis of modified diphenyl diselenides, ideally
3

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combining beneficial features found in diselenides, ebselen and yet unexplored of
selenocyanates.
The key intermediate 2-(chloroseleno)benzoyl chloride (2), obtained from the reaction of
2
,2`-diselanediyldibenzoic acid (1) with thionyl chloride, possesses two electrophilic centers
suitable for N-nucleophilic substitution.[27] The primary aromatic amines 4-
aminophenylselenocyanate (3) and 4-(2-(4-aminophenyl)diselanyl)benzenamine (4) were
selected as selenium-based key synthons that enable access to symmetrical diselenides. In this
context, the selenenylationacylation of 3 and 4 with 2 afforded the corresponding ebselen
selenocyanate 5 and ebselen diselenide 6 in good yields (71% and 68%, Scheme 1).[9,28,29]
Interestingly, 2,2`-diselanediyldibenzoic acid (1) has not been involved in any isocyanide
based multicomponent reaction (IMCR) before. This promoted us to investigate it in Ugi four
component reaction (U4CR) which in turn would give access to highly functionalized
diaryldiseleno-peptidomimetic scaffolds (Scheme 1), different in stability and redox properties
than selenocystein-based diselenide.
The U4CR comprises the one pot synthesis of α-aminoacyl amides from an aldehyde, amine,
acid and isonitrile.[30] The amine components employed in this exploratory investigation
were tryptamine and benzylamine, whereas in this initial study, tert-butylisocyanide, was chosen
because it is highly reactive and commercially available. Isobutyraldehyde and 4-
methylbenzaldehyde were used as examples of aliphatic and aromatic aldehyde components
(Scheme 1).
<
Scheme 1 >
Further efforts were directed into the preparation of organoselenium cyclic imides which
recently received much attention in drug discovery and development.[31] Organoselenium
cyclic imides have been used for the synthesis of different prodrugs and drug delivery systems,
to estimate the characteristics of dual-acting drugs and to evaluate the receptor-binding with
respect to their relative spatial arrangements.[32][31,33-35]
Cyclic imides form an integral part of different naturally occurring compounds (e.g.,
alkaloids like rebeccamycin) and therapeutically or otherwise biologically interesting compounds
(e.g., thalidomide, chlorophthalim, isogranulatimide) (Figure 1).[36] Among these are herbicidal,
antimicrobial, anti-inflammatory, anticancer and antidepressant compounds.[37] The observed
biological properties usually refer to their ability to inhibit protoporphyrinogen IX oxidase which
4

ACCEPTED MANUSCRIPT
in turn is essential for chlorophyll and heme biosynthesis.[38,39] Furthermore, these compounds
are easily absorbed by cells (in vivo) due to their hydrophobic and neutral nature. Moreover, the
electrophilicity and size of the imide ring substituent(s) are pivotal in determining the bioactivity
via modulation of their steric properties.[40,41]
Regardless of the vast application of N-substituted cyclic imides, only few synthetic
procedures are available to date. Condensation of an amine with anhydrides usually is the typical
method of choice. Herein we report the synthesis of selenium containing cyclic imides by
dehydrative condensation of an anhydride (maleic, succinic or glutaric anhydride) and selenium-
containing
amines
namely:
4-aminophenylselenocyanate
3
and
4-(2-(4-
aminophenyl)diselanyl)benzenamine 4 (Schemes 2 & 3). The later amines 3 and 4 were selected
as selenium-based key synthons that enable access to selenocyanate- and symmetrical diselenide-
containing cyclic imides.[11]
<
Scheme 2 >
The synthesis of selenium-containing maleimides, succinimides and glutarimides was carried
out in two steps. Reactions of maleic, succinic and glutaric anhydrides with 3 and 4 in toluene
and at room temperature afforded the corresponding N-substituted maleanilic (10 and 16),
succinanilic (12 and 18) and glutaranilic acids (14 and 20) in approximately quantitative yields.
Cyclic imides (11, 13, 15, 17, 19 and 21) were obtained from their corresponding monoamidic
acids (10, 12, 14, 16, 18 and 20) via dehydration and subsequent ring-closure up on gentle
heating with acetic anhydride and sodium acetate (Schemes 2 & 3). The reaction was
accomplished in 30 minutes and the product was easily isolated by ice-water precipitation.
Purification was achieved by simple washing with water. If necessary, further purification can be
accomplished by column chromatography.
<
Scheme 3 >
2
.2. Pharmacology, toxicity and antioxidant profiles
2
.2.1. Cytotoxic activity of redox active compounds in Hep G2 cancer cells
The cytotoxic activity of the synthesized compounds was assayed using Hep G2 cells in
order to check if any of them would be useful for in-depth biochemical investigations. It’s worth
noting that Hep G2 cells are a suitable model for the studying of intracellular trafficking,
hepatocarcinogenesis and drug targeting in vitro.[42] Recently, these cells have been employed
5

ACCEPTED MANUSCRIPT
extensively as they exhibit large part of cellular functions similar to those of normal hepatocytes,
such as expression of hepatocyte-specific cell surface receptors and synthesis and secretion of
plasma proteins.[43,44] The cytotoxicity of the compounds was determined using an MTT assay
using 5-fluorouracil (5-Fu) and ebselen as positive control. The IC50 values were estimated from
the respective dose response curves and are summarized in Table 1.
<
Table 1 >
Interestingly, the substitution pattern plays a distinct role in the activity of the diselenides,
showing that it is not general selenium cytotoxicity. The compounds under investigation could be
divided into two classes: 1) cytotoxic compounds, able to reduce the viability of Hep G2 tumor
*
cells (with higher IC than ebselen) and 2) non-cytotoxic compounds. Class 1 compounds were
5
0
further divided into two subclasses based on their extent of cytotoxicity i.e. compounds with
†
good cytotoxicity (IC50 < 8 µM) (10, 11, 16 and 14) and compounds with modest to very low
cytotoxicity (17, 19 and 21).
In culture, cells became rounded, cell layer partially condensed forming cell-free areas, and
cells were detached from the culture plate. On the other hand, compounds falling under the
second class i.e. non-cytotoxic ones (5, 6, 7, 8, 9, 12, 13, 15, 18 and 20) were unable to reduce
the survival of Hep G2 cells at all.
A basic SAR shows a correlation between chemical structures and the cytotoxic activities.
Maleimides (11 and 17) and their corresponding N-substituted maleanilic acids (10 and 16) were
among the most potent cytotoxic compounds compared to their succinimides (13 and 19),
glutarimides (15 and 21), N-substituted succinanilic (12 and 18) and glutaranilic acids (14 and
2
0) analogues.
Contrary to our expectations, ebselen like compounds 5 and 6 showed moderate to low
cytotoxicity. This may be attributed to their poor solubility and lipophilicity. The same holds true
in case of peptidomimetic compounds 7, 8 and 9. The degree of bulkiness and low solubility
together with their high molecular weight and large molecular volumes may affect their action on
various levels, cell penetration and localization or at the receptors/enzymes binding sites.
2
.2.2. Assessment of antioxidant activity
*
†
The cytotoxicity threshold is at IC50 < 14 µM.
Having lower IC50 than 5-Fu i.e. IC50 < 8 µM.
6

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The last decade has witnessed a growing interest in the development of selenium and
selenium- based quinones redox modulators because of their role in the etiology of tumorigenesis
via modulation of oxidative stress (OS). Interestingly, the mechanism of action of
organoselenium compounds depends on their environment, as they have the possibility to
function as antioxidant catalysts in normal cells and pro-oxidants in cells exposed to oxidative
stress. These agents do not themselves change the redox balance, but their activities are
depending upon the cellular redox state in which they are placed.
Mount evidence suggested that regular uptake of antioxidants is required to scavenge ROS
and RNS to reduce the risk of cancer, diabetes as well as aging and neurodegeneration related
diseases. This ultimately accentuates the role of antioxidants in disease prevention.[45,46] The
same holds true for electrophiles (diselenides and selenocyanates are electrophiles, the latter not
unlike isothiocyanates responsible for the broccoli anticancer effect).
As part of our research, we herein aim to evaluate the potency of the newly synthesized
organoselenium compounds as reliable antioxidants. DPPH, GPx-like activity and bleomycin
dependent DNA damage assays were used for the assessment of the compounds antioxidant
activities.
2.2.2.1. DPPH free radical scavenging assay
The DPPH method is a standard to estimate the (chemical) antioxidant property of nutritional
products and chemicals like novel organoselenium compounds. It is one of the most efficient
methods for evaluating the radical-scavenging action by a chain-breaking mechanism.[47,48]
.
This assay is based on the ability of DPPH to be decolorized in the presence of antioxidants. The
corresponding DPPH radical-scavenging activity was determined by the decrease in absorbance
.
.
at 517 nm due to reduction by the antioxidant (AH) or reaction with a radical species:DPPH + R
DPPH-R.
The method is fast, efficient and as being standard, comparative data are abundant. However,
→
one must be aware that chemical antioxidant activity does not necessarily translate into in vivo
action, and that ADME factors are not covered either. The results of the DPPH free radical
scavenging activity compiled in Figure 2.
7

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Compound 16 was the most active compound in this assay with free radical scavenging
activity higher than ebselen and similar activity to ascorbic acid. Furthermore, compounds 10,
1
8, 20, 5, 6, 12 and 17 showed a moderate radical scavenging activity. The rest of the
compounds showed low radical scavenger activity i.e. 8, 7, 9, 11, 13, 14, 15, 19 and 21.
Figure 2>
<
In general, it was found that the amide acids (10, 12, 14, 16, 18 and 20) show considerable
better free radical-scavenging activity more than their corresponding cyclic imides (11, 13, 15,
1
7, 19 and 21). Surprisingly, the corresponding ebselen selenocyanate 5 and ebselen diselenide 6
show only moderate antioxidant activity in this assay.
2.2.2.2. Glutathione peroxidase-like activity assay
GPx probably is the best studied selenoenzyme. In conjunction with its thiol cofactor
glutathione (GSH), it catalyzes the reduction of peroxides and protects cellular components and
lipid membranes against OS. The enzyme catalytic site includes selenium which undergoes a
redox cycle affording the selenol (RSeH) active form that is able to reduce peroxides (hydrogen
peroxides and organic peroxides). It’s also worth mentioning that all the GPx family [classical
cytosolic GPx (cGPx), phospholipid hydroperoxide GPx (PHGPx), plasma GPx (pGPx) and
gastrointestinal GPx (giGPx)] all require selenium at their active sites to exercise their catalytic
activity.[49]
Ever since the initial observations by Wendel et al.[50,51] and Sies et al. that ebselen mimics
the activity of GPx and many organoselenium compounds have been devised as synthetic GPx
mimics.[52][16,53][54,55] This includes benzoselenazolinones, selenenamides, selenosubtilisin
and diaryl diselenides. The latter have recently attracted considerable attention as it behaved as
good mimics of GPx in biological systems in vitro.[56-60][61,62] Therefore we examined novel
symmetrical diselenides and their selenocyanate analogues for their GPx mimicking activity.
Different methods are reported in the literature to estimate catalytic activities of
organoselenium compounds as the GPx mimics, these include high-performance liquid
chromatography (HPLC) and NMR based dithiol-disulfide conversion assays.[63,64]
In this study, we have evaluated the GPx activity by the NADPH-reductase coupled
assay.[65] The principle of this assay relies on the transformation of the oxidized glutathione
8

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(GSSG), which is produced upon reduction of peroxides by GPx, to its reduced state by
+
glutathione reductase. This results in the oxidation of NADPH to NADP which is accompanied
by decrease in the cofactors absorbance at 340 nm providing a spectrophotometric means for
monitoring GPx like activity of the investigated compounds (Figure 3).[65] The values
represented in figure 3 are expressed after background correction for the reaction with H O and
2
2
GSH.
<
Figure 3>
An inspection of the data indicates that compound 16 exhibited the most potent GPx-like
activity, compared to ebselen, followed by 17, 7, 8, 18, 19, 20 and 21. On the other hand,
compounds 9, 11, 14 and 15 showed moderate GPx activity.
In contrast to expectation, ebselen selenocyanate 5 and ebselen diselenide 6 were not active
in this assay. This unexpected behavior may be due to the electronic nature of the substituents at
the phenyl groups in the para position that, unlikely as it seems, diminish the GPx-like activity.
2.2.2.3. Bleomycin DNA damage Assay
The pro-oxidant activities of the aforementioned compounds were assessed via bleomycin
induced DNA damage assay. This assay has been endorsed as a specific, yet sensitive, method to
estimate the pro-oxidant activity of drugs and food antioxidants.[66,67] The bleomycins are
2
+
family of antibiotics that exhibit antitumor activity by Fe assisted cleavage of DNA. Briefly, If
+
3
+2
the compounds are able to reduce the bleomycin- Fe to bleomycin-Fe , DNA degradation
in this system would be stimulated, resulting in a positive test for pro-oxidant activity.
Among the tested compounds (Table 2), compounds 10, 12 and 17 manifest the lowest DNA
degradation potency induced by bleomycin-Fe complex. Furthermore, compounds 6, 13 and 14
show activity similar to ascorbic acid. This ultimately reveals that these manifested poor
prooxidant activity.
<Table 2>
2
.2.3. Antimicrobial evaluation
9

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To study the cytotoxic activity beyond a human cell line we also studied the effect on lower
organisms i.e. fungi and bacteria. Thus the antimicrobial activity of the compounds was
evaluated against gram-negative Escherichia coli (E. coli) and gram-positive Staphylococcus
aureus (S. aureus) as well as against the pathogenic fungus Candida albicans (C. albicans). A
standard agar diffusion assay was used and the diameters [mm] of inhibition zones are
summarized in Table 3.
<Table 3>
In general, most compounds exhibited toxicity against gram-positive (S. aureus) bacteria,
more than the gram-negative (E. coli) bacteria. In this context, compounds 13 and 16 were the
most active ones against S. aureus with up to 95 % relative activity (compared to the known
drug, ampicillin). Compounds 14, 18, 20 and 21 showed good-moderate activity (90 % activity
index). Against gram-negative E. coli only compound 17 showed a moderate toxicity.
In the case of C. albicans, compounds 10 and 13 exhibited activity similar to the antifungal
drug colitrimazole with activity indices of 96 and 100%, respectively.
2
.2.4. Physicochemical parameters and drug-likeness assessment
Whilst the spectrum of activities associated with some of the compounds against Hep G2
cell, bacteria but especially against C. albicans are promising, it should be further checked
whether these compounds might have the required bioavailability. One such test is compliance
with the traditional Lipinski Rule of Five which indicates if a chemical substance can be orally
active in humans by estimating molecular properties which are important for
drug's pharmacokinetics.[68] The overall physicochemical properties of the test compounds and
the drug-likeness model score (collective property of physicochemical properties,
pharmacokinetics and pharmacodynamics of a compound is represented by a numerical value)
were computed employing the Molinspiration online property calculation toolkit and MolSoft
software (MolSoft, 2007).[69,70]
Lipinski’s Rule states that most molecules with good membrane permeability should have
partition coefficient values (logP) of the compounds in the octanol-water system ≤ 5, molecular
weight ≤ 500, number of hydrogen bond acceptors ≤ 10 and number of hydrogen bond donors ≤
5
.[71]
1
0

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In a physicochemical perspective (Table 4), all of the studied compounds have hydrogen
bond acceptors (HBA) far fewer than 8 and hydrogen bond donors (HBD) far fewer than 4. This
in turn fits with the Lipinski’s rule of five indicating that these compounds may have good
absorption or permeability properties through the biological membranes. Furthermore, most of
the compounds’ lipophilicity, expressed as logP, was found to be below 5 confirming their drug-
relevant properties.
<Table 4>
Only compounds 6, 7, 8 and 9 violate the rule as they were found to be too lipophilic (logP
>
5) and their molecular masses were beyond the limit i.e. > 700 Daltons.
3
. Conclusion
The facile synthesis of novel organoselenocyanates and symmetrical diselenides was
described. Most compounds were readily synthesized in two steps in good to moderate yields
and were evaluated for their in vitro cytotoxic activity against Hep G2 cell line, gram-negative,
gram-positive bacteria and a pathogenic yeast. In general, compound 16 was considered to be the
most interesting with a free radical-scavenging activity comparable to ascorbic acid and a GPx-
like activity similar to ebselen. On the other hand, the simple amides and cyclic imides proved to
be better toxins than the ebselen and Ugi-derivatives. Compounds 10, 11 and 14 were able to
reduce the viability of Hep G2 tumor cells already after 48 h incubation at concentrations below
1
0 µM, especially the activity of some cyclic imide selenocyanates (10, 14) against Candida
fungus is of relevance, as the activity values are similar to those of reference antimycotics. These
initial results suggest further investigations using a wider arsenal of cancer and primary cells and
against different organisms, especially humanopathogenic fungi.
Also the free radical scavenging ability, as measured by DPPH assay, was more enhanced in
the simple amides and imides (schemes 2 & 3). Compounds 10, 18 and 20 were the strongest
radical scavengers among the tested compounds, followed by compounds 5, 6, 12 and 17.
Furthermore, the pro-oxidant activity was estimated using bleomycin dependent DNA damage.
Compared to the standard antioxidant ascorbic acid, compounds 10, 12 and 17 showed the
highest protection. Compounds 13, 14 and 6 showed activity similar to ascorbic acid. The GPx-
like activity was evaluated in a NADPH-reductase coupled assay. Compound 16 was the most
1
1

ACCEPTED MANUSCRIPT
potent one, followed by 7, 8, 17, 18, 19, 20 and 21. The larger and more lipophilic molecules
synthesized initially (Scheme 1) are mostly outside the realm guarded by Lipinski’s Rule of Five,
whereas the simpler amides and imides (schemes 2 & 3) comply with Lipinski’s Rule of Five.
However, this cannot explain the equally lower activity in the non cell-based assays.
While it is premature to estimate why the diseleno-N-substituted maleanilic acid 16 was
particularly the most active organoselenium compound in the most of performed assays, one may
speculate that this compound may hit specific cellular target(s) and cause a widespread
modification of proteins and enzymes for the benefit of activation. Furthermore, it’s likely that
that such compound might also be taken up by cells and modified in vivo into active metabolic
intermediates. As this is an immensely unknown class of compounds and hence it is too early to
speculate over details on their exact metabolism, pharmacokinetics in animals and enrichment of
such compounds in specific tissues or degradation, although these issues are clearly important
and will form part of future studies. Ultimately, as the structure of this compound provides
considerable scope for modifications, and the synthesis of derivatives is now straightforward,
this will become a promising starting point to future modulations of its activity through the
design, synthesis and evaluation of structural variants of compound 16. It will also be necessary
to produce a wider range of such compounds, including some tellurium-containing analogues,
and to screen for further activities and selectivity, also in order to derive reliable structure–
activity relationships.
Our studies clearly demonstrate differential behavior of diselenides, dependent on the
substitution pattern. We see evidence that such compounds can not only play a role in anticancer
redox processes but also may have even more value in antifungal, but clearly at this point the
compound base is still too meager to jump to conclusions. Structurally it may be wise to not stick
any longer to the old gold standard of ebselen, which had the advantage of a first player. A clear
QSAR will require a larger and diverse set of compounds to obtain a better understanding of the
mode(s) of action. Eventually, this justifies the realization of more in-depth studies and
additional experiments to investigate the exact mode(s) of action responsible for the pronounced
biological activity apparently exhibited by this compound and to identify possible intracellular
targets (such as specific organelles, membranes or proteins). Additionally, we are fully aware
that these findings raise wealth of more questions. For example, what are the possible
1
2

ACCEPTED MANUSCRIPT
applications and the corresponding pharmacological and pharmacokinetic properties of such
compound?
We see plenty of room for further, multi-disciplinary studies involving synthetic, bioorganic
and medicinal chemistry, cell biology, and pharmacology in order to develop a strategy to treat
cancer or yeast infections by applying organoselenium compounds.
4
. Experimental protocols
4
.1. Material and methods
All chemical reagents for the synthesis of compounds were purchased from Sigma-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 techniques. Silica
gel 60 (Macherey-Nagel, 50-200 ꢀm) was used for column chromatography. Unless noted
otherwise, the dimensions of columns used were 2.5 cm (diameter) and 25-30 cm (height of
silica gel). TLC plates (silica gel 60 F , 0.20 mm) were purchased from Merck. NMR
2
54
1
13
spectroscopy: H NMR spectra were recorded at 400 MHz, C NMR spectra at 100 MHz on a
Bruker DRX 500 or Avance 500 spectrometer. Chemical shifts are reported in δ (ppm),
1
expressed relative to the solvent signal at 7.26 ppm (CDCl , H NMR) and at 77.16 ppm (CDCl ,
3
3
1
3
1
13
C NMR), as well as 3.31 ppm ( H NMR, CD
3
OD) and 49.00 ppm ( C NMR, CD
3
OD).
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). HRMS: high-resolution mass spectrometry was performed on an Accela
UPLC-system (Thermo-Fisher) coupled to a linear trap-FT-Orbitrap combination (LTQ-
Orbitrap), operating in positive ionization mode. These spectra indicated ≥ 99% MS-purity of the
prepared compounds.
DNA (Calf Thymus type1), bleomycin sulfate, thiobarbituric acid (TBA), 1,1-diphenyl-1-
picrylhydrazyl (DPPH), ethylenediaminetetraacetic acid (EDTA), and ascorbic acid were
obtained from Sigma. All other chemicals were of analytical grade. Compound 1, 3 and 4 were
synthesized according to the literature reported method.[72-74]
4
.2.Synthesis and characterization
1
3

ACCEPTED MANUSCRIPT
Preparation of sodium diselenide
A mixture of sodium hydroxide (4.4 g, 110 mmol), selenium powder (8.8 g, 111.40 mmol) and
3
rongalite (sodium formaldehyde sulfoxylate) (8.8 g, 74.50 mmol) in 60 cm of water was stirred
under nitrogen for 30 min. After the selenium had completely dissolved a brownish red solution
was generated.
Synthesis of 2,2’-diselenobis[benzoic acid] (1)
3
A stirred solution containing o-aminobenzoic acid (14.0 g, 34.91 mmol), 20 cm of conc.
3
o
hydrochloric acid and 20 cm of water in an ice bath (under 5 C), was added dropwise to sodium
3
nitrite (9.0 g, 134.31 mmol) in 20 cm of water. A solution of 2- carboxybenzodiazonium
chloride resulted; this was added dropwise to a stirred solution containing sodium diselenide
which was prepared in advance by the reaction of selenium powder (8.8 g, 111.40 mmol),
3
rongalite (8.8 g, 74.50 mmol) and sodium hydroxide (4.4 g, 110 mmol) in 60 cm of water. The
mixture was stirred for an additional 2 h after an initial evolution of nitrogen gas stopped. It was
confirmed that the solution was basic using litmus paper. The reaction mixture was acidified
with hydrochloric acid and the precipitated solid was collected by filtration, washed with water,
and dried in a desiccators to give 2,2’-diselenobis[benzoic acid]. Yield: 20.0 g, 90%. M.p. 293
o
C.
General procedure for the preparation of ebselen derivatives 5 and 6
Thionyl chloride (2.5 mL, 34.0 mmol) was added to diselenide 1 (1.0 g, 2.5 mmol) in a three
necked round bottomed flask. The reaction mixture was heated to reflux for 6 h. Excess thionyl
chloride was removed under vacuum. The obtained residue was dissolved in dichloromethane
(10 mL). A solution of amine (5.0 mmol) and triethylamine (2.1 mL, 15 mmol) in
o
dichloromethane (15 mL) was added to the reaction mixture at 0 C. The ice bath was removed
and the reaction mixture was stirred for additional 3 h. The reaction mixture was poured into
water and extracted with dichloromethane (2x20 mL). The organic layer was washed with HCl
(
1 N), an aqueous solution of NaHCO (5 %w/v), and water. The organic layer was dried over
3
sodium sulfate and evaporated to give a dark brown solid, which was purified by column
chromatography on silica gel.
General procedure for the preparation of peptide derivatives 7, 8 and 9
1
4

ACCEPTED MANUSCRIPT
As a general procedure, a mixture of 1 (1 mmol), aldehyde (2 mmol), amine (2 mmol) and
isonitrile (2.2 mmol) in 2 mL methanol was stirred at room temperature overnight. Upon
completion (monitored by TLC), 10 mL water were added to dissolve the sticky product. The
2 2
aqueous layer was extracted three times with CH Cl , the organic layers were combined, dried
over Na SO and concentrated to yield a sticky product which was purified by chromatography
2
4
on silica gel.
General procedure for the preparation of amide-acids 10, 12, 14, 16, 18 and 20
To a stirring solution of anhydride (1 mmol) in dry toluene (5 mL) was added dropwise a
solution of amine (1 mmol) at room temperature. The mixture was vigorously stirred for 3 h, and
the formed precipitate was separated by s filtration. The cake was washed with toluene and dried
under reduced pressure.
General procedure for the preparation of cyclic imides 11, 13, 15, 16, 19 and 21.
A reaction mixture containing the appropriate acid (cf. schemes 2, 3) in 5 mL of acetic anhydride
and 100 mg of sodium acetate was heated for 2 h under reflux. The reaction was cooled and
quenched with water. The aqueous solution was extracted with CH Cl , dried with Na SO ,
2
2
2
4
filtered, and the solvent was evaporated.
4
.2.1. 2-(4-selenocyanatophenyl)benzo[d][1,2]selenazol-3(2H)-one (5)
1
Yield: 0.266 g (71%). HMNR (400 MHz, DMSO-d ) δ ppm: 7.46-7.53 (m, 1H, phenyl SeCN),
6
7
8
1
=
.68-7.73 (d, 1H, J= 8.06, PhSeCN), 7.73-7.82 (d, 4H, J= 7.6), 7.89- 7.94 (m, 1H, PhSeCN),
1
3
.06-8.12 (m, 1H, phenyl SeCN); C NMR (100 MHz, DMSO-d ) δ ppm: 165.23, 140.97,
6
38.70, 134.58, 132.56, 128.37, 128.04, 126.39, 125.87, 125.57, 119.93, 105.31; MS (ESI): m/z
+
+
+
found 381.2 [M +1], 403 [M +Na]; calcd. 379.9; HRMS calcd. for C H N OSe [M +Na]:
14
8
2
2
+
4
02.8898, found 402.88586 [M +Na].
4
.2.2. 2, 2`(4-( diseleno)phenyl)benzo[d][1,2]selenazol-3(2H)-one (6)
1
Yield: 0.481 g (68%). HMNR (400 MHz, DMSO-d ) δ ppm: 7.44-7.50 (m, 2H, PhSe), 7.53-
6
7
.64 (m, 2H, PhSe), 7.66-7.75 (m, 8H), 7.88-7.93 (d, 2H, J= 7.80), 8.08-8.14 (d, 2H, J= 8.00);
1
3
C NMR (100 MHz, DMSO-d
6
) δ ppm: 165.12, 139.88, 138.80, 133.60, 132.35, 132.32, 128.51,
+
1
27.95, 126.58, 126.28, 125.93, 125.40, 125.18; MS (ESI): m/z = found 709.0 [M +1], 731.8
+
+
[M +Na]; calcd. 707.79; HRMS calcd. for
26 16 2 2 4
C H N O Se [M +Na]: 730.7798, found
+
7
30.77648 [M +Na].
1
5

ACCEPTED MANUSCRIPT
4
.2.3. N,N`-(1-(tert-butylcarbamoyl)-2-methylpropyl)-N-(2-(1H-indol-3-yl)ethyl)-2-
(diseleno)benzamide (7)
1
Yield: 0.697 g (70%). HMNR (400 MHz, CDCl ) δ ppm: 0.89-0.95 (d, 6H, J= 5.04, iso-propyl),
3
1
2
1
7
1
1
2
1
1
.03- 1.07 (d, 6H, J= 6.05, iso-propyl), 1.28-1.31 (s, 9H, t-butyl), 1.32-1.34 (s, 9H, t-butyl), 2.49-
.78 (m, 6H, CH ), 2.98-3.19- (m, 4H, CH ), 3.39-3.47- (t, 2H, J= 10.70), 3.57-3.65- (t, 2H, J=
2
2
1.60, CH), 4.27-4.40 (s, 2H), 6.50-6.62 (s, 1H), 6.79-6.86 (m, 3H), 7.02-7.11 (m, 5H), 7.13-
.22 (m, 3H), 7.48-7.55 (s, 1H), 7.84-7.92 (t, 2H, J= 7.96), 8.11-8.19 (d, 2H, 5.49), 8.23-8.32 (s,
1
3
6
H); C NMR (100 MHz, DMSO-d ) δ ppm: 172.22, 169.85, 136.04, 131.71, 131.65, 130.68,
30.23, 127.00, 126.83, 126.74, 122.12, 121.87, 119.32, 118.36, 118.31, 111.01, 60.37, 51.20,
+
8.61, 28.56, 28.52, 26.16, 25.55, 21.03, 20.07, 18.89; MS (ESI): m/z = found 997.5 [M +1],
+
+
035.4 [M +K]; calcd. 996.33; HRMS calcd. for C H N O Se [M +Na]: 1019.3198, found
52
64
6
4
2
+
019.3112 [M +Na].
4
.2.4. N,N -̀ ((tert-butylcarbamoyl)(p-tolyl)methyl)-N-(2-(1H-indol-3-yl)ethyl)-2-
(diseleno)benzamide (8)
1
Yield: 0.753 g (69%). HMNR (400 MHz, CDCl ) δ ppm: 1.27-1.35 (s, 18H, t-butyl), 2.29-2.36
3
(
s, 6H, P-CH
3
), 4.40 -4.71 (m, 4H, CH
2 2
), 5.24-5.49 (m, 4H, CH ), 5.71-5.91 (s, 2H, CH), 6.94-
1
3
7
1
1
2
.18 (m, 22H, Ph), 7.35-7.41 (s, 2H), 7.62-7.77 (s, 2H); C NMR (100 MHz, DMSO-d ) δ ppm:
71.61, 171.56, 168.36, 138.41, 138.38, 136.52, 136.46, 132.20, 132.16, 132.10, 132.07, 130.47,
29.65, 129.61, 129.40, 128.24, 128.21, 127.42, 126.72, 64.82, 64.77, 60.36, 52.84, 52.78, 51.59,
6
+
8.55, 28.34, 21.11, 21.02, 14.17; MS (ESI): m/z = found 1114.7 [M +Na]; calcd. 1092.33;
+
HRMS calcd. for C H N O Se [M +Na]: 1115.3198, found 1013.7488.
6
0
64
6
4
2
4
.2.5. N,N-
̀
(1-(tert-butylcarbamoyl)-2-methylpropyl)-N-benzyl-2-(diseleno)benzamide (9)
1
Yield: 0.685 g (77%). HMNR (400 MHz, CDCl
3
) δ ppm: 0.96-1.04 (d, 12H, J= 6.3, iso-propyl),
1
6
1
1
8
9
.31- 1.38 (s, 18H, t-butyl), 2.69-2.77 (s, 2H), 3.87-4.02 (m, 2H), 4.44-4.65 (m, 4H, phenyl),
1
3
.95- 7.24 (m, 16H), 7.59-7.72 (s, 2H, phenyl); C NMR (100 MHz, DMSO-d ) δ ppm: 172.32,
6
69.22, 136.23, 136.17, 135.83, 131.81, 130.94, 130.67, 130.02, 128.35, 128.29, 128.18, 128.14,
27.51, 126.71, 126.67, 126.47, 51.08, 28.71, 28.63, 27.14, 19.91, 19.63; MS (ESI): m/z = found
+
+
+
91.4 [M +1], 913.5 [M +Na]; calcd. 890.28; HRMS calcd. for C H N O Se [M +Na]:
4
6
58
4
4
2
+
13.2698, found 913.2681 [M +Na].
4
.2.6. (Z)-3-(4-selenocyanatophenylcarbamoyl)acrylic acid (10)
1
Yield: 0.269 g (91%). HMNR (400 MHz, DMSO-d
6
) δ ppm: 6.30-6.35 (dd, 1H, J= 11.95,
CH=), 6.46-6.50 (dd, 1H, J= 12.01, =CH), 7.68-7.70 (s, 4H, Ph), 10.32-10.72 (s, 1H, COOH);
1
3
6
C NMR (100 MHz, DMSO-d ) δ ppm: 166.81, 163.41, 139.82, 134.78, 131.53, 130.18, 120.67,
1
6

ACCEPTED MANUSCRIPT
+
1
20.58, 117.11, 114.99, 105.34; MS (ESI): m/z = found 297.1 [M +1]; calcd. 295.97; HRMS
+
+
calcd. for C H N O Se [M +1]: 296.97794, found 296.97709 [M +1].
1
1
8
2
3
4
.2.7. 1-(4-selenocyanatophenyl)-1H-pyrrole-2,5-dione (11)
1
Yield: 0.144 g (52%). HMNR (400 MHz, DMSO-d ) δ ppm: 7.23-7.31- (s, 2H, CH=CH), 7.43-
6
1
3
7
1
(
.55 (d, 2H, J=8.43, Ph), 7.84-7.96 (d, 2H, J= 8.35, Ph); C NMR (100 MHz, DMSO-d ) δ ppm:
69.59, 143.05, 134.81, 134.22, 134.01, 132.52, 129.88, 127.97, 124.76, 123.10, 105.28; MS
ESI): m/z = found 279.2 [M +1]; calcd. 277.96; HRMS calcd. for C H N O Se [M +NH ]:
95.99894, found 296.96283 [M +1+ NH ].
6
+
+
1
1
6
2
2
4
+
2
4
4
.2.8. 3-(4-selenocyanatophenylcarbamoyl)propanoic acid (12)
1
Yield: 0.265 g (89%). HMNR (400 MHz, DMSO-d
(
6
) δ ppm: 2.51-2.65 (m, 4H, CH
2
), 7.53-7.76
1
3
s, 4H, Ph), 9.94-10.42 (s, 1H, COOH); C NMR (100 MHz, DMSO-d ) δ ppm: 173.66,170.54,
6
1
2
3
70.45, 140.45, 134.85, 120.15, 120.06, 115.99, 105.35, 31.00, 28.58; MS (ESI): m/z = found
+
+
+
99.3 [M +1]; calcd. 297.99; HRMS calcd. for C H N O Se [M +1]: 298.99794, [M +Na]:
1
1
10
2
3
+
+
20.9798, found 298.99273 [M +1], 320.97473 [M +Na].
4
.2.9. 1-(4-selenocyanatophenyl)pyrrolidine-2,5-dione (13)
1
Yield: 0.134 g (48%). HMNR (400 MHz, DMSO-d ) δ ppm: 2.77-2.80 (s, 4H, CH ), 7.25-7.42
6
2
1
3
(
1
6
d, 2H, J= 8.49, Ph), 7.74-7.88 (d, 2H, J= 8.56, Ph); C NMR (100 MHz, DMSO-d ) δ ppm:
76.69, 133.89, 133.57, 128.52, 123.75, 105.28, 28.52; MS (ESI): m/z = found 280.30 [M +1];
+
+
+
8 2 2
calcd. 279.98; HRMS calcd. for C11H N O Se [M ]: 279.98, found 278.96779 [M ].
4
.2.10.
4-(4-selenocyanatophenylcarbamoyl)butanoic acid (14)
1
Yield: 0.290 g (93%). HMNR (400 MHz, DMSO-d ) δ ppm: 1.75-1.86 (p, 2H, J =7.38, J =
6
1
2
1
4.70, CH ), 2.20-2.32 (t, 2H, J= 7.28, CH ), 2.33-2.42 (t, 2H, J=7.41, CH ), 7.51-7.79 (m, 4H,
2 2 2
1
3
Ph), 9.66-10.49 (s, 1H, COOH); C NMR (100 MHz, DMSO-d ) δ ppm: 174.05, 171.08,
6
1
40.44, 136.41, 134.83, 120.31, 120.22, 116.05, 105.34, 35.36, 32.87, 20.25; MS (ESI): m/z =
+
+
found 313.00 [M +1], 335.2 [M +Na]; calcd. 312.00; HRMS calcd. for C H N O Se
12
12
2
3
+
+
[M +Na]: 334.9898, found 334.99078 [M +Na].
4
.2.11.
1-(4-selenocyanatophenyl)piperidine-2,6-dione (15)
1
Yield: 0.126 g (43%). HMNR (400 MHz, DMSO-d
(
6
) δ ppm: 2.08-2.28 (m, 6H, CH
2
), 7.72-7.91
1
3
d, 2H, J=8.42, CH ), 7.24-7.46 (d, 2H, J=8.45, Ph); C NMR (100 MHz, DMSO-d ) δ ppm:
2
6
1
72.26, 172.14, 140.48, 134.07, 131.45, 130.86, 130.76, 130.11, 124.44, 105.21, 26.73, 21.00;
+
+
MS (ESI): m/z = found 311.1 [M +NH ], 318.1 [M +Na]; calcd. 293.99; HRMS calcd. for
C H N O Se [M +1]: 294.99794, [M +K]: 334.09624, found 294.00063 [M +1], 334.99084
12 10 2 2
4
+
+
+
+
[M +K].
1
7

ACCEPTED MANUSCRIPT
4
.2.12.
(Z, Z)-3, 3 ̀- (4-(diseleno)phenylcarbamoyl)acrylic acid (16)
1
Yield: 0.489 g (91 %). HMNR (400 MHz, DMSO-d
6
) δ ppm: 3.32-3.43 (s, 2H, NH), 6.43-6.53
(
d, 2H, J=11.95, CH=), 6.19-6.37 (d, 2H, J=12.01, =CH), 7.08-7.32 (m, 2H, Ph), 7.52-7.62 (m,
1
3
6
1
5
5
H, Ph), 10.02-10.88 (s, 2H, COOH); C NMR (100 MHz, DMSO-d
6
) δ ppm: 166.79, 163.23,
+
36.01, 133.04, 131.56, 130.23, 124.41, 120.14; MS (ESI): m/z = found 540.5 [M +1]; calcd.
+
+
16 2 6 2
39.93; HRMS calcd. for C20H N O Se [M +1]: 540.93794, [M +Na]: 562.9198, found
+
+
40.94191 [M +1], 562.92402 [M +Na].
4
.2.13.
1, 1`-(4-(diseleno)phenyl)-1H-pyrrole-2,5-dione (17)
1
Yield: 0.227 g (45%). HMNR (400 MHz, DMSO-d
7
6
) δ ppm: 7.22-7.26 (s, 4H, CH=CH), 7.36-
1
3
.41 (d, 4H, J=8.42, Ph), 7.59-7.67 (m, 2H, Ph), 7.79-7.84 (d, 2H, J= 8.40, Ph); C NMR (100
MHz, DMSO-d
6
) δ ppm: 169.69, 134.74, 131.37, 130.10, 127.53, 119.69; MS (ESI): m/z = found
+
+
+
5
5
21.2 [M +NH ]; calcd. 521.2 [M +NH ]; HRMS calcd. for C H N O Se [M +1+NH ]:
22.95688, found 522.94966 [M +1+NH ].
4
4
20 12
2
4
2
4
+
4
4
.2.14.
3, 3`-(4-(diseleno)phenylcarbamoyl)propanoic acid (18)
1
Yield: 0.511 g (94%). HMNR (400 MHz, DMSO-d ) δ ppm: 2.51-2.65 (m, 8H, CH ), 7.40-7.65
6
2
1
3
(
m, 8H, Ph), 10.03-10.19 (s, 1H, COOH); C NMR (100 MHz, DMSO-d ) δ ppm: 173.70,
6
1
70.31, 170.22, 139.59, 139.48, 133.29, 123.38, 119.65, 119.56, 31.05, 31.00, 28.67; MS (ESI):
+
+
m/z = found 543.2 [M ]; calcd. 543.97; HRMS calcd. for C H N O Se [M +1]: 544.97794,
2
+
0
20
2
6
2
+
+
[
M +Na]: 566.9598, found 544.97367 [M +1], 566.95533 [M +Na].
.2.15. 1, 1-(4-(diseleno)phenyl)pyrrolidine-2,5-dione (19)
Yield: 0.208 g (41%). HMNR (400 MHz, DMSO-d ) δ ppm: 2.70-2.80 (s, 8H, CH ), 7.20-7.29
4
̀
1
6
2
1
3
(d, 4H, J= 8.48, Ph), 7.69-7.82 (d, 4H, J= 8.46, Ph); C NMR (100 MHz, DMSO-d
6
) δ ppm:
+
1
5
76.76, 132.31, 131.14, 129.68, 127.98, 28.46; MS (ESI): m/z = found 531.1 [M +Na]; calcd.
+
+
07.94; HRMS calcd. for C20
.2.16. 4, 4-(4-(methylselanyl)phenylcarbamoyl)butanoic acid (20)
Yield: 0.526 g (92%). HMNR (400 MHz, DMSO-d ) δ ppm: 1.76-1.84 (m, 4H, CH ), 2.25-2.29
16 2 4 2
H N O Se [M +Na]: 530.9298, found 530.93427 [M +Na].
4
̀
1
6
2
(
2 2
m, 4H, CH ), 2.33-2.39 (m, 4H, CH ), 6.42-6.59 (s, 1H), 7.16-7.29 (s, 1H), 7.32-7.41 (s, 1H),
1
3
7
1
1
.45- 7.64 (m, 8H, Ph), 9.99-10.10 (s, 1H); C NMR (100 MHz, DMSO-d ) δ ppm: 174.07,
6
74.03, 170.95, 170.86, 139.56, 139.45, 136.06, 134.22, 134.06, 133.32, 133.19, 123.45, 119.81,
19.72, 119.63, 114.39, 35.41, 35.36, 32.91, 32.72, 20.33, 19.94; MS (ESI): m/z = found 573.4
+
+
+
[M +1], 594.8 [M +Na]; calcd. 572.00; HRMS calcd. for C H N O Se [M +1]: 573.00794,
22 24 2 2
+
+
+
[M +Na]: 594.9898, found 573.00407 [M +1], 594.98679 [M +Na].
4
.2.17. 1, 1`-(4-(methylselanyl)phenyl)piperidine-2,6-dione (21)
1
8

ACCEPTED MANUSCRIPT
1
Yield: 0.493 g (92%). HMNR (400 MHz, DMSO-d
7
6 2
) δ ppm: 2.01-2.25 (m, 12H, CH ), 7.14-
1
3
.33- (d, 4H, J= 8.35, Ph), 7.68-7.86- (d, 4H, J= 8.39, Ph); C NMR (100 MHz, DMSO-d ) δ
6
ppm: 172.24, 139.21, 131.44, 130.10, 119.77, 119.68, 38.86, 26.69; MS (ESI): m/z = found
+
+
+
5
5
37.8 [M +1], 558.7 [M +Na]; calcd. 535.98; HRMS calcd. for C H N O Se [M +K]:
22 20 2 4 2
+
75.0783, found 574.9896 [M +K].
4
.3. Biological assays
.3.1. Cytotoxicity assay
4
The Hep G2 human liver carcinoma cell line was purchased from American Type Culture
Collection (HTB‑37; Rockville, MD, USA). The cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% (v/v) calf serum (Hyclone Laboratories,
0
Ogden, UT), 60mg/mL penicillin G and 100mg/mL streptomycin sulfate maintained at 37 C in a
humidified atmosphere containing about 15% (v/v) CO in air.
2
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
-
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% CO and allowed to grow for two
2
days. Then 60 µL of serial dilutions of the test compounds were added. After 48h of incubation
o
at 37 C and 10% CO
2
, 75 µL MTT in phosphate buffered saline (PBS) were added to a final
-1
concentration of 0.5 mg mL . After 2 h the precipitate of formazan crystals was centrifuged and
the supernatant discarded. The precipitate was washed with 100 µL PBS and dissolved in 100 µL
DMSO. The resulting color was measured at 590 nm using an ELISA plate reader. All
investigations were carried out in two parallel experiments. The IC values were determined as
5
0
the concentrations of tested materials, which showed 50% of the absorbance of untreated control
cells as estimated from the dose-response curves. 5-Fluorouracil (5-Fu) and ebselen were used as
a positive control.
4
.3.2. In vitro studies
.3.2.1. DPPH free radical scavenging activity
4
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 diphenylpicrylhydrazyl (DPPH).
This spectrophotometric assay uses stable radical diphenylpicrylhydrazyl (DPPH) as a reagent.
1
9

ACCEPTED MANUSCRIPT
The test compounds were dissolved in methanol to obtain a final concentration of 1 mg/ml. 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 was read against a blank at 517 nm. Ascorbic acid
(vitamin C) and ebselen were used as standard antioxidant (positive control). Blank sample was
run without DPPH and using methanol instead of sample. Negative control sample was run with
methanol instead of tested compound. The radical scavenging activity was calculated from the
following equation:
I%= (Ablank-Asample)/(Ablank)x100
4
.3.2.2. Glutathione peroxidase like activity
GPx kit (Biodiagnostic, Egypt) was used for the determination of GPx according to Paglia et
al.[71] 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
reductase and 4.8 µmol NADPH) and 0.01 ml (41 µM) tested compounds and the reaction was
started by the addition of H O (0.8 mM). The contents were mixed well and the absorbances
2
2
were recorded at 340 nm over a period of 3 min against deionized water. The change of
absorbance per minute (A340nm / min) was estimated using ebselen (41 µM) as the positive
control. The values represented in figure 3 are expressed after background correction for the
reaction with H O and GSH. In case of colored compounds, their activities were estimated after
2
2
subtracting their own absorbances at the used wave length.
.3.2.3. Bleomycin-dependent DNA damage
4
The reaction mixture contained calf thymus DNA (0.5 mg/ml), bleomycin sulfate (0.05 mg/ml),
MgCl (5 mM), FeCl (50 mM), samples to be tested (2 mM) and L-ascorbic acid was used as
2
3
o
positive control. The mixture was incubated at 37 C 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 80
0
C 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.
2
0

ACCEPTED MANUSCRIPT
4
.3.2.4. Antimicrobial activity
Chemical compounds were individually tested against gram positive (Staphylococcus aureus)
and gram negative (Escherichia coli) bacterial pathogens as well as Candida albicans fungus
(
yeast) strain. Antimicrobial tests were carried out by the agar well diffusion method using 100
8
4
µL of suspension containing 1x10 CFU/mL of pathological tested bacteria and 1x10 spores/mL
of fungi spread on nutrient agar (NA), and potato dextrose agar (PDA) medium respectively.
After the media had cooled and solidified, paper discs of 6 mm diameter soaked with 20 µl of the
test compounds (1mg/ml) were added to the agar plates and incubated at 30°C. After incubation
time, antimicrobial activity was evaluated by measuring the zone of inhibition against the test
organisms and compared with that of the standard. The antibacterial activity of a common
standard antibiotic ampicillin and the antifungal coltrimazole were chosen as positive control
using the same procedure as above at the same concentration. The relative (%) activity index was
calculated as shown below:
%
activity index= (inhibition zone of the test compounds/ inhibition zone of the standard drug)
×
100.
Acknowledgements
The authors thank the Egyptian Ministry of Higher Education, Deutscher Akademischer
Austauschdienst (DAAD), Leibniz Institute of Plant Biochemistry and Mansoura University for
financial support. We would like further to thank Mr Ahmed Abbass, Faculty of Pharmacy,
Mansoura University, Egypt for his help in performing the biological assays.
2
1

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