Enhancing the chemosensitivity of HepG2 cells towards cisplatin by organoselenium pseudopeptides

Article abstract of DOI:10.1016/j.bioorg.2021.104713

Despite all recent advances in the treatment of hepatocellular carcinoma (HCC), chemotherapy resistance still represents a major challenge in its successful clinical management. Chemo-sensitization offers an attractive strategy to counter drug resistance. Herein we report the identification of novel organoselenium-based pseudopeptides as promising highly effective chemo-sensitizers in treating HCC with cisplatin. A series of functionalized pseudopeptide- (5–9 and 17–19), peptidomimetic- (10–12 and 20–23), and tetrazole-based (13–16 and 24–27) organoselenium compounds were synthesized via isonitrile-based multicomponent reactions from two novel selenium-containing isocyanides. All compounds were evaluated for their cytotoxicity against HepG2 and the non-cytotoxic doses were used to restor the sensitivity of the cells to cisplatin. New organoselenium compounds (7, 9, 15, or 23) led to an effective chemo-sensitization of HepG2 cells towards cisplatin (up-to 27-fold). Cell cycle studies indicate that the most potent peptidomimetic diselenide 23 arrested cells at the S phase and induced apoptosis via ROS modulation.

Full text of DOI:10.1016/j.bioorg.2021.104713

Bioorganic Chemistry 109 (2021) 104713
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Enhancing the chemosensitivity of HepG2 cells towards cisplatin by
organoselenium pseudopeptides
a
,
b
,
*
c
a
d
Saad Shaaban
, Sameh M. Shabana , Yasair S. Al-Faiyz , Georg Manolikakes ,
e,**
Fardous F. El-Senduny
a
Department of Chemistry, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa 31982, Saudi Arabia
Organic Chemistry Division, Department of Chemistry, Faculty of Science, Mansoura University, Egypt
Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt
b
c
d
e
Department of Chemistry, Technical University Kaiserslautern, 67663 Kaiserslautern, Germany
Department of Chemistry, Biochemistry Division, Faculty of Science, Mansoura University, Mansoura, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Despite all recent advances in the treatment of hepatocellular carcinoma (HCC), chemotherapy resistance still
represents a major challenge in its successful clinical management. Chemo-sensitization offers an attractive
strategy to counter drug resistance. Herein we report the identification of novel organoselenium-based pseu-
dopeptides as promising highly effective chemo-sensitizers in treating HCC with cisplatin. A series of function-
alized pseudopeptide- (5–9 and 17–19), peptidomimetic- (10–12 and 20–23), and tetrazole-based (13–16 and
Hepatocellular carcinoma
Organoselenium
Multicomponent reactions
Isocyanide
Chemo-sensitization
2
4–27) organoselenium compounds were synthesized via isonitrile-based multicomponent reactions from two
novel selenium-containing isocyanides. All compounds were evaluated for their cytotoxicity against HepG2 and
the non-cytotoxic doses were used to restor the sensitivity of the cells to cisplatin. New organoselenium com-
pounds (7, 9, 15, or 23) led to an effective chemo-sensitization of HepG2 cells towards cisplatin (up-to 27-fold).
Cell cycle studies indicate that the most potent peptidomimetic diselenide 23 arrested cells at the S phase and
induced apoptosis via ROS modulation.
1
. Introduction
Primary liver cancer is among the most frequent cancer types and
short timeframe after an initial response [9]. Due to its low inherent
chemosensitivity and its acquired drug resistance, there is an urgent
need to identify novel compounds, sensitizing HCC towards therapy
with common therapeutic agents [10].
one of the leading causes of cancer-related deaths globally [1,2]. He-
patocellular carcinoma (HCC) is the most frequent kind of primary liver
cancer with a high incidence in Africa and Asia [3]. Despite its spread,
the successful treatment of HCC still represents a tremendous challenge,
leading to a poor long-term prognosis, particularly for patients with
advanced stages of HCC. Whereas radical therapeutic approaches, such
as surgical tumor removal or liver transplantation, are indicated only for
early stages, therapies for advanced HCC are still lacking. Currently,
sorafenib is the only approved drug for systemic chemotherapy of HCC,
unfortunately with only a modest efficacy [4]. Treatment of HCC with
standard chemotherapeutic agents [5-7], such as cisplatin or 5-fluoro-
uracil, fails in the majority of patients [8]. Due to its genomic insta-
bility and complex morphology, HCC cancer cells are either inherently
resistant to common chemotherapeutics or develop resistance within a
Chemo-sensitization represents an attractive and very effective
approach to counter the development of chemoresistance in tumors
[11,12]. Tumor cells can become more susceptible to the initial
chemotherapeutic drug using an additional agent, a so-called chemo-
sensitizer. This is where organic selenides come into play. In recent
years, organic selenides have evolved as an attractive compound class in
medicinal chemistry and drug development [13]. They are being used as
chemopreventive, chemoprotective, and antioxidant agents, as
confirmed in several epidemiological studies and pre-clinical trials (e.g.,
SELEBLAT, SELECT, and NPC) [14,15]. For example, the two synthetic
organoselenium compounds, methylseleninic acid (I) and diphe-
nylmethyl selenocyanate (II) exhibited good chemoprotective and che-
mopreventive activities against different cancer types in combination
*
Corresponding author at: Department of Chemistry, College of Science, King Faisal University, Saudi Arabia.
** Corresponding author at: Biochemistry Division, Department of Chemistry, Faculty of Science, Mansoura University, 35516, Mansoura, Egypt.
E-mail addresses: sibrahim@kfu.edu.sa, dr_saad_chem@mans.edu.eg (S. Shaaban), fkaneer@mans.edu.eg (F.F. El-Senduny).
https://doi.org/10.1016/j.bioorg.2021.104713
Received 3 December 2020; Received in revised form 24 January 2021; Accepted 30 January 2021
Available online 9 February 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
with cisplatin, cyclophosphamide or paclitaxel therapy (Fig. 1) [16,17].
Furthermore, ebselen (III) has shown excellent antioxidant and anti-
inflammatory activities and currently being explored as a neuro-
protective agent in clinical trials [18-20].
2. Results and discussion
2.1. Synthesis
Moreover, the selenocyanate-containing compounds BSC (IV) and p-
XSC (V) have shown excellent cancer chemopreventive activity [14,21].
Indeed, we have reported several organoselenium compounds (VI-VIII)
with exciting and, at the same time, highly selective anti-HCC activities
IMCRs enable the expeditious generation of molecular diversity and
structural complexity from simple starting building blocks in an atom-
convergent and economical fashion. They are a versatile tool in me-
dicinal chemistry, and plenty of biologically active small molecules have
been synthesized via the Passerini, Ugi, or Groebke–-
Blackburn–Bienayme reactions over the years [41,42,45,46]. Although
IMCRs provide straightforward and practical access to drug-like scaf-
folds, their utility is significantly hampered by the low structural di-
versity of readily available isocyanide building blocks, which restricts
the accessible structural space. Isonitriles bearing an additional func-
tional group handle, such as the chalcogens (e.g., sulfur, selenium,
tellurium) functionality, are not commercially available and are limited
to few examples the literature. Since our initial report on the first three
phenyl-sulfide, -selenide, and -telluride containing isocyanides, no
further chalcogen-based isonitriles were reported in the literature
[23,26]. In continuation of our previous work, we set out to expand the
chemical space accessible via isocyanide-based multicomponent re-
actions (ICMR) by establishing novel chalcogen-containing isonitriles.
Therefore we studied the conversion of the two selenium-based
[
18,22-28]. Interestingly, all these compounds exhibited superior cyto-
toxicity to the traditional anticancer drug fluorouracil, up-regulated the
expression levels of CD40 protein and interleukin 2 and 6, and down-
regulated the expression of Bcl-2 in HepG2 cells. Recently, we also re-
ported cytoprotective selenorganic compounds with potential gluta-
thione peroxidase (GPx)-like activity (IX) and antiapoptotic properties
(
X) for the 158 N myelin-forming cells (Fig. 1) [29]. Despite the inter-
esting chemopreventive and anticancer activities of organoselenium
compounds, their chemo-sensitization effect with chemotherapeutic
drugs has just begun.
Diversity-oriented synthesis (DOS) is a robust chemical tool that al-
lows the construction of skeletally diverse small molecules efficiently
and deliberately [30-33]. Furthermore, it enables the understanding of
the biological mechanism of sophisticated diseases (e.g., cancer and
cardiovascular diseases) and frequently employed for drug discovery. In
this context, lead candidates are identified via small-molecule libraries’
high-throughput screening, which can be optimized to be used as probes
or modulators for specific biological targets [30-33]. Multicomponent
reactions (MCRs) are among the most explored DOS pathways in terms
of combinatorial synthesis and the context of molecular diversity and
complexity [34,35]. Fortunately, isocyanide-based Multicomponent re-
actions (IMCRs) are among the most well-known and explored MCR
subclass [36-41]. The isonitrile (a.k.a. isocyanide) group’s extraordinary
nature accounts for the great potential of IMCRs. They are stable organic
compounds with a formally divalent carbon that readily reacts with both
′
amines, 4-selenocyanatoaniline (1) and 4,4 -diselanediyldianiline (2),
[22,25,47] into the corresponding isonitriles 5 and 6 via a two-step
formylation/dehydration procedure [48,49].
4-Selenocyanatoaniline (1) was prepared directly from aniline ac-
cording to an established procedure and further converted into the
diselenide 2. Formylation was initially performed using formic acid
under neat conditions; however, the corresponding formamides 3 and 5
were obtained in low yields (<40%). Next, formylation was performed
employing formic acid/acetic anhydride mixture. Unfortunately, a
mixture of the corresponding acetanilides and formamides was ob-
tained. Further formylation of 1 and 2 was achieved with acetic formic
anhydride, prepared in situ from acetyl chloride and sodium formate in
dry ether, delivering the desired formamides 3 and 4 in quantitative
yields [50]. We then directed our attention to the dehydration of both
formamides. Reaction with various dehydrating agents, such as phos-
electrophiles and nucleophiles to give -adducts [38,41,42]. Amongst all
α
known IMCRs, Groebke–Blackburn–Bienayme (GBB), Ugi, and Passerini
reactions are by far the most popular [34,38,43,44]. These one-pot re-
actions give fused imidazole, peptidomimetic, and pseudopeptide scaf-
folds in a high diversity and combinatorial-controlled fashion from
readily available components such as amines, acids, and oxo compounds
3
phorus oxychloride (POCl ), cyanuric chloride, triphenylphosphine, 4-
(
e.g., aldehydes or ketones). While these reactions are a convenient tool
toluenesulfonyl chloride, Burgess reagents, or phosgene were per-
formed [41,42,51-54].
for constructing skeletally diverse small molecules, they suffer from the
lack of commercially available isonitriles. Unlike the other utilized
building blocks, isonitriles have an unpleasant odor and are of limited
Interestingly, the formation of nonpolar products (using TLC) with
the characteristic isonitriles odor was only observed in the case of POCl3.
Initial attempts to isolate the observed nonpolar products failed. Due to
the reported instability of isonitriles, we suspected that isonitriles 5 and
6 might be unstable at room temperature. Indeed, both the desired
selenocyanate isocyanide 5 and diselenide diisonitrile 6 were obtained
in 92 and 94% yields after performing purification and isolation at low
1
commercial availability, i.e., fewer than three-dozen can be bought . In
2
009, we reported the synthesis of sulfur-, selenium- and tellurium-
based isonitriles and their applications in IMCRs [22].
In continuation of our previous projects, we herein report an
expansion of the chemical space of isocyanides via designing and syn-
thesizing two novel selenium-based isocyanides. The newly synthesized
isonitriles’ reactivity was explored in different IMCRs, leading to a
structurally diverse library of highly functionalized organoselenium
compounds. The latter was tested for cytotoxicity against HepG2 cells.
Furthermore, the chemo-sensitizing and chemoprotective efficacy of the
synthesized novel organoselenium compounds was investigated, and
their potential mode of action was evaluated by cell cycle analysis,
apoptosis analysis, and evaluation of reactive oxygen species (ROS)
level. Our studies revealed a so far unprecedented sensitization of HCC
cells against the standard anticancer drug cisplatin with organoselenium
agents.
◦
temperatures (≤0 C) (Scheme 1). Both compounds show a character-
istic isocyanide carbon triplet peak at 165 ppm in the 13C NMR spectra
(see supporting information). It is worth noting that isocyano-4-
selenocyanatobenzene (5) is not stable and must be prepared directly
before usage.
With established access to the two novel isocyanides 5 and 6, we set
out to incorporate the selenocyanate and diselenides functionality into a
small set of structurally diverse molecules using different IMCRs. In
accordance with our aim, the reactivity of the selenocyanate isocyanide
5 and diselenide diisonitrile 6 was explored in three typical IMCRs, the
Passerini, the Ugi, and the azido-Ugi reaction (Scheme 2 and Scheme 3).
By variation of the other building blocks, we prepared a small library
of 21 different organoselenium compounds (Scheme 4).
We selected four different aldehyde (paraformaldehyde, iso-
butyraldehyde, isovaleraldehyde, and 4-nitrobenzaldehyde) for our
initial study and three different amines (benzyl amine, aniline, and 4-
methoxyaniline) components. As an acid component, only acetic acid
1
Twenty-six isocyanides are commercially available at sigmaaldrich.com.
3
(Passerini/Ugi) or trimethylsilyl azide (TMSN ) (azido-Ugi reaction)
2

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
Fig. 1. Structures of selected organoselenium compounds (I-X) with anticancer, chemopreventive, and antioxidant properties.
Scheme 1. Synthesis of selenocyanate and diselenide isocyanides 5 and 6.
Scheme 2. Schematic overview of the participation of the selenocyanate isocyanide 5 in Passerini, Ugi, and azido-Ugi multicomponent reactions.
3

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
Scheme 3. Schematic overview of the participation of the diselenide diisonitrile 6 in Passerini, Ugi, and azido-Ugi multicomponent reactions.
were used. The Passerini reaction was performed in dichloromethane,
whereas the Ugi and azido-Ugi reactions were performed in methanol.
Peptidomimetic selenocyanates 7–16 and diselenides 17–26 were syn-
thesized in moderate-excellent yields (up to 94%).
sensitizing/chemoprotective effects of the non-cytotoxic compounds.
Cisplatin is one of the main chemotherapeutic drugs used for the
treatment of different cancer types. Its cytotoxic mechanism is the for-
mation of platinum-DNA adducts and ROS induction, thereby triggering
DNA damage response, subsequent cell cycle arrest, and finally
apoptosis [56]. Moreover, cisplatin is effective in combination with
radiotherapy and immunotherapy [57]. Unfortunately, in particular
HCC cells, cancer cells can develop multiple mechanisms of resistance
towards cisplatin [58]. It has been reported that a selenium-containing
diet can restore prostate cancer sensitivity to tumor necrosis factor-
related apoptosis-inducing ligand (TRAIL)-induced apoptosis [16]. Be-
sides, diphenylmethyl selenocyanate II has been used to sensitize a
murine tumor model to cisplatin via the overproduction of reactive
oxygen species, p53 activation, downregulation of the antiapoptotic
protein Bcl-2, and overexpression of apoptotic protein Bax [17]. Based
on this literature precedence, we decided to investigate further the use
of our novel organoselenium compounds as chemo-sensitizers to
improve the treatment of HCC with cisplatin using HepG2 cells as a
model system.
2
2
.2. Biological evaluation
.2.1. Organoselenium anti-HepG2 activity
After preparing a small set of structurally diverse, novel organo-
selenium compounds, we started to assay their biological activities.
Based on our previously established broad-spectrum cytotoxicity
screening, we evaluated the cytotoxic activity of compounds 7–27
against HepG2 cells, a simple model system for hepato-carcinogenesis in
vitro drug targeting studies [3,19,29,47,55]. Cell viability was evaluated
using MTT assay, and IC50 values were calculated after 48 h using
GraphPad prism (Table 1).
Most of the tested organoselenium agents did not exhibit any cyto-
toxic effect (IC50 ≥ 50
μM) against HepG2 cells in the tested concen-
tration range used, except for pseudopeptides 10, 17, and 19. The latter
two exhibited pronounced cytotoxic activities (IC50 = 7 ± 0.1 for
compound 17 and 9 ± 1.5 µM for compound 19), which are approx. two-
fold higher than cisplatin (IC50 = 16.3 ± 0.5 µM). Furthermore, pseu-
dopeptide 10 showed moderate activity with an IC50 = 28 ± 0.4 µM.
Observed morphological alterations in cells treated with compounds 17
or 19, as depicted in Fig. 2, S1 and S2 (Supplementary Materials
Figure S1 and S2), indicate that both organoselenium compounds induce
apoptosis as observed in the formation of black round apoptotic bodies.
In our previous work, we could already show that peptidomimetic
organoselenides can induce apoptosis via arresting cells at the G0/G1
phase and disruption of endoplasmic reticulum and cytoskeleton orga-
nization (stress fibers) [23,26].
All fourteen organoselenium compounds displaying no cytotoxic
effect in the initial MTT assay were evaluated for their chemo-sensitizing
activities combined with cisplatin. Therefore, HepG2 cells were initially
treated with 20 µM of the selected organoselenium compounds for 4 h,
and then serial dilutions of cisplatin were applied (50, 25, 12.5, 6.25,
3
.125, 1.56 µM) (Table 2). IC50 values of the respective combinations
were determined by MTT assay after 48 h overall treatment (Fig. 3).
To our delight, cells pretreated with the organoselenium compounds
7
, 9, 15, and 23 were more susceptible towards treatment with cisplatin
(
IC50 = 3 ± 1.8, 3 ± 0.2, 6 ± 0.5, 0.6 ± 0.3 µM, respectively), leading to a
significantly increased response compared to treatment with cisplatin
alone (IC50 = 16.3 ± 0.5 µM) (Table 2). The peptidomimetic diselenide
2
3 displayed the most promising chemo-sensitizing effect (27-fold in-
From a chemistry point of view, compounds 10, 17, and 19 share
some common structural features other than being organoselenium de-
rivatives. Compounds 17 and 19 are both Ugi adducts and diselenides at
the same time. Furthermore, compounds 10 and 17 have a common
isopropyl fragment. Collectively, the herein reported novel selenium-
containing pseudopeptides 17 and 19 warrant further investigation as
potential anticancer agents.
crease; Table 2). Pretreatment experiments were carried out following
the literature reported procedures [59-63] to find out the lowest doses
with the best chemo-sensitization effect. Our experiments started by
treating HepG2 cells with compound 23 (40, 20, 10, 5 or 2.5 µM) for four
h followed by incubation with cisplatin (50, 25, 12.5, 6.25, 3.125, 1.56
µM) for an additional 44 h. A drastic cell growth inhibition was observed
after 20 µM of compound 23 where the IC50 of cisplatin was declined
from 16.3 to 0.6 µM (Fig. 4). Next, the optimum time of pre-incubation
was investigated at different time points (1, 2, 3, 4, 5, or 6 h). Inter-
estingly, only one-hour pretreatment of HepG2 cells with compound 23
2
.2.2. Chemo-sensitization/chemoprevention of HepG2 cells to cisplatin
cytotoxicity
After the initial evaluation of the organoselenium compounds’
cytotoxicity, we turned our attention towards potential chemo-
(
20 µM) was sufficient to sensitize the cells towards cisplatin (IC50 = 0.6
4

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
Scheme 4. Organoselenium pseudopeptides, 7–27, synthesized employing the Passerini, Ugi, and azido-Ugi multicomponent reactions.
µM) toxicity (Fig. 4 and Supplementary Material Figure S3). The short
time sensitizing activity indicates the modulation of antiapoptotic
components in HepG2 cells, leading to increased sensitivity towards
cisplatin, according to Chakraborty et al., [17]. It worth mentioning that
the study presented here is only an entry point for considerably broader
and more in-depth investigations, which ultimately will consider the
concomitant and posttreatment group as well as mechanistic aspects
underlying the chemosensitization of compound 23.
staining for the early apoptotic marker phosphatidylserine with Annexin
V-PE by flow cytometry revealed that cisplatin alone was less efficient in
inducing apoptosis (2% early apoptotic cells vs. 36.6% with combined
treatment), but preincubation for just four h increased the sensitivity of
HepG2 cells to cytotoxicity of cisplatin (11.8% apoptotic cells vs. 3.7%
with single-drug treatment).
The results showed that the apoptosis index in the sensitized cells
was higher than the cells treated with cisplatin alone or the DMSO-
treated cells (36.3%±0.8 vs. 2.3%±0.3 and 0.4%±0.2, respectively
(Fig. 6).
Most anticancer drugs act by perturbating the cell cycle, either by
overexpressing cell cycle inhibitors (p15, p16, p21, or p27), down-
regulating cyclin-dependent kinases, damaging DNA, or affecting cyto-
skeleton organization [64,65]. Therefore, we further investigated the
sensitizing mechanism of compound 23 by cell cycle analysis employing
flow cytometry. Cisplatin was reported to induce HL-60 cells to be
initially arrested at S-phase, and then over time, gradually accumulated
and arrested at the sub-G1 phase [66]. Our results also showed that
cisplatin induces cell arrest at G1-phase. Interestingly, HepG2 cells
sensitized with the peptidomimetic diselenide 23 were arrested at S-
phase (Fig. 5), which indicates the induction of early apoptosis. There-
fore, the level of apoptotic cells was evaluated after treatment with
cisplatin alone and with cells pretreated with compound 23. The
In our former studies, we could confirm possible mechanism(s) for
some organoselenium agent’s cytotoxicity against different types of
cancer, such as modulation of the ROS and GSH levels, activation of
caspase 3/7, cell cycle arrest, and subsequent induction of apoptosis
[3,19,22,23,29,38]. The peptidomimetic diselenide 23 could therefore
chemo-sensitize HepG2 cells via an analogous perturbation of the cell’s
redox system. To evaluate this possible mode of action, the intracellular
ROS levels were estimated using H2-DCFDA assay by flow cytometry in
sensitized HepG2 cells and compared to treatment with cisplatin alone.
H2-DCFDA is a lipophilic probe used to detect several types of ROS, such
as peroxides, peroxynitrites, and lipid hydroperoxides. As expected,
5

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
Table 1
to oxidize redox-sensitive cellular compartments leading to cellular
malfunction and subsequently cell death [67-72]. It is also reported that
the cisplatin cytotoxic mechanism is correlated to ROS’s overproduction
[73-75].
Selenorganic compounds effect on the viability of
a
HepG2 cells.
b
Compounds
IC50 (µM)
c
In the case of the HepG2, compound 23 did not exhibit any cyto-
toxicity (IC50 > 50 µM), whereas cisplatin cytotoxicity was in the
micromolar scale, i.e., 16.3 µM (Table 1). Surprisingly,
a drastic inhibition of HepG2 cell growth was observed upon treat-
ment with 20 µM of compound 23 and 0.6 µM cisplatin (Figs. 4 and 5). It
is likely that the ROS levels (HepG2 pre-existing and cisplatin-induced)
are now enough to activate compound 23. The latter facilitates ROS’s
Cisplatin
16.3 ± 0.5
7
8
9
-d
-d
-d
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
0
1
2
3
4
5
7
8
9
0
1
2
3
4
5
6
7
28 ± 0.4
-d
-d
-d
-d
-d
7 ± 0.1
Table 2
-d
9 ± 1.5
Effect of the selenorganic compounds on the modulation of the cytotoxicity of
a
-d
-d
-d
-d
-d
-d
-d
-d
cisplatin against HepG2 cells.
b
c
Compounds
Chemo-sensitization IC50 (µM)
Chemo-sensitization fold
Cisplatin
16.3 ± 0.5
3 ± 0.2
–
7
5
5
1
1
2
2
3
1
1
1
27
1
1
2
9
3 ± 0.2
11
12
11.6 ± 0.2
17.8 ± 0.2
8 ± 0.5
1
1
1
3
4
5
a
The cytotoxicity was assessed after 48 h treat-
10 ± 0.4
6 ± 0.5
ment of HepG2 cells with various concentrations of
b
the organoselenium agents using the MTT assay.
20
21
22
20 ± 0.4
17.8 ± 0.2
13.8 ± 0.2
0.6 ± 0.1
29.2 ± 1.5
29.2 ± 0.2
8 ± 0.7
IC50 values in µM were estimated, and the standard
c
deviation (±) is for three parallel experiments. The
2
3
positive control used in this experiment is cisplatin;
d
25
no apparent cytotoxicity was observed at the
2
2
6
7
employed concentration range.
a
HepG2 cells were pretreated for four h with 20
μ
M of organoselenium
cisplatin-induced significant ROS elevation (24.9%± 0.26). On the other
hand, the peptidomimetic diselenide 23 alone could attenuate the ROS
level (1.1%± 0.21). Notably, the organoselenium compound 23 dis-
played a similar attenuating effect in combination with cisplatin (5.4%±
compounds, and then serial dilutions of cisplatin (50, 25, 12.5, 6.25, 3.125, 1.56
b
µM) was added, and the viability was determined after 48 h by MTT assay. IC50
values of cisplatin in µM were estimated, and the standard deviation (±) is for
c
three parallel experiments. Chemo-sensitization fold was calculated as follows:
0
.17) (Fig. 7).
IC50 value of cisplatin (16.3
μ
M)/cytotoxicity resulted from a combination of
Organoselenium compounds were reported to use pre-existing ROS
selenium compounds with cisplatin.
Fig. 2. Morphological changes of HepG2 cells after
treatment with compound DMSO vehicle (panel a),
cisplatin (panel b), compound 17 (panel c), and
compound 19 (panel d). Cells were treated with
different concentrations, and morphology was
observed after 48 h of incubation. The presented fig-
ures show the morphological alterations (red arrow)
induced after treatment with 6.25
μ
M of compound
1
7, 19, or cisplatin. The cells were round and show
apoptotic debris (black round cells indicated by a red
arrow). (For interpretation of the references to colour
in this figure legend, the reader is referred to the web
version of this article.)
6

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
tetrazole-based (13–16 and 24–27) organoselenium compounds from
two novel isocyanide building blocks (5 and 6) via common IMCRs.
Among the twenty-one novel organoselenium compounds, 17 and 19
exhibited promising cytotoxic activity against HepG2 cells, presumably
by inducing apoptosis. Interestingly, most organoselenium compounds
(
7–9, 11–15, 18, and 20–27) did not display any cytotoxic effect.
Further studies revealed that four compounds 7, 9, 15, or 23 exhibited
an enhanced chemo-sensitizing effect on HepG2 cells to cisplatin (up-to
2
7-fold increase in cisplatin activity). The peptidomimetic diselenide 23
lead to an effective chemo-sensitization of HepG2 cells in 20 µM con-
centration within only one hour.
Fig. 3. Chemo-sensitization experiment design. A) HepG2 cells were treated for
forty-eight h with cisplatin; B) HepG2 cells were treated for four h with 20 µM
of the organoselenium compounds, and then serial dilutions of cisplatin (50, 25,
Cell cycle analysis revealed a cell cycle delay at S-phase in HepG2
cells treated with cisplatin combined with the peptidomimetic dis-
elenide 23, which indicates a sensitizing effect via enhancing the
cisplatin-induced apoptosis. Additionally, peptidomimetic diselenide 23
enhanced the chemo-sensitizing effect by exerted its cytotoxicity via
amplification of the ROS severity. In summary, the present study entails
a novel efficient synthetic avenue towards structurally diverse organo-
selenium compounds, which significantly increases the chemical space
accessible via ICMRs. The herein synthesized compounds can act as lead
structures for the development of novel chemotherapeutics and chemo-
sensitizers for the effective treatment of HCC. Furthermore, our work
provides numerous chances for future studies at the biology/chemistry
interface. This research is worth further investigations and probably
follow-up studies in different scientific areas such as medicinal chem-
istry, pharmacology, and drug development.
1
2.5, 6.25, 3.125, 1.56 µM) were applied.
reaction- pre-existing in HepG2 and induced by cisplatin-with redox-
sensitive cellular compartments and subsequently triggers cellular
injury and apoptosis [67-72]. However, the exact organoselenium
compounds’ potential has not been thoroughly studied in the context of
cancer chemosensitization research.
These results reveal compound 23 as a new chemo-sensitizer to
cisplatin to overcome the emergence of resistance and improve the
clinical outcomes for patients. Notably, the exact underlying mechanism
is still not understood and merit further studies.
3
. Conclusion
4
. Experimental protocols
HCC is the most common and aggressive kind of liver cancer with an
overall poor long-term prognosis. Treatment of HCC with classical
cytotoxic drugs is limited due to its inherent or readily acquired che-
moresistance. Chemo-sensitization is a desirable strategy to overcome
this limitation and provide a still lacking general systemic therapy for
HCC. Herein, we disclose the synthesis of small set of novel pseudo-
peptide- (5–9 and 17–19), peptidomimetic- (10–12 and 20–23), and
4
.1. Material and methods
All chemicals (e.g., solvents, reagents) were purchased from Sigma-
Aldrich and used directly. TLC was performed on aluminum sheets,
SiO coated, (60 F254) and ultraviolet light was employed to visualize
spots were by. Column chromatography was performed silica 60.
2
Fig. 4. Pretreatment with compound 23 restored the sensitivity of HepG2 to cisplatin. Top left panel: cells were pre-treated with different concentrations (40, 20, 10,
, 2.5 µM) of compound 23 for 4 h then treated with serial dilutions of cisplatin (50, 25, 12.5, 6.25, 3.125, 1.56 µM) for total 48 h. MTT evaluated the cell viability.
Lower left panel: cells were pretreated with 20 M of compound 23 for the indicated time points then treated with a serial dilution of cisplatin (50, 25, 12.5, 6.25,
M (panel a) or 3.125 M (panel b) of cisplatin alone shows fewer apoptotic
M for four h increased the apoptosis in cells treated with 6.25 M (panel c) or 3.125 M (panel d) of cisplatin. (For
5
μ
3
.125, 1.56 µM). Right panel: morphological changes of cells treated either with 6.25
μ
μ
cells (red arrow) while cells pretreated with 20
μ
μ
μ
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
7

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
Fig. 5. Cell cycle arrest at S phase after combination treatment with both cisplatin and diselenide 23. The cells were treated either with 0.6 µM cisplatin alone or
treated with 20 µM compound 23 for four h then treated with 0.6 µM cisplatin for a total of 48 h. The DNA content was evaluated by flow cytometry. The induction of
apoptosis was analyzed by staining cells with Annexin-V. Statistical analysis was performed using GraphPad prism 8.0 by one-way ANOVA test. **** p < 0.0001 and
** p < 0.0069.
triplet, d = doublet and s = singlet. The structure and purity (≥97%) of
the compounds were established using HRMS spectrometry and NMR
′
spectroscopy. 4-Selenocyanatoaniline (1) and 4,4 -diselanediyldianiline
(
2) were synthesized according to literature reported methods
3,19,29].
[
4
4
.2. Biological assays
.2.1. Cell lines and reagents
HepG2 cells were obtained from VACSERA company, Egypt. Cells
were grown in a modified medium (Eagle’s Dulbecco) (BioWhittaker™)
◦
and kept at 5% CO
2
and 37 C. Bovine fetal serum (10%) was supple-
mented to the medium (Life Science Group L UK, Cat No: S-001B-BR)
and (100 IU/mL) penicillin/ streptomycin (100 µg/mL) (Lonza, 17-
6
02E).
4
.2.2. Dose selection and sample preparation
Cisplatin was obtained from Sigma-Aldrich, solubilized in saline
◦
(
0.9%), and then kept at –20 C as 1666 µM stock solution. Ten mM stock
Fig. 6. Apoptosis index increased in the sensitized cells with compound 23. The
apoptotic index was calculated as the percentage of Annexin V-positive and PI-
negative cells (early apoptosis) divided by the total number of cells in the gated
region (viable cells).
solutions of organoselenium compounds were prepared in Dimethyl
sulfoxide (DMSO Cat. No. 20385.02, Serva, Heidelberg, Germany) and
◦
stored at –20 C. The compounds were diluted to the desired concen-
tration using a modified medium so that the DMSO final concentration is
less than 0.5%.
Melting points are reported uncorrected. Electrospray ionization (ESI)
techniques were used to measure the mass spectra (MS), while, matrix-
assisted laser desorption/ionization (MALDI) techniques were used to
In the case of chemosensitization experiment: Five different con-
centrations of compound 23 (40, 20, 10, 5, 2.5 µM) were tested together
with six different concentrations of cisplatin (50, 25, 12.5, 6.25, 3.125,
1
13
measure the high-resolution mass spectra (HRMS). H NMR and
C
1
.56 µM). The best chemosensitization effect was observed using 20 µM
of compound 23 for 4 h and applied in cell cycle, apoptosis, and ROS
NMR spectra were recorded at 300.03 MHz, 500.18 MHz, 600.31 MHz
1
13
(
H), 75.44 MHz, 125.77 MHz, 150.95 MHz ( C), respectively. Chem-
1
3
assays.
ical shifts (δ) are relative to the residual CDCl
3
(δ = 77.16 ppm for
C
1
and δ = 7.26 ppm for H). Coupling constants (J) are given in Hz and
multiplicities of the signals are abbreviated as follows: dt = doublet of
triplets, dd = doublet of doublets, m = multiplet, q = quartett, t =
4
.2.3. Cytotoxicity assay
Liver cancer cells (HepG2) were treated with the new organo-
selenium pseudo peptides to screen for the most active compound. Cells
8

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
Fig. 7. Estimation of the intracellular ROS levels was estimated using H
2
-DCFDA by flow cytometry. Pretreatment of HepG2 cells with compound 23 decreased the
ROS levels. The cells were treated either with 0.6 µM cisplatin alone, 20 µM of compound 23 alone or treated with 20 µM of compound 23 for four h then treated with
0
.6 µM cisplatin for a total of 48 h. ROS level was evaluated using DCFDA as an indicator for oxidative stress by flow cytometry, the experiment was repeated 3X, and
statistical significance was calculated by One-way ANOVA test by Prism software.
were seeded in a 96-well plate (3*104 cells/mL, 100 µL/well). After
500 µL) containing RNase (1 mg/mL) for 30 min [78]. The cells were
analyzed by Accuri C6 Flow cytometer within one hour.
◦
overnight incubation at 37 C and 5% CO
2
, the cells were incubated with
5
0 µM (final concentration in the assay) of the tested compounds. DMSO
(
0.5% V/V) was used as a negative control while cisplatin (50 µM) was
4.2.6. Apoptosis analysis
◦
used as a positive control. After 48 h of 37 C and 5% CO
2
incubation,
Cells were treated either with 0.6 µM cisplatin alone or treated with
20 µM compound 23 for 4 h then treated with 0.6 µM cisplatin for total
48 h. The cells were combined after trypsinization and washed two times
with 1x PBS. The induction of apoptosis was analyzed by staining cells
with PE-Annexin-V [79] in presence of propidium iodide (BD Phar-
mingen™, Cat. No. 556547) and analyzed by Accuri C6 Flow cytometer.
The apoptotic index was calculated as the percentage of Annexin V-
positive and PI-negative cells (early apoptosis) divided by the total
number of cells in the gated region (viable cells).
MTT (5 mg/mL PBS) was added and the plate was incubated for 4 h [76].
Afterward, acidified sodium dodecyl sulfate solution (10% SDS in 1x
PBS/0.01 N HCl) was used to solubilize the produced formazan crystals.
The absorbance was measured at λ570-630 nm by Biotek plate reader
◦
(
Gen5™) after fourteen h of incubation at 37 C and 5% CO
2
Based on the initial screening assay results, a serial dilution of the
most cytotoxic organoselenium pseudo peptides was used for the eval-
uation of its IC50. The HepG2 cells were seeded as above then were
treated with different concentrations (50, 25, 12.5, 6.25, 3.125, and
1
.56 µM) of the most active compound. After 48 h of incubation, the
4.2.7. Reactive oxygen species determination
viability of the cells was determined by using MTT assay as described
above. IC50 of compounds were calculated by GraphPad Prism 8
software.
Cells were treated either with 0.6 µM cisplatin alone or treated with
20 µM compound 23 for 4 h then treated with 0.6 µM cisplatin for total
48 h. After that, the cells were collected, washed then stained with 20
◦
μ
M DCFDA (Abcam®, ab113851) and incubated for 30 min at 37 C. The
4
.2.4. Chemo-sensitization activity
stained cells were analyzed by Accuri C6 Flow cytometer.
The cells were seeded as mentioned above, and on the next day, the
cells were initially pretreated with 20 or 40 µM of the non-toxic com-
pounds (the concentration that showed 100% cell viability, data not
shown) for 4 h then treated with a serial dilution of cisplatin (50, 25,
4
4
.3. Synthesis and characterization
.3.1. Preparation of acetic formic anhydride [50]
1
2.5, 6.25, 3.125, and 1.56 µM) for another 44 h. After incubation at 37
◦
In a dry, three-necked flask, 1.88 mol (133 mL) acetyl chloride is
2
C and 5% CO , the viability was determined by MTT reagent. The
added to 2.2 mol (150 g) sodium formate in 125 mL of diethyl ether
viability was calculated by GraphPad Prism 8 software. The compound
that should chemo-sensitizing activity was further used in the study
where different concentrations and time points of preincubation were
tested [77,80,81].
◦
(
anhydrous) while the temperature is maintained at 0 C. The mixture is
◦
then allowed to stir for 6 h at 26 C and filtered. The residue solid is
washed twice with ether (2 X 50 mL). The ether organic solvent is
removed by under low pressure, and the resulted acetic formic anhy-
dride residue is further used without any purification.
4
.2.5. Cell cycle analysis
General procedure I: Preparation of isocyanide derivatives 5 & 6
Cells were treated either with 0.6 µM cisplatin alone or treated with
0 µM compound 23 for 4 h then treated with 0.6 µM cisplatin for a total
(
Scheme 1)
2
Freshly prepared acetic formic anhydride (1.2 mmol) was added
of 48 h. Then the cells were fixed by using ethanol (ice-cold) for two h at
◦
dropwise at room temperature to a solution of selenoamine (1 mmol) in
THF solution (10 mL). The reaction progress was monitored with TLC,
ꢀ
20 C. Cells were then washed with PBS and stained with PI (20 µg/mL,
9

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
and after the reaction completion, the corresponding N-formyl de-
purified employing “flash” chromatography using petrol ether: ethyl
rivatives obtained quantitatively was used without any purification.
acetate = 4:3. White solid; Yield: 133 mg (45%). IR (KBr) ν = 490 (Se-C),
–
–
ꢀ 1
1
POCl
3
(1.2 mmol) in THF (5 mL) was added carefully, drop-by-drop,
1587 (C
MHz, CDCl
2.15 (s, 3H, H
159.43, 138.85, 134.48, 119.76, 116.17, 102.03, 63.14, 20.76. MS
–
O), 1684 (C
–
O), 2238 (CN), 3665 (NH) cm ; H NMR (300
H), 7.52 (m, 4H, Ar-H), 4.61 (s, 2H, CH ),
O); C NMR (75 MHz, CDCl ) δ 169.72, 165.68,
to a solution of N-formyl derivatives (1 mmol) and Et
3
N (3 mmol) in THF
3
) δ 8.22 (s, 1H, N
–
13
2
◦
–
(
5 mL) at 00 C. The reaction progress was monitored with TLC, and after
3
CC
–
3
the reaction completion, a saturated Na
2
CO
3
aqueous cold solution was
◦
+
+
added, and the THF layer was washed with brine (2 X 50 mL). At 0 C, the
THF layer was removed under low pressure and purified by flash “col-
umn” chromatography (petroleum ether/ ethyl acetate = 8/1).
Isocyano-4-selenocyanatobenzene (5)2
(ESI): m/z = found 297.02 [M ꢀ H]; calcd 297.98 [M ]; HRMS calcd.
+
+
for C11
H
10
N
2
O
3
Se [M ]: 297.98566, found 297.98491 [M ]. Anal.
Calcd for C11
10 2 3
H N O Se (calcd 297.98): C, 44.46; H, 3.39; N, 9.43.
Found: C, 44.48; H, 3.41; N, 9.46.
Compound 5 synthesized following procedure I from 4-selenocyana-
toaniline 1 (198 mg, 1 mmol). TLC was used to monitor its formation
1-(4-nitrophenyl)-2-oxo-2-((4-selenocyanatophenyl)amino)ethyl ace-
tate (9)
petrol ether: ethyl acetate = 10:1, R
f
= 0.35, purified employing “flash”
Compound 9 synthesized following procedure II from isocyano-4-
selenocyanatobenzene (5) (229 mg, 1.1 mmol), acetic acid (63 µL, 1
mmol), and p-nitrobenzaldehyde (151 mg, 1 mmol). TLC was used to
chromatography using petrol ether: ethyl acetate = 8:1. White solid;
1
Yield: 191 mg (92%). H NMR (300 MHz, DMSO) δ 7.89–7.82 (dd, J =
1
3
7
.8, 1.3 Hz, 2H, Ar-H), 7.69–7.62 (dd, J = 7.8, 1.3 Hz, 2H, Ar-H);
C
monitor its formation petrol ether: ethyl acetate = 2:1, R
rified employing “flash” chromatography using petrol ether: ethyl ace-
tate = 4:3. White solid; Yield: 163 mg (39%). IR (KBr) = 504 (Se-C),
f
= 0.29, pu-
NMR (75 MHz, DMSO) δ 166.15, 134.43, 128.22, 127.06, 105.37. MS
+
+
(
ESI): m/z = found 182.23 [M ꢀ CN], 324.12 [M +3K]; calcd. 207.98
ν
1
+
–
–
ꢀ 1
[
M ].
1610 (C
MHz, CDCl
(m, 2H, Ar-H), 7.53 (m, 2H, Ar-H), 7.51 (m, 2H, Ar-H), 6.17 (s, 1H), 2.23
–
O), 1730 (C
–
O), 2245 (CN), 3681 (NH) cm ; H NMR (300
1
,2-bis(4-isocyanophenyl)diselane (6)
Compound 6 synthesized following procedure I from 4,4 -dis-
elanediyldianiline 2 (342 mg, 1 mmol). TLC was used to monitor its
formation petrol ether: ethyl acetate = 10:1, R = 0.33, purified
3
) δ 8.32 (s, 1H, N
–
H), 8.19–8.12 (m, 2H, Ar-H), 7.65–7.60
′
1
3
(s, 3H, CH
3 3
); C NMR (75 MHz, CDCl ) δ 169.05, 165.60, 159.15,
f
141.64, 138.64, 134.39, 128.23, 124.01, 121.71, 101.85, 74.66, 20.97.
+
+
employing “flash” chromatography using petrol ether: ethyl acetate =
MS (ESI): m/z = found 419.79 [M ꢀ H]; calcd 419.00 [M ]; HRMS
1
+
+
8
7
4
1
:1. Yellow solid; Yield: 342 mg (94%). H NMR (300 MHz, CDCl
.56–7.46 (dd, J = 7.7, 1.4 Hz, 4H, Ar-H), 7.23–7.14 (dd, J = 7.7, 1.4 Hz,
H, Ar-H); 13C NMR (75 MHz, CDCl
) δ 165.68, 132.02, 132.19, 131.84,
3
) δ
calcd. for C17
H
13
3
N O
5
Se [M ]: 419.00204, found 419.00149 [M ].
Anal. Calcd for C17
H N O Se (calcd 419.00): C, 48.82; H, 3.13; N,
13 3 5
3
10.05. Found: C, 48.80; H, 3.10; N, 10.03.
+
+
27.14. MS (ESI): m/z = found 365.90 [M +2H]; calcd 363.90 [M ];
((diselanediylbis(4,1-phenylene))bis(azanediyl))bis(3-methyl-1-oxobu-
tane-1,2-diyl) diacetate (17)
+
HRMS calcd. for C14
H
8
N
2
Se
2
[M ]: 363.90179, found 366.16024
+
[
M +2H]. Anal. Calcd for C14
H
8
N
2
Se
2
(363.90): C, 46.43; H, 2.23; N,
Compound 17 synthesized following procedure II from 1,2-bis(4-iso-
cyanophenyl)diselane (6) (200 mg, 0.55 mmol), acetic acid (63 µL, 1
mmol), and isopropyl aldehyde (91 µL, 1 mmol). TLC was used to
7
.74. Found: C, 46.40; H, 2.20; N, 7.71.
General procedure II: Synthesis of functionalized pseudopep-
tide organoselenium derivatives (7–9 & 17–19) via Passerini reac-
tion (Scheme 2 and 3)
monitor its formation petrol ether: ethyl acetate = 2:1, R = 0.27, pu-
f
rified employing “flash” chromatography using petrol ether: ethyl ace-
1
Isonitrile (1.1 mmol) was added to a mixture of acid component (1
tate = 4:3. Yellow solid; Yield: 528 mg (84%). H NMR (300 MHz,
mmol), and aldehyde (1 mmol) in CH
2
Cl
2
(1 mL), and the mixture was
3
CDCl ) δ 8.11 (s, 2H, 2 N H), 7.41–7.25 (m, 8H, Ar-H), 4.96 (d, J = 5.2
–
allowed to stir for 12 h at room temperature. Water was added and the
organic layer was separated, dried with Sodium sulfate, and evaporated
under low pressure. The residue obtained was purified by column “flash”
chromatography using petroleum ether/ ethyl acetate = 5/3.
Hz, 2H, 2CH), 2.21 (ddd, J = 14.2, 7.1, 5.8 Hz, 2H, 2CH), 2.10 (s, 6H,
–
13
2H
3
CC
–
O), 0.91 (d, J = 6.8 Hz, 12H, 4CH
3 3
). C NMR (75 MHz, CDCl )
δ 170.30, 167.95, 137.13, 133.47, 126.39, 120.92, 78.56, 60.47, 30.77,
+
20.98, 18.67, 17.33, 14.20. MS (ESI): m/z = found 628.97 [M ]; calcd
+
+
3
-Methyl-1-oxo-1-((4-selenocyanatophenyl)amino)butan-2-yl acetate
628.06 [M ]; HRMS calcd. for C26
H
32
N
2
O
6
Se
2
[M ]: 628.05908, found
+
(
7)
628.05876 [M ]. Anal. Calcd for C26
H
32
N
2
O
Se
6 2
(calcd 628.06): C,
Compound 7 synthesized following procedure II from isocyano-4-
49.85; H, 5.15; N, 4.47. Found: C, 49.83; H, 5.12; N, 4.45.
((diselanediylbis(4,1-phenylene))bis(azanediyl))bis(2-oxoethane-2,1-
diyl) diacetate (18)
selenocyanatobenzene (5) (229 mg, 1.1 mmol), acetic acid (63 µL, 1
mmol), and isopropyl aldehyde (91 µL, 1 mmol). TLC was used to
monitor its formation petrol ether: ethyl acetate = 2:1, R
f
= 0.30, pu-
Compound 18 synthesized following procedure II from 1,2-bis(4-iso-
cyanophenyl)diselane (6) (200 mg, 0.55 mmol), acetic acid (63 µL, 1
mmol), and paraformaldehyde (60 mg, 1 mmol). TLC was used to
rified employing “flash” chromatography using petrol ether: ethyl ace-
tate = 4:3. White solid; Yield: 342 mg (86%). IR (KBr) = 506 (Se-C),
ν
1
–
–
ꢀ 1
1
590 (C
–
O), 1695 (C
) δ 8.31 (s, 1H, N
.2 Hz, 1H, CH-O), 2.27–2.15 (m, 1H, CH), 2.12 (s, 3H, H
–
O), 2222 (CN), 3565 (NH) cm ; H NMR (300
monitor its formation petrol ether: ethyl acetate = 2:1, R = 0.27, pu-
f
MHz, CDCl
3
–
H), 7.54–7.36 (m, 4H, Ar-H), 4.91 (d, J =
rified employing “flash” chromatography using petrol ether: ethyl ace-
–
–
1
5
3
CC
) δ 170.53, 168.33,
39.15, 134.38, 121.75, 115.69, 102.30, 78.55, 30.76, 20.88, 18.64,
O),
tate = 4:3. Yellow solid; Yield: 479 mg (88%). H NMR (300 MHz,
1
3
0
.98–0.87 (m, 6H, 2CH
3
); C NMR (75 MHz, CDCl
3
CDCl ) δ 7.79 (s, 2H, 2 N H), 7.54–7.44 (m, 4H, Ar-H), 7.42–7.33 (m,
3
–
13
1
4H, Ar-H), 4.61 (s, 4H, 2CH
CDCl
2
), 2.15 (s, 6H, 2CH
3
); C NMR (75 MHz,
+
+
1
7.34. MS (ESI): m/z = found 340.87 [M ]; calcd 340.03 [M ]; HRMS
3
) δ 169.32, 165.08, 136.82, 133.65, 126.68, 120.75, 63.30, 20.78.
+
+
+
+
calcd. for C14
H
16
N
2
O
3
Se [M ]: 340.03261, found 340.03156 [M ].
Se (calcd 340.03): C, 49.57; H, 4.75; N, 8.26.
Found: C, 49.54; H, 4.74; N, 8.28.
-oxo-2-((4-selenocyanatophenyl)amino)ethyl acetate (8)
MS (ESI): m/z = found 544.87 [M ]; calcd 543.96 [M ]; HRMS calcd.
+
+
Anal. Calcd for C14
H
16
2
N O
3
for C20
H
20
N
2
O
Se
6 2
[M ]: 543.96518, found 544.96363 [M +H]. Anal.
Se (calcd 543.96): C, 44.30; H, 3.72; N, 5.17.
Calcd for C20
H
20
N
2
O
6
2
2
Found: C, 44.32; H, 3.75; N, 5.20.
Compound 8 synthesized following procedure II from isocyano-4-
selenocyanatobenzene (5) (229 mg, 1.1 mmol), acetic acid (63 µL, 1
mmol), and paraformaldehyde (30 mg, 1 mmol). TLC was used to
((diselanediylbis(4,1-phenylene))bis(azanediyl))bis(1-(4-nitrophenyl)-
2-oxoethane-2,1-diyl) diacetate (19)
Compound 19 synthesized following procedure II from 1,2-bis(4-iso-
cyanophenyl)diselane (6) (200 mg, 0.55 mmol), acetic acid (63 µL, 1
mmol), and para nitrobenzaldehyde (151 mg, 1 mmol). TLC was used to
monitor its formation petrol ether: ethyl acetate = 2:1, R = 0.31,
f
monitor its formation petrol ether: ethyl acetate = 2:1, R = 0.25, pu-
f
2
rified employing “flash” chromatography using petrol ether: ethyl ace-
Isocyano-4-selenocyanatobenzene (5) is not stable and must be prepared
directly before usage and therefore our trials to obtain HRMS was not possible.
1
tate = 4:3. Yellow solid; Yield: 637 mg (81%). H NMR (300 MHz,
1
0

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
CDCl
3
) δ 8.60 (s, 2H, 2 N
–
H), 7.97 (d, J = 8.8 Hz, 4H, Ar-H), 7.53 (d, J
cm 1; 1H NMR (300 MHz, CDCl
ꢀ
) δ 9.68 (s, 1H, N H), 7.62–7.48 (m,
–
3
=
8.9 Hz, 4H, Ar-H), 7.24 (d, J = 8.6 Hz, 4H, Ar-H), 7.15 (d, J = 8.8 Hz,
4H, Ar-H), 7.38–7.28 (m, 3H, Ar-H), 7.17 (ddd, J = 5.5, 2.8, 1.2 Hz, 2H,
–
–
13
4
H, Ar-H), 6.08 (s, 2H, 2CH), 2.13 (s, 6H, 2H
3
CC
O); C NMR (75
Ar-H), 4.26 (d, J = 11.2 Hz, 1H, CH), 2.41 (ddq, J = 19.5, 13.0, 6.6 Hz,
–
MHz, CDCl
3
) δ 169.54, 165.97, 148.07, 141.84, 136.59, 132.95, 128.24,
1H, CH), 1.83 (s, 3H, H
3
CC
–
O), 1.01 (d, J = 6.5 Hz, 3H, CH
3
), 0.92 (d, J
1
26.98, 123.88, 121.15, 74.71, 20.88. MS (ESI): m/z = found 803.61
= 6.6 Hz, 3H, CH
3
); 13C NMR (75 MHz, CDCl
3
) δ 173.21, 169.33,
+
+
+
[
M +H
2
O]; calcd 785.99 [M ]; HRMS calcd. for C32
H
26
N
4
O
10Se
2
[M ]:
10Se
141.63, 140.27, 134.67, 129.77, 128.73, 128.38, 121.36, 115.06,
+
7
85.99794, found 787.09301 [M +H]. Anal. Calcd for C32
H
26
N
4
O
2
101.76, 72.11, 27.00, 23.87, 20.23, 19.74. MS (ESI): m/z = found
+
+
+
+
(
calcd 785.99): C, 48.99; H, 3.34; N, 7.14. Found: C, 48.97; H, 3.32; N,
.11.
General procedure III: Synthesis of functionalized peptidomi-
415.88 [M ]; calcd 415.07 [M ]; HRMS calcd. for C20
H
21
N
3
O
2
Se [M ]:
7
415.08, found 438.06710 [M +Na]. Anal. Calcd for C20
21 3 2
H N O Se
(calcd 415.08): C, 57.97; H, 5.11; N, 10.14. Found: C, 57.99; H, 5.13; N,
10.16.
metic organoselenium derivatives (10–12 & 20–23) via Ugi reaction
′
(
Scheme 2 and 3)
N,N -(diselanediylbis(4,1-phenylene))bis(3-methyl-2-(N-phenyl-
Isonitrile (1.1 mmol) was added to a mixture of acid components (1
acetamido)butanamide) (20)
mmol), aldehyde (1 mmol), and amine (1 mmol) in CH
3
OH (1 mL), and
Compound 20 synthesized following procedure III from 1,2-bis(4-
isocyanophenyl)diselane (6) (200 mg, 0.55 mmol), acetic acid (63 µL,
1 mmol), aniline (46 µL, 1 mmol), and isopropyl aldehyde (91 µL, 1
mmol). TLC was used to monitor its formation petrol ether: ethyl acetate
the mixture was allowed to stir for 12 h at room temperature. Water was
added and extracted with 3x10 mL dichloromethane. The organic layer
was separated, dried with anhydrous Sodium sulfate, and evaporated
under low pressure. The residue obtained was purified by column “flash”
chromatography using petroleum ether/ ethyl acetate = 5/3.
= 2:1, R
petrol ether: ethyl acetate = 5:2. Yellow solid; Yield: 638 mg (82%). H
NMR (300 MHz, CDCl ) δ 9.40 (s, 2H, 2 N H), 7.53–7.41 (m, 8H, Ar-H),
7.37–7.27 (m, 6H, Ar-H), 7.20–7.12 (m, 4H, Ar-H), 4.21 (d, J = 11.2 Hz,
f
= 0.33, purified employing “flash” chromatography using
1
2
-(N-(4-methoxyphenyl)acetamido)-3-methyl-N-(4-selenocyanato-
3
–
phenyl)butanamide (10)
–
Compound 10 synthesized following procedure III from isocyano-4-
selenocyanatobenzene (5) (229 mg, 1.1 mmol), acetic acid (63 µL, 1
mmol), p-anisidine (123 mg, 1 mmol), and isopropyl aldehyde (91 µL, 1
mmol). TLC was used to monitor its formation petrol ether: ethyl acetate
2H, 2CH), 2.44 (tt, J = 12.8, 6.6 Hz, 2H, 2CH), 1.82 (s, 6H, 2H
3
CC
–
O),
); C NMR
) δ 173.09, 168.98, 141.76, 138.48, 133.79, 129.72,
128.59, 128.33, 125.75, 120.41, 72.34, 26.92, 23.87, 20.19, 19.80. MS
1
3
1.00 (d, J = 6.5 Hz, 6H, 2CH
3
), 0.93 (d, J = 6.6 Hz, 6H, 2CH
3
(75 MHz, CDCl
3
+
+
=
3:1, R
f
= 0.36, purified employing “flash” chromatography using
(ESI): m/z = found 779.17 [M +H]; calcd 778.15 [M ]; HRMS calcd.
+
+
petrol ether: ethyl acetate = 5:3. White solid; Yield: 396 mg (89%). IR
for C38H
42
4
N O
4
Se
2
[M ]: 778.15365, found 801.13910 [M +Na]. Anal.
Se (calcd 778.15): C, 58.76; H, 5.45; N, 7.21.
–
–
–
–
(
KBr)
ν = 494 (Se-C), 1592 (C
O), 1744 (C
cm ; H NMR (300 MHz, CDCl ) δ 9.46 (s, 1H, N
H, Ar-H), 7.08–7.00 (m, 2H, Ar-H), 6.86–6.78 (m, 2H, Ar-H), 4.26–4.12
m, 1H, CH), 3.74 (s, 3H, H
CO), 2.50–2.26 (m, 1H, CH), 1.86 (s, 3H,
O), 1.01 (d, J = 6.5 Hz, 3H, H C), 0.93 (d, J = 6.6 Hz, 3H, H C);
C NMR (75 MHz, CDCl ) δ 173.77, 169.31, 159.46, 140.24, 134.74,
29.29, 121.32, 114.94, 101.65, 55.50, 26.71, 23.74, 20.13, 19.71. MS
O), 2256 (CN), 3623 (NH)
Calcd for C38
H
42
N
4
O
4
2
ꢀ
1 1
–
H), 7.62–7.51 (m,
Found: C, 58.74; H, 5.43; N, 7.22.
3
′
4
N,N -(diselanediylbis(4,1-phenylene))bis(2-(N-benzylacetamido)-3-
(
3
methylbutanamide) (21)
–
–
H
1
3
3
CC
3
3
Compound 21 synthesized following procedure III from 1,2-bis(4-
isocyanophenyl)diselane (6) (200 mg, 0.55 mmol), acetic acid (63 µL,
1 mmol), benzylamine (108 µL, 1 mmol), and isopropyl aldehyde (91 µL,
1 mmol). TLC was used to monitor its formation petrol ether: ethyl ac-
3
1
+
+
(
ESI): m/z = found 445.91 [M ]; calcd 445.09 [M ]; HRMS calcd. for
+
+
C
21
H
23
N
H
3
O
3
Se [M ]: 445.09046, found 446.09742 [M ]. Anal. Calcd
Se (calcd 445.09): C, 56.76; H, 5.22; N, 9.46. Found: C,
etate = 2:1, R
petrol ether: ethyl acetate = 5:2. Yellow solid; Yield: 645 mg (80%). H
NMR (300 MHz, CDCl ) δ 9.29 (s, 2H, 2 N H), 7.42–7.38 (m, 3H, Ar-H),
7.29–7.24 (m, 5H, Ar-H), 7.13–7.02 (m, 10H, Ar-H), 4.74–4.61 (d, J =
17.1 Hz, 2H, 2CH), 4.52 (d, J = 17.1 Hz, 4H, 2CH ), 2.48 (tt, J = 12.9,
O), 0.91 (d, J = 6.5 Hz, 6H,
f
= 0.34, purified employing “flash” chromatography using
1
for C21
23
N
3
O
3
5
6.78; H, 5.25; N, 9.49.
-(N-(4-methoxyphenyl)acetamido)-4-methyl-N-(4-selenocyanato-
3
–
2
phenyl)pentanamide (11)
2
–
–
13
Compound 11 synthesized following procedure III from isocyano-4-
selenocyanatobenzene (5) (229 mg, 1.1 mmol), acetic acid (63 µLl, 1
mmol), p-anisidine (123 mg, 1 mmol), and isovaleraldehyde (110 µl, 1
mmol). TLC was used to monitor its formation petrol ether: ethyl acetate
6.3 Hz, 2H, 2CH), 2.03 (s, 6H, 2H
2CH
3
CC
3
), 0.81 (d, J = 6.6 Hz, 6H, 2CH
3
); C NMR (75 MHz, CDCl ) δ
3
173.56, 168.53, 138.29, 137.51, 136.76, 133.57, 128.81, 127.46,
126.25, 125.82, 120.91, 120.60, 66.80, 50.40, 26.95, 22.71, 19.82,
+
+
=
3:1, R
f
= 0.29, purified employing “flash” chromatography using
19.08. MS (ESI): m/z = found 807.24 [M +H]; calcd 806.18 [M ];
+
petrol ether: ethyl acetate = 5:3. White solid; Yield: 367 mg (80%). IR
HRMS calcd. for C40
H
46
N
4
O
4
Se
2
[M ]: 806.18495, found 829.17511
–
–
–
–
+
(
KBr)
ν = 504 (Se-C), 1654 (C
O), 1739 (C
cm ; H NMR (300 MHz, DMSO) δ 10.37 (s, 1H, N
H, Ar-H), 7.33 (d, J = 8.5 Hz, 2H, Ar-H), 6.98 (dd, J = 7.8, 1.3 Hz, 2H,
CO), 1.75 (s, 3H,
), 1.41–1.25 (m, 1H, CH), 0.87 (dd, J
); C NMR (75 MHz, DMSO) δ 170.84, 159.17,
O), 2243 (CN), 3601 (NH)
[M +Na]. Anal. Calcd for C40
46 4 4 2
H N O Se (calcd 806.18): C, 59.70; H,
ꢀ
1 1
–
H), 7.80–7.64 (m,
5.76; N, 6.96. Found: C, 59.68; H, 5.74; N, 6.93.
′
4
N,N -(diselanediylbis(4,1-phenylene))bis(2-(N-(4-methoxyphenyl)acet-
Ar-H), 5.21 (t, J = 7.2 Hz, 1H, CH), 3.79 (s, 3H, H
3
amido)-3-methylbutanamide) (22)
–
H
3
CC
–
O), 1.59–1.43 (m, 2H, CH
2
Compound 22 synthesized following procedure III from 1,2-bis(4-
isocyanophenyl)diselane (6) (200 mg, 0.55 mmol), acetic acid (63 µL,
1 mmol), p-anisidine (123 mg, 1 mmol), and isopropyl aldehyde (91 µL,
1 mmol). TLC was used to monitor its formation petrol ether: ethyl ac-
1
3
=
12.2, 6.2 Hz, 6H, 2CH
3
1
40.79, 135.12, 132.60, 131.55, 121.45, 116.96, 114.57, 105.57, 57.95,
+
5
5.72, 24.89, 23.52, 23.00. MS (ESI): m/z = found 481.87 [M +Na];
+
+
calcd 459.10 [M ]; HRMS calcd. for C22
H
25
N
3
O
3
Se [M ]: 459.10611,
etate = 2:1, R
petrol ether: ethyl acetate = 5:2. Yellow solid; Yield: 511 mg (61%). H
NMR (300 MHz, CDCl
) δ 7.54–7.32 (m, 8H, Ar-H), 7.09–7.02 (m, 4H,
Ar-H), 6.87–6.76 (m, 4H, Ar-H), 4.22 (d, J = 11.7 Hz, 2H, 2CH), 3.74 (s,
6H, 2H
CO), 2.36 (dd, J = 11.3, 5.7 Hz, 2H, 2CH), 1.81 (s, 6H, 2
f
= 0.30, purified employing “flash” chromatography using
+
1
found 482.09533 [M +H + Na]. Anal. Calcd for C22
25 3 3
H N O Se (calcd
4
59.10): C, 57.64; H, 5.50; N, 9.17. Found: C, 57.62; H, 5.53; N, 9.20.
-methyl-2-(N-phenylacetamido)-N-(4-selenocyanatophenyl)butana-
mide (12)
Compound 12 synthesized following procedure III from isocyano-4-
3
3
3
–
H
3
CC
–
O), 0.99 (d, J = 6.5 Hz, 6H, 2CH
3
), 0.93 (d, J = 6.6 Hz, 6H,
1
3
selenocyanatobenzene (5) (229 mg, 1.1 mmol), acetic acid (63 µL, 1
mmol), aniline (91 µL, 1 mmol), and isopropyl aldehyde (91 µL, 1
mmol). TLC was used to monitor its formation petrol ether: ethyl acetate
2CH ). C NMR (75 MHz, CDCl ) δ 173.54, 168.96, 159.38, 138.45,
3 3
137.70, 133.86, 129.38, 125.85, 120.49, 114.76, 55.49, 26.69, 23.73,
+
20.13, 19.75. MS (ESI): m/z = found 837.25 [M ꢀ H]; calcd 838.17
+
+
=
3:1, R
f
= 0.29, purified employing “flash” chromatography using
[M ]; HRMS calcd. for C40
H
46
N
4
O
6
Se
2
[M ]: 838.17478, found
+
petrol ether: ethyl acetate = 5:3. White solid; Yield: 367 mg (80%). IR
838.17561 [M ]. Anal. Calcd for C40
H
46
N
4
O
6
Se
2
(calcd 838.17): C,
–
–
–
O), 2239 (CN), 3591 (NH)
(
KBr)
ν
= 502 (Se-C), 1644 (C
–
O), 1733 (C
57.42; H, 5.54; N, 6.70. Found: C, 57.39; H, 5.51; N, 6.68.
1
1

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
′
N,N -(diselanediylbis(4,1-phenylene))bis(2-(N-benzylacetamido)acet-
N-(3-methyl-1-(1-(4-selenocyanatophenyl)-1H-tetrazol-5-yl)butyl)ani-
amide) (23)
Compound 23 synthesized following procedure III from 1,2-bis(4-
line (15)
Compound 15 synthesized following procedure IV from isocyano-4-
selenocyanatobenzene (5) (229 mg, 1.1 mmol), TMSN
isocyanophenyl)diselane (6) (200 mg, 0.55 mmol), acetic acid (63 µL,
3
(145 µL, 1.1
1
1
mmol), benzylamine (108 µL, 1 mmol), and paraformaldehyde (30 mg,
mmol). TLC was used to monitor its formation petrol ether: ethyl ac-
mmol), isovaleraldehyde (110 µL, 1 mmol), and aniline (46 µL, 1 mmol).
TLC was used to monitor its formation petrol ether: ethyl acetate = 3:1,
etate = 2:1, R
petrol ether: ethyl acetate = 5:2. Yellow solid; Yield: 570 mg (79%). H
NMR (300 MHz, CDCl ) δ 8.90 (s, 2H, 2 N H), 7.36–7.29 (m, 4H, Ar-H),
.26–7.18 (m, 10H, Ar-H), 7.09 (d, J = 6.6 Hz, 4H, Ar-H), 4.56 (d, J =
6.5 Hz, 4H, 2CH ), 2.11 (s, 6H,
), 3.95 (d, J = 23.6 Hz, 4H, 2CH
O); C NMR (75 MHz, CDCl ) δ 172.63, 167.28, 138.06,
f
= 0.20, purified employing “flash” chromatography using
R
f
= 0.20, purified employing “flash” chromatography using petrol
1
ether: ethyl acetate = 5:3. White solid; Yield: 222 mg (54%). IR (KBr)
ν
–
–
–
–
ꢀ 1
;
–
= 506 (Se-C), 1651 (C
C), 1633 (C
H NMR (600 MHz, CDCl
) δ 7.72–7.62 (m, 2H, Ar-H), 7.30–7.24 (m,
1H, Ar-H), 7.19–7.15 (m, 1H, Ar-H), 7.09–6.94 (m, 2H, Ar-H), 6.71–6.59
N), 2251 (CN), 3650 (NH) cm
3
1
7
1
2
1
1
3
2
2
–
–
13
–
H
3
CC
3
(m, 1H, Ar-H), 6.40–6.29 (m, 2H, Ar-H), 4.90 (s, 1H, N H), 3.90–3.73
(m, 1H, CH), 1.88–1.69 (m, 2H, CH ), 1.64–1.50 (m, 1H, CH), 0.80 (ddd,
35.58, 133.53, 129.03, 128.24, 127.92, 126.74, 125.72, 120.65,
2
13
+
20.33, 53.74, 50.96, 21.47. MS (ESI): m/z = found 745.08 [M +Na];
J = 29.1, 15.1, 7.1 Hz, 6H, 2CH
3 3
). C NMR (75 MHz, CDCl ) δ 157.11,
+
+
calcd 722.09 [M ]; HRMS calcd. for C34
H
34
4
N O
4
Se
2
[M ]: 722.09105,
145.45, 136.88, 134.90, 134.30, 133.63, 133.20, 131.91, 129.55,
+
found 723.09540 [M +H]. Anal. Calcd for C34
34 4 4 2
H N O Se (calcd
127.36, 126.37, 125.49, 119.32, 113.79, 47.42, 43.78, 24.92, 22.32. MS
+
+
7
22.09): C, 56.67; H, 4.76; N, 7.78. Found: C, 56.65; H, 4.74; N, 7.76.
General procedure IV: Synthesis of functionalized
(ESI): m/z = found 457.12 [M +2Na]; calcd 412.09 [M ]; HRMS calcd.
+
+
for C19 Se [M ]: 412.09147, found 412.10317 [M +H]. Anal.
H
20
N
6
organoselenium-based tetrazoles (13–16 & 24–27) via azido-Ugi
reaction (Scheme 2 and 3)
Calcd for C19H N Se (calcd 412.09): C, 55.48; H, 4.90; N, 20.43. Found:
20 6
C, 55.50; H, 4.92; N, 20.45.
Isonitrile (1.1 mmol) was added to a mixture of TMSN
3
(1.1 mmol),
4-methoxy-N-(3-methyl-1-(1-(4-selenocyanatophenyl)-1H-tetrazol-5-
yl)butyl)aniline (16)
aldehyde (1 mmol), and amine (1 mmol) in CH OH (1 mL), and the
3
mixture was allowed to stir for 12 h at room temperature. Water was
added and extracted with 3x10 mL dichloromethane. The organic layer
was separated, dried with anhydrous Sodium sulfate, and evaporated
under low pressure. The residue obtained was purified by column “flash”
chromatography using petroleum ether/ ethyl acetate = 5/3.
N-(2-methyl-1-(1-(4-selenocyanatophenyl)-1H-tetrazol-5-yl)propyl)an-
iline (13)
Compound 16 synthesized following procedure IV from isocyano-4-
selenocyanatobenzene (5) (229 mg, 1.1 mmol), TMSN
3
(145 µL, 1.1
mmol), isovaleraldehyde (110 µL, 1 mmol), and p-anisidine (123 mg, 1
mmol). TLC was used to monitor its formation petrol ether: ethyl acetate
= 3:1, R
f
= 0.20, purified employing “flash” chromatography using
petrol ether: ethyl acetate = 5:3. White solid; Yield: 155 mg (35%). IR
–
3
–
–
N), 2249 (CN), 3648 (NH)
(KBr)
ν = 497 (Se
–
C), 1643 (C
cm ; H NMR (600 MHz, CDCl
(d, J = 8.6 Hz, 2H, Ar-H), 6.68–6.58 (m, 2H, Ar-H), 6.38–6.29 (m, 2H,
Ar-H), 4.68 (d, J = 7.3 Hz, 1H, N H), 4.05 (s, CH O), 1.87–1.72 (m, 2H,
CH
), 1.63–1.54 (m, 1H, CH), 1.25–1.13 (m, 1H, CH), 0.85–0.74 (dd, J
C), 1596 (C
ꢀ
1 1
Compound 13 synthesized following procedure IV from isocyano-4-
) δ 7.71 (t, J = 10.3 Hz, 2H, Ar-H), 7.27
selenocyanatobenzene (5) (229 mg, 1.1 mmol), TMSN
3
(145 µL, 1.1
mmol), isopropyl aldehyde (91 µL, 1 mmol), and aniline (91 µL, 1
mmol). TLC was used to monitor its formation petrol ether: ethyl acetate
–
3
2
1
3
=
3:1, R
f
= 0.25, purified employing “flash” chromatography using
= 6.6, 3.9 Hz, 6H, 2CH
3 3
). C NMR (151 MHz, CDCl ) δ 157.29, 153.70,
petrol ether: ethyl acetate = 5:3. White solid; Yield: 334 mg (84%). IR
139.23, 134.94, 133.05, 127.32, 125.34, 116.19, 115.03, 100.10, 55.66,
–
3
–
+
(
KBr)
ν
= 489 (Se-C), 1660 (C
–
C), 1633 (C
–
N), 2239 (CN), 3586 (NH)
49.04, 43.76, 24.95, 22.32. MS (ESI): m/z = found 443.02 [M +H];
ꢀ
1
1
+
+
cm ; H NMR (250 MHz, CDCl
) δ 7.72–7.60 (m, 2H, Ar-H), 7.25–7.12
calcd 442.10 [M ]; HRMS calcd. for C20
H
22
N
6
OSe [M ]: 442.10203,
+
(
m, 2H, Ar-H), 6.99 (dd, J = 8.3, 7.5 Hz, 2H, Ar-H), 6.63 (t, J = 7.3 Hz,
found 442.100982 [M ]. Anal. Calcd for C20
H
22
N
6
OSe (calcd 442.10):
1
H, Ar-H), 6.36 (d, J = 7.7 Hz, 2H, Ar-H), 4.52 – (s, 1H, N
–
H), 4.01 (q, J
),
) δ 156.81,
C, 54.42; H, 5.02; N, 19.04. Found: C, 54.39; H, 5.00; N, 19.01.
′
=
7.1 Hz, 1H, CH), 2.26–2.04 (m, 1H, CH), 0.98 (d, J = 6.6 Hz, 3H, CH
3
N,N -(((diselanediylbis(4,1-phenylene))bis(1H-tetrazole-1,5-diyl))bis
1
3
0
1
1
3
3
.83 (d, J = 6.7 Hz, 3H, CH
3
); C NMR (75 MHz, CDCl
3
(2-methylpropane-1,1-diyl))dianiline (24)
45.99, 134.72, 133.18, 129.50, 127.66, 125.66, 119.25, 114.06,
Compound 24 synthesized following procedure IV from 1,2-bis(4-
+
00.61, 55.32, 33.28, 19.40. MS (ESI): m/z = found 398.94 [M ]; calcd
isocyanophenyl)diselane (6) (200 mg, 0.55 mmol), TMSN
3
(145 µL,
+
+
98.07 [M ]; HRMS calcd. for C18
H
18
N
6
Se [M ]: 398.07582, found
1.1 mmol), isopropyl aldehyde (91 µL, 1 mmol), and aniline (91 µL, 1
mmol). TLC was used to monitor its formation petrol ether: ethyl acetate
+
98.08613 [M ]. Anal. Calcd for C18
H
18
N
6
Se (calcd 398.07): C, 54.41;
H, 4.57; N, 21.15. Found: C, 54.43; H, 4.60; N, 21.17.
-methoxy-N-(2-methyl-1-(1-(4-selenocyanatophenyl)-1H-tetrazol-5-
= 3:1, R
petrol ether: ethyl acetate = 5:3. White solid; Yield: 640 mg (86%). H
NMR (250 MHz, CDCl
) δ 7.72–7.60 (m, 4H, Ar-H), 7.25–7.12 (m, 4H,
Ar-H), 6.99 (dd, J = 8.3, 7.5 Hz, 4H, Ar-H), 6.63 (t, J = 7.3 Hz, 2H, Ar-
H), 6.36 (d, J = 7.7 Hz, 4H, Ar-H), 4.52 (s, 2H, 2 N H), 4.01 (q, J = 7.1
Hz, 2H, 2CH), 2.26–2.04 (m, 2H, 2CH), 0.98 (t, J = 6.6 Hz, 6H, 2CH ),
) δ 156.67,
f
= 0.23, purified employing “flash” chromatography using
1
4
yl)propyl)aniline (14)
3
Compound 14 synthesized following procedure IV from isocyano-4-
selenocyanatobenzene (5) (229 mg, 1.1 mmol), TMSN
3
(145 µL, 1.1
–
mmol), isopropyl aldehyde (91 µL, 1 mmol), and p-anisidine (123 mg, 1
mmol). TLC was used to monitor its formation petrol ether: ethyl acetate
3
1
3
0.83 (d, J = 6.7 Hz, 6H, 2CH
3 3
); C NMR (75 MHz, CDCl
=
3:1, R
f
= 0.20, purified employing “flash” chromatography using
145.99, 133.66, 133.22, 131.79, 129.43, 126.71, 119.26, 114.19, 55.18,
+
petrol ether: ethyl acetate = 5:3. White solid; Yield: 385 mg (90%). IR
33.42, 19.38. MS (ESI): m/z = found 745.16 [M +H]; calcd 744.14
–
3
–
–
+
+
(
KBr)
ν
= 505 (Se-C), 1656 (C
–
C), 1623 (C
) δ 7.72–7.65 (m, 2H, Ar-H), 7.18–7.09
m, 2H, Ar-H), 6.66–6.58 (m, 2H, Ar-H), 6.42–6.32 (m, 2H, Ar-H),
CO),
.27–2.07 (m, 1H, CH), 1.24–1.15 (m, 1H, CH), 1.08–0.99 (d, J = 6.4 Hz,
N), 2221 (CN), 3614 (NH)
[M ]; HRMS calcd. for
C
34
H
36
N10Se
2
[M ]: 744.14548, found
ꢀ
1
1
+
cm ; H NMR (300 MHz, CDCl
783.10836 [M +K]. Anal. Calcd for C34
36 2
H N10Se (calcd 744.14): C,
(
54.99; H, 4.89; N, 18.86. Found: C, 54.97; H, 4.86; N, 18.83.
′
4
2
3
1
1
4
.28–4.18 (d, J = 6.7 Hz, 1H, N
–
H), 3.68–3.63 (s, 3H, H
3
N,N -(((diselanediylbis(4,1-phenylene))bis(1H-tetrazole-1,5-diyl))bis
(2-methylpropane-1,1-diyl))bis(4-methoxyaniline) (25)
1
3
H, CH
3
), 0.83 (d, J = 6.4 Hz, 3H, CH
3
); C NMR (75 MHz, CDCl
3
) δ
Compound 25 synthesized following procedure IV from 1,2-bis(4-
57.03, 153.83, 139.78, 134.80, 133.08, 127.67, 125.43, 117.01,
isocyanophenyl)diselane (6) (200 mg, 0.55 mmol), TMSN
3
(145 µL,
14.92, 100.16, 57.51, 55.68, 33.35, 19.42. MS (ESI): m/z = found
1.1 mmol), isopropyl aldehyde (91 µL, 1 mmol), and p-anisidine (123
mg, 1 mmol). TLC was used to monitor its formation petrol ether: ethyl
+
+
29.29 [M +H]; calcd 428.08 [M ]; HRMS calcd. for C19
H
20
N
6
OSe
+
+
[
M ]: 428.08638, found 429.08873 [M +H]. Anal. Calcd for
OSe (calcd 428.08): C, 53.40; H, 4.72; N, 19.66. Found: C,
3.38; H, 4.69; N, 19.64.
acetate = 3:1, R
f
= 0.20, purified employing “flash” chromatography
19 20 6
C H N
using petrol ether: ethyl acetate = 5:3. White solid; Yield: 699 mg (87%).
1
5
H NMR (300 MHz, CDCl
3
) δ 7.66 (d, J = 8.5 Hz, 4H, Ar-H), 7.04 (d, J =
1
2

S. Shaaban et al.
Bioorganic Chemistry 109 (2021) 104713
8
.5 Hz, 4H, Ar-H), 6.57–6.46 (m, 4H, Ar-H), 6.39–6.24 (m, 4H, Ar-H),
.24 (d, J = 7.9 Hz, 2H), 3.82 (s, 2H, 2 N H), 3.56 (s, 6H, H CO),
), 0.78 (d, J = 6.7
) δ 156.97, 153.41, 140.01,
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+
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