Discovery of phenylselenoether-hydantoin hybrids as ABCB1 efflux pump modulating agents with cytotoxic and antiproliferative actions in resistant T-lymphoma

Article abstract of DOI:10.1016/j.ejmech.2020.112435

Multidrug resistance (MDR) in cancer cells is a crucial aspect to consider for a successful cancer therapy. P-gp/ABCB1, a member of ABC transporters, is involved in the main tumour MDR mechanism, responsible for the efflux of drugs and cytotoxic substances. Herein, we describe a discovery of potent selenium-containing ABCB1 MDR efflux pump modulators with promising anticancer activity. On three groups of selenoethers comprehensive studies in terms of design, synthesis, and biological assays, including an insight into cellular mechanisms of anticancer action as well as an ADMET-screening in vitro were performed, followed by in-depth SAR analysis. Among the investigated new phenylselenoether hybrids, four compounds showed significant cytotoxic and anti-proliferative effects, in particular, in resistant cancer cells. Hydantoin derivatives (5–7) were significantly more effective than the reference inhibitor verapamil (up to 2.6-fold at a 10-fold lower concentration) modulating ABCB1-efflux pump, also possessing a good drug-drug interaction profile. The best compound (6) was further evaluated in human JURKAT T-lymphocytic cancer cells for its impact on cell proliferation rate. Mechanistically, the expression of cyclin D1, an enhancer of the cell cycle, decreases, while p53, an inhibitor of cell proliferation, was up-regulated upon the treatment with compound 6 alone or in combination with the chemotherapeutic agent doxorubicin. In summary, a new chemical space of highly active selenium-containing anticancer agents has been discovered, with a new lead compound 6 that warrants more in-depth biological evaluation and further pharmacomodulation.

Full text of DOI:10.1016/j.ejmech.2020.112435

Journal Pre-proof
Discovery of phenylselenoether-hydantoin hybrids as ABCB1 efflux pump modulating
agents with cytotoxic and antiproliferative actions in resistant T-lymphoma
Wesam Ali, Gabriella Spengler, Annamária Kincses, Márta Nové, Cecilia Battistelli,
Gniewomir Latacz, Małgorzata Starek, Monika Dąbrowska, Ewelina Honkisz-
Orzechowska, Annalisa Romanelli, Manuela Monica Rasile, Ewa Szymańska, Claus
Jacob, Clemens Zwergel, Jadwiga Handzlik
PII:
S0223-5234(20)30406-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112435
EJMECH 112435
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 February 2020
Revised Date: 23 April 2020
Accepted Date: 6 May 2020
Please cite this article as: W. Ali, G. Spengler, Annamá. Kincses, Má. Nové, C. Battistelli, G. Latacz,
Mał. Starek, M. Dąbrowska, E. Honkisz-Orzechowska, A. Romanelli, M.M. Rasile, E. Szymańska, C.
Jacob, C. Zwergel, Jadwiga Handzlik, Discovery of phenylselenoether-hydantoin hybrids as ABCB1
efflux pump modulating agents with cytotoxic and antiproliferative actions in resistant T-lymphoma,
European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112435.
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Discovery of phenylselenoether-hydantoin hybrids as ABCB1 efflux pump modulating
agents with cytotoxic and antiproliferative actions in resistant T-lymphoma
Wesam Alia,b, Gabriella Spenglerc, Annamária Kincsesc, Márta Novéc, Cecilia Battistellid,
Gniewomir Latacza, Małgorzata Stareke, Monika Dąbrowskae, Ewelina Honkisz-
Orzechowskaa, Annalisa Romanellif, Manuela Monica Rasilef, Ewa Szymańskaa, Claus
Jacobb*, Clemens Zwergel,b,f,g* Jadwiga Handzlika*
a Department of Technology and Biotechnology of Drugs, Jagiellonian University,
Medical College, Medyczna 9, PL 30-688 Kraków, Poland
bDivision of Bioorganic Chemistry, School of Pharmacy, Saarland University, Campus B 2.1,
D-66123 Saarbruecken, Germany
c Institute of Medical Microbiology and Immunobiology, Faculty of Medicine, University of
Szeged, Dóm tér 10, H-6720 Szeged, Hungary
d Istituto Pasteur Italia, Fondazione Cenci-Bolognetti, Department of Molecular Medicine,
Sapienza University of Rome, Viale Regina Elena 324, 00161, Rome, Italy.
e Department of Inorganic Chemistry, Jagiellonian University, Medical College, Medyczna 9,
PL 30-688 Cracow, Poland
f Department of Drug Chemistry and Technologies, Sapienza University of Rome, Piazzale
Aldo Moro 5, 00185 Rome, Italy
g Department of Precision Medicine, “Luigi Vanvitelli” University of Campania, Via L. De
Crecchio 7, 80138 Naples, Italy.
* Corresponding authors:
(J. Handzlik) E-mail address: j.handzlik@uj.edu.pl; (C. Jacob) E-mail address:
c.jacob@mx.uni-saarland.de;(C. Zwergel) E-mail address: clemens.zwergel@uniroma1.it
1

Graphical Abstract
Research Highlights:
•
•
•
•
•
Design and synthesis of new phenyl selenium ether hybrids.
ABCB1 efflux pump modulating assays in mouse T-lymphoma cancer cells.
Cytotoxic and anti-proliferative activity assays in sensitive and MDR cancer cells
Effects on proliferation rate and cell cycle in human JURKAT cancer T-cells
ADME-Tox profile in vitro for most active agents investigated
2

Abstract
Multidrug resistance (MDR) in cancer cells is a crucial aspect to consider for a successful
cancer therapy. P-gp/ABCB1, a member of ABC transporters, is involved in the main tumour
MDR mechanism, responsible for the efflux of drugs and cytotoxic substances. Herein, we
describe a discovery of potent selenium-containing ABCB1 MDR efflux pump modulators
with promising anticancer activity. On three groups of selenoethers comprehensive studies in
terms of design, synthesis, and biological assays, including an insight into cellular
mechanisms of anticancer action as well as an ADMET-screening in vitro were performed,
followed by in-depth SAR analysis. Among the investigated new phenylselenoether hybrids,
four compounds showed significant cytotoxic and anti-proliferative effects, in particular, in
resistant cancer cells. Hydantoin derivatives (5-7) were significantly more effective than the
reference inhibitor verapamil (up to 2.6-fold at a 10-fold lower concentration) modulating
ABCB1-efflux pump, also possessing a good drug-drug interaction profile. The best
compound (6) was further evaluated in human JURKAT T-lymphocytic cancer cells for its
impact on cell proliferation rate. Mechanistically, the expression of cyclin D1, an enhancer of
the cell cycle, decreases, while p53, an inhibitor of cell proliferation, was up-regulated upon
the treatment with compound 6 alone or in combination with the chemotherapeutic agent
doxorubicin. In summary, a new chemical space of highly active selenium-containing
anticancer agents has been discovered, with a new lead compound 6 that warrants more in-
depth biological evaluation and further pharmacomodulation.
Keywords: MDR; selenother; ABCB1, P-glycoprotein inhibitor, T-lymphoma, JURKAT
3

1. Introduction
During the last decades, numerous new classes of anti-cancer drugs emerged, which
usually support the natural body defence. At the same time, the cancer cells developed
different mechanisms to face the cytotoxic effect of these drugs. In this field, the term of
multidrug resistance (MDR) is defined as the low sensitivity for certain cells against drugs,
often found in cancer for tumour therapy [1-5]. MDR is now a deeply investigated issue in the
field of chemotherapy. It can result in the upregulation of drug efflux/ drug efflux pumps, cell
growth, and survival signalling. MDR can also be related to the down-regulation of apoptosis
signals or drug uptakes.
Nevertheless, key players of the MDR action are ATP-binding cassette (ABC)
transporters, such as the P-glycoprotein (Pgp, ABCB1), MRP1 (ABCC1) and BCRP
(ABCG2) ones [6], which are involved in essential processes in cancer cells’ defence.
Moreover, the inhibition of these transporters could be beneficial to overcome drug resistance
[7]. In more detail, P-glycoprotein/ ABCB1 (P-gp/ABCB1) is a member of the ABC
transporter family, which is encoded by the MDR1 (ABCB1) gene, found in a plethora of
healthy and cancerous cells. In the latter ones, they are often overexpressed and work as a
self-defence against cytotoxic substances [8, 9] by acting as an efflux pump for various drugs,
including anticancer agents.
One critical key in the battle against cancer is to overcome drug resistance thus to
resume the activity of current or well-known anti-cancer drugs, e.g. topotecan, doxorubicin,
vincristine, cisplatin, cyclophosphamide, methotrexate or 5-fluorouracil. A successful and
well-accepted method in this field is the inhibition of P-gp function during chemotherapy
[10]. The co-administration of efflux pump inhibitors (EPIs), which are specific compounds
able to modulate the efflux action of MDR drug transporters, has been reported and well-
reviewed [1-5]. Due to the complexity of cancer aetiology, a combined approach targeting
these MDR proteins as well as hitting known antitumoral pathways such as p53 is more and
more considered to be a successful strategy to fight cancer.
Recently, plenty of compounds that modulate P-gp in various assays conditions were
synthesized and studied. Mainly, three generations of reversal MDR agents, e.g. verapamil (I),
dexniguldipine (II) and zosuquidar (III) (Fig. 1A) have been widely investigated [1, 3, 7, 11].
Furthermore, a group of inhibitors for other ABC family members involved in cancer MDR,
i.e. MRP1 (MK571, LY171883 and BAYu9773, Fig. 1B) and BCRP (Ko143 and FTC, Fig.
4

1C), have been evaluated [6]. However, their therapeutic usage is restricted by insufficient
pharmaceutical profile, in particular, in the field of safety. Thus, further search for EPIs
among new chemical groups is in great medical importance, but it is worth tu emphasize that
rational design of new EPIs is a high challenge. Although recent scientific achievements
provided crystal structures of some MDR ABC transporters, the computer aided structure-
based design is significantly limited in this protein family due to a variety of key-points that
could be targeted by inhibiotors, and thus providing various structurally different “binding
pockets”. For instance, some structures can inactivate pump as enrgy users acting on ATP,
while different structures can act via a competition with a drug-substrate to one of the binding
sites.
(A)
O
N+
-O
N
F
O
O
O
O
N
O
N
F
O
O
N
O
O
N
OH
N
N
H
Verapamil (I)
Dexniguldipine (II)
Zosuquidar (III)
(B)
OH
O
S
O
O
S
OH
OH
O
N
Cl
N
S
N
O
OH
N
N
N
H
HO
O
MK571
LY171883
BAY u9773
(C)
O
O
O
O
O
H
N
H
N
N
N
H
N
NH
O
O
O
Ko143
Fumitremorgin C (FTC)
Fig. 1. Selected structures of cancer efflux ABC efflux pump inhibitors; (A) three generations (I-III) of ABCB1
inhibitors; (B) inhibitors of MRP1: MK571, LY171883 and BAYu9773; (C) inhibitors of BCRP: Ko143 and
FTC [6].
On the other hand, the structure-activity relationship analysis (SAR) in the vast group
of Pgp-modulators allowed to indicate a beneficial role for hydrophobic/aromatic ending
5

within their structures and also some flexibility in the middle (Fig. 1 A). Although the number
of modulators found for either MRP1 or BCRP is much smaller, SAR for their several agents
shows that both groups differ from the Pgp pharmacophores. In contrast to bulky aromatic
ends of members of the three generations of Pgp-inhibitors (Fig. 1A), the inhibitors MRP1
(MK571, LY171883, BAY u9773, Fig. 1B) tend to have an amphiphilic structure with the
clear hydrophilic moiety at the end opposite to the aromatic/hydrophobic one, while BCRP
inhibitors (Ko143, FTC, Fig. 1C) seem to prefer 4- or 5-fused ring structures. Thus, the
ligand-based design is still a predominant approach in the rational design of cancer EPIs, in
particular in the case of ABCB1, thanks to the rich library of modulators identified so far.
In the last decade, our research team described numerous compounds targeting
ABCB1 inhibitors in cancer cell lines belonging to the types of inhibitors shown in Fig. 1A
[12-16]. In more detail, we were able to show highly potent ABCB1-inhibitory properties for
aryl-piperazine hydantoin compounds [12, 14, 15]. The 5-aromatic substituted hydantoins
were particularly potent [12, 15], while the potency decreased when the aromatic moiety was
moved into position 3 [13]. Among the most active hydantoin-compounds found so far, the
5,5-diphenylhydantoin derivative 1 and spiro-fluorene derivative 2 (Fig. 2) were 4-7-fold
more potent than the reference P-gp inhibitor verapamil, but they did not display either
cytotoxic or antiproliferative activity towards cancer cell lines, i.e., T-lymphoma [15] and
NCI60 cell line panel (https://dtp.cancer.gov/discovery_development/nci-60/, data not
published). In contrast, recent evidence indicates an increasing role of the triazine scaffold in
potent hybrid anticancer agents [16], including 1,3,5-triazine-2,4-diamines [17, 18],
especially, piperazine and morpholine derivatives displayed strong cytotoxic effects in vitro
against NCI60 cancer cell lines [18].
Furthermore, our recent follow-up study was focused on aromatic selenoesters and
seleno-anhydrides, possessing both anticancer and P-gp inhibitory properties [10]. The most
active compounds 3 and 4 (Fig. 2), were tested in MDR mouse T-lymphoma cells
overexpressing P-gp, displaying at the same time up to 3.4-fold more potent P-gp modulatory
effect compared to the reference verapamil [19]. The main drawback of those selenoesters and
seleno-anhydride lies in their low chemical stability and their very unpleasant intensive smell,
limiting their further evaluation and potential therapeutic application.
6

Fig. 2 Hydantoin Pgp-modulators, 1 and 2, and seleno-compounds (3 and 4) with both anticancer and Pgp-
modulatory properties found previously
.
Taking into consideration the conclusions of previous studies [10, 12-19], and the fact
that selenium in the form of ethers is chemically much more stable and devoid of any
unpleasant smell, we decided to design and investigate a series of new phenylselenoethers
with different aromatic/heterocyclic termination. In this study, a new series of compounds
was designed as hybrid structures of both selenoethers and moieties found in the previous
active P-gp inhibitors. We designed phenylselenoether derivatives, which can be divided into
three main groups: (i) aryl-hydantoins and their bioisosteres with variable alkyl chain lengths
(5-7, group A), (ii) (aryl)piperazines with various substituents at N-piperazine, including
aromatic, alkyl and ester moieties (8-13, group B), as well as (iii) the 1,3,5-triazine scaffold
with small C1-linker branches (5-17, group C, Table 2). The new hybrid compounds were
synthesized and tested against P-gp in mouse T-lymphoma cells, then, their possible ability to
regulate the cell proliferation rate and their cytotoxic effects were evaluated, as well. The best
compounds 5-7 were further studied regarding the mechanism of ABCB1 modulation. For
the most active compound (6), we extended our studies from the mouse cells to a cellular
model of human T-lymphocytic cancer (JURKAT) [20], in order to evaluate its effects alone
or in combination with the reference chemotherapeutic agent doxorubicin, as well as potential
molecular mechanisms of action for the selenoether at a cellular level. Selected compounds,
active in lymphocytic cancer cells, were also examined on their antiproliferative action in
neuroblastoma cancer cell lines. Furthermore, we evaluated their drug-like properties,
including lipophilicity, possible metabolic pathways, and potential drug-drug interactions
(DDI) risks. Finally, qualitative structure-activity relationships (SAR) were discussed.
Thus, the goal of this work was a comprehensive study regarding differently
substituted seleno-ethers (5-17, Table 1), in order to find new lead structure(s) in search of
innovative anticancer drugs to overcome cancer MDR mechanisms.
7

Table 1 Chemical structures of compounds 5-17
Cpd
5
Gr.
A
Ar
R
n
-CH2COOCH3
4
-CH2COOCH3
-CH3
6
4
6
7
A
A
-
-
-
-
-
Ph
-
-
-
-
-
8
B
B
B
B
B
2-MeO-Ph
4-F-Ph
9
10
11
12
3,4-diCl-Ph
-CH3
O
-
-
13
B
H3C
O
-
-
-
-
-H
-
-
-
-
14
15
16
17
C
C
C
C
-CH3
C2H5
diCH3
2. Results and discussion
2.1. Chemical synthesis
Compounds 5-17 (Table 1) were obtained according to the synthesis route shown in Scheme
1. The hydantoin derivatives 5-7 (group A) were prepared applying a one-pot synthesis
method, including Se-alkylation of previously described hydantoin intermediates 18-20 [14,
21-23]. In more detail, a reduction of the diphenyl diselenide to produce sodium selenolate
was performed using sodium borohydride in water/tetrahydrofuran mixture (THF) 1/1 under a
nitrogen atmosphere [23]. Upon decolourization of the solution, a suitably substituted
hydantoin in dichloromethane (DCM) was injected without any need of a deprotonation salt to
8

produce the corresponding phenyl selenium hydantoins 5-7 in moderate yields and with
excellent purity, within 3-4 days. (Scheme 1A).
Scheme 1. Synthesis pathways of the final compound 5-17 and the relative intermediates 18-30.
9

In the case of the phenyl selenium piperazine derivatives (8-13), the appropriate
commercially available piperazines were alkylated using 1-bromo-3-chloropropane in the
presence of anhydrous potassium carbonate in acetone as solvent at room temperature [24]
.
The so obtained crude intermediates were used applying the Se-alkylation method described
for 5-7 (Scheme 1B).
1,3,5-Triazine selenium derivatives (14-17) were obtained as follows: firstly, the
preparation of the sodium selenoate was carried out in a similar way as described for group A.
The subsequent Se-alkylation was performed within 3-4 hours, without any need to use higher
temperatures or deprotonation salt. The next step involved cyclic condensations in basic
conditions with methyl piperazine biguanide, as previously described [23, 24].
Spectral and chromatographic analytical methods confirmed the structures and purity of the
final compounds 5-17. The intermediates (21-30) were either bought (22 and 24) or
synthesized in the case of compounds 21, 23, and 25-30 according to the previous methods
[24-29]. The compounds 9-17 have been transformed into the corresponding crystalline
hydrochloric salts for the relative biological studies.
2.2. Pharmacology
2.2.1. Efflux modulating effects
Compounds 5-17 (Table 1) were evaluated for their efflux modulating effects in mouse T-
lymphoma cell line transfected with the human MDR1 gene that codes for the ABC
transporter ABCB1 measuring the accumulation of rhodamine 123, which is a substrate for
ABCB1 [12, 30-32]. The percentage of mean fluorescence intensity was calculated for the
treated MDR cells compared to the untreated cells, and then a fluorescence activity ratio
(FAR) was determined. Verapamil was tested at the commonly used concentration (20 μM).
All compounds (5-17) were investigated at the 10-fold lower concentration (2 μM) while
weakly-active ones (8-10, 12-17) also at the concentration of verapamil. In the case of the
active modulators at 2 μM (FAR>2, 5-7, 11), the FAR values were determined at a low
concentration of 0.2 μM, as well. Results are presented in Table 2.
10

Table 2 Effects of compounds 5-17 on rhodamine 123 retention by human MDR1 (ABCB1)
gene-transfected mouse lymphoma cells
FAR (Control)
DMSO (2 v/v%)
0.84
FAR (2 μM)
-
Compounds
Verapamil
FAR (0.2 μM)
FAR (20 μM)
-
17.59
-
2.47
2.77
1.09
-
-
-
42.91
24.20
45.05
1.07
0.92
1.07
2.27
0.97
0.75
0.82
5
6
7
8
-
-
1.57
1.02
4.25
-
0.85
0.96
0.87
0.95
1.13
1.40
9
10
11
12
13
14
15
16
17
1.07
-
-
-
-
-
-
0.83
0.93
1.11
FAR, Fluorescence activity ratio was calculated using the following equation:
MDRtreated MDRcontrol
FAR=
parentaltreated parentalcontrol
The inhibition of ABCB1 transporter is evident when FAR >1. [32] Since the treatment had no effect on
the parental cells (PAR) lacking the overexpressed ABCB1 system, the intensity of fluorescence did not
change in the treated cells compared to the control ones, for this reason, the denominator was considered
as 1.
Among the evaluated derivatives, the compounds 5-7 (tested at 2 μM) displayed strong
inhibitory potency, being up to 2.6-fold more effective than the reference inhibitor verapamil
(at 20 μM). Other compounds (10, 11), had significantly lower inhibitory activities (FAR
2.27 at 2 μM or 4.25 at 20 μM), while compounds 8, 9 and 12-17 were inactive (Table 2).
2.2.1.1. Studies on ABCB1 modulating mechanisms of compounds 5-7
The most active compounds found in the rhodamine 123 accumulation assay (5-7) were
tested at 100 µM on their influence on ATPase activity of Pgp pump in the luminescence Pgp-
Glo™ Assay, according to previously described methods and protocols [33-35] (Fig. 3). In
this assay, the Pgp-dependent decreases in luminescence are measured to reflect its increased
ATP consumption. The ABCB1 basal ATP consumption (basal activity) is considered as a
difference between the luminescent signal of samples treated with sodium orthovanadate, the
selective and potent Pgp inhibitor (100% inhibition observed), and the luminescence of
untreated Pgp samples. Thus, inhibitors give lower values (<100% of the basal activity), while
the pump stimulators/substrates cause a statistically significant increase of the basal activity.
11

Verapamil at 200 µM was used as a reference modulator with the substrate/ATPase
stimulatory mode of action, and caffeine as a reference inactive towards ABCB1 transporter
to give negative control.
The obtained results showed that all compounds 5-7 significantly increased (p<0.001,
p<0.0001) the Pgp basal activity in a similar way, but stronger, than verapamil (Fig. 3). These
results are in line with those from the rhodamine 123 efflux assay, where stronger effects for
5-7 in respect verapamil were also found.
Fig. 3 The effect of the ABCB1 substrate verapamil (VER) (200 μM), ABCB1-negative
compound caffeine (CFN) (100 μM) and compounds 5, 6 and 7 (100 μM) on ABCB1 basal
activity. The compounds are recognized as an ABCB1 substrate if they stimulate their basal
activity (>100%). Data are presented as the mean ± SD. Statistical significance was
evaluated by one-way ANOVA, followed by Bonferroni's comparison test (***p<0.001,
****p<0.0001 compared with the basal activity).
Summing up, compounds 5-7 did not inhibit the work of Pgp pump in this assay but displayed
a substrate property. Thus, the putative mechanism of the dye-substrate efflux inhibition
caused by 5-7 (Table 2) was the most probably associated with a competitive displacement in
the substrate-binding site of this MDR transporter.
2.2.2. Cytotoxic and anti-proliferative effects
The whole series (5-17) was also examined on their cytotoxic and anti-proliferative activity in
both sensitive (PAR) and resistant (MDR) mouse T-lymphoma cell lines (Table 3).
12

Table 3 Cytotoxic and antiproliferative effect of the compounds 5-17 on sensitive parental
(PAR) and resistant (MDR) mouse T-lymphoma cells
Antiproliferative effects
Cytotoxic effects
PAR mean IC50
MDR mean IC50
PAR mean IC50
MDR mean IC50
(µM) ± SD
Compound
(µM) ± SD
(µM) ± SD
(µM) ± SD
16.75 ± 1.37
7.82 ± 0.57
2.91 ± 0.18
3.39 ± 0.09
0.90 ± 0.07
4.21 ± 0.40
44.35 ± 2.88
>100
5
3.84 ± 0.05
12.53 ± 0.95
28.96 ± 2.30
>100
1.34 ± 0.05
4.67 ± 0.38
13.07 ± 0.97
71.94 ± 4.30
7.67 ± 0.84
3.66 ± 1.23
23.40 ± 1.22
37.67 ± 1.95
>100
0.67 ± 0.03
3.25 ± 0.11
21.50 ± 0.24
>100
6
7
8
9
13.92 ± 0.07
17.04 ± 2.22
62.91 ± 3.03
53.72 ± 1.71
>100
6.55 ± 0.53
2.27 ± 0.26
60.59 ± 0.98
51.56 ± 1.30
>100
29.09 ± 0.97
3.50 ± 0.29
98.56 ± 0.64
75.70 ± 3.31
>100
10
11
12
13
14
15
16
17
28.93 ± 1.89
48.47 ± 2.56
8.28 ± 1.47
>100
>100
38.12 ± 1.74
65.16 ± 1.36
35.29 ± 1.07
84.93±1.36
>100
>100
>100
The tested compounds 5-7 and 11 showed very high cytotoxic effects in both MDR and PAR
cell lines (IC50< 5 µM), with compound 6 presenting the highest activity among the tested
selenoethers. Most compounds of the series displayed lower cytotoxic concentrations for PAR
T-lymphoma cells in comparison to MDR ones. This difference is particularly evident in the
case of compound 10 (>4-fold) and 8 (>2-fold), and only slight in the case of the most
cytotoxic compounds (5-7 and 11). Compounds 12, 13 with low cytotoxic effects in PAR cell
lines were less potent in MDR cells too, while compounds 9 and 14-17 displayed no
cytotoxicity in both PAR and MDR cell lines, even at a concentration of 100 µM (84 µM for
16 tested in PAR).
The series demonstrated unexpectedly interesting antiproliferative properties in MDR mouse
T-lymphoma cells, as each member caused more potent antiproliferative effects in MDR than
in PAR cells. Almost the whole series displayed IC50 (MDR) lower than 100 µM, while 5-7,
10, 11, and 17 lower than 10 µM. Similar to the cytotoxicity test results, compound 6 also
displayed the most potent antiproliferative action for both PAR (IC50= 3.83 µM) and MDR
(IC50 = 1.33 µM) cells, being the only member with IC50<10µM in PAR cells. To sum up,
13

most of the compounds showed moderate antiproliferative properties in PAR cells, with IC50
in the range of 12-65 µM, excluding 9 and 14, which were inactive up to a concentration of
100 µM.
2.2.3. Anticancer properties in JURKAT human T-lymphoblastic leukaemia cell line.
2.2.3.1. Effects of compound 6 on proliferation rate in JURKAT cells.
Compound 6 was the best compound of our series so far in terms of antiproliferative and
cytotoxic activities, as well endowed with significant efflux pump inhibitory properties in
mouse T-lymphoma. In order to extend the analysis to a human cell context, representing an
immortalized T lymphocyte cell line used as a model for acute lymphoblastic leukaemia
(ALL) [36], we decided to explore the effect of compound 6 on proliferating JURKAT
leukaemia cells. This cellular system represents a useful model for testing the effects of
different compounds on cell metabolism [37] and proliferation rate [38]. The effects of
compound 6 on either proliferation rate or cell cycle-related gene expression were examined
alone or in combination with doxorubicin (a chemotherapeutic agent able to block tumour
growth in vivo) in order to find whether the co-treatment should enhance the effect of
doxorubicin [38-40]. Doxorubicin is a well-known and successful antineoplastic drug
commonly used as a chemotherapeutic agent in various cancers, including ALL, but, at
present, drug resistance is common [41, 42]
.
For these reasons, we performed proliferation assays in JURKAT cells treated with
compound 6 at 0.1- 0.5 and 2 μM concentrations, alone or in combination with doxorubicin,
tested at 50 and 250 nM at two distinct time points (24 and 72 hours).
While at early time compound 6 did not significantly affect cell proliferation rate,
doxorubicin alone was able to block proliferation, especially at higher concentrations (Fig.
4A). Notably, at 72 hours post-treatment, a significant reduction in proliferation was observed
in cells treated with compound 6 alone and with doxorubicin alone, but a more significant
effect was detected in cells co-treated with both compound 6 and doxorubicin. These data
highlighted a synergistic effect of compound 6 with doxorubicin, especially when compound
6 was added to cell cultures at a higher dose (2 μM) (Fig. 4B). In summary, these results
indicate that the co-treatments with compound 6 and doxorubicin counteracted cell
proliferation in JURKAT cells and that the co-treatment with compound 6 enhances the effect
of doxorubicin.
14

Fig. 4. Dose-response curves for anti-proliferation activity of compound 6 and doxorubicin in a dose
range (0.1, 0.5, and 2 μM for compound 6 and 50 and 250 nM for doxorubicin) on JURKAT cells after
24 h (A) and 72 h (B) of treatment. The results represent the average cell number ± SEM of three
independent experiments. Significance is represented as *p < 0.05 and **p < 0.01 related to control
groups.
2.2.3.2. Effects of compound 6 on cell cycle-related gene expression in JURKAT
In order to estimate a potential molecular mechanism for the anticancer activity found for 6,
we focused on the expression levels of cyclin D1, a well-known gene related to cell cycle
progression contributing to uncontrolled proliferation and found to be negatively regulated in
a previous study by the treatment with a selenium-containing compound in gastric cancer
[20]. In addition, we analysed the expression level of p53 involved in apoptotic response, cell
cycle arrest, and senescence [43].
In order to understand whether compound 6 was able to limit cell proliferation through gene
expression regulation of cyclin D1 and p53, we treated JURKAT cells with compound 6 at
15

three concentrations (0.1, 0.5 and 2 μM) and doxorubicin (50 nM), analysing with RT-PCR
the mRNA transcripts levels of cyclin D1 and p53. DMSO was used as a negative control.
The results depicted in Fig. 5 show that compound 6 was able to significantly reduce cyclin
D1 expression and increase the level of p53 (Fig. 5A), already after 24 hours of treatment.
Moreover, the co-treatment with both compound 6 and doxorubicin enhanced the effect of the
single treatments on gene expression.
Fig. 5. (A-C) JURKAT cells were treated with compound
6 (0,1 - 0,5 or 2 μM) alone or (B-D) in combination
with doxorubicin (50nM) or with DMSO (CTR), as a control, for 24 hours. Expression of cyclinD1 (A-B) and
p53 (C-D) mRNAs were evaluated on total RNA by qRT-PCR. Bars represent means ± SEM of 6 experiments.
To sum up, these results indicate that compound 6 inhibited cell cycle progression through the
reduction of the expression of cyclin D1 and inhibited cell proliferation by inducing p53
expression.
2.2.4. Anticancer action against neuroblastoma cancer cells
16

In order to estimate a broader range of anti-cancer potency, including also non-lymphocytic
cancer cells, an influence of the most active selenoethers 5-7 on neuroblastoma cancer
proliferation was investigated in SH-SY5Y cells affecting their viability (Fig. 6). The results
show that all tested compounds at a concentration of 10 μM significantly inhibited cell
proliferation up to 60%. Compounds 6 and 7 were the most active ones, inhibiting cell growth
by 12% and 8%, respectively, already at a concentration of 0.5 μM, while the compound 5
was active only at 10 μM.
**
***
****
****
****
****
****
Fig. 6. Effect of selenoethers 5-7 on neuroblastoma cell viability. SH-SY5Y cells were incubated for
72 h in the presence of the compounds 5-7 at a concentration of 0.1, 0.5, 2, 10 [μM]. Cell viability was
measured by MTS assay. Each point (mean ± SEM of two independent experiments, each of which
consisted of six replicates per treatment group) represents absorbance units and is expressed as a
percentage of control compared to 0.1% DMSO control cells (set as 100%). Statistical analysis by
one-way ANOVA showed significant differences between the groups (P<0.05) and was followed by
the Bonferroni multiple comparison test. Data indicated with ****P≤0.0001, ***P≤0.001, **P≤0.01
reflects statistically significant differences between control and experimental groups.
2.4. Drug-likeness in vitro
2.4.1. Lipophilicity
In the presented work, the lipophilicity of the selenoethers was estimated using standard RP-
TLC method. Retention parameters for the compounds were designated and analysed to give
17

RM0 values that reflect the lipophilic properties of the tested compounds (details in Table S1,
Supplementary).
The RM0 values of the tested compounds ranged from 0.79 to 6.05 (Fig. 7) and were
corresponding to structural differences of the compounds. Thus, group A of hydantoin
derivatives (5-7) was more lipophilic than both, group B of phenylpiperazine selenoethers (9-
13) and C of 1,3,5-triazine derivatives (14-17). In the case of group B, the significant internal
diversity of lipophilic properties between aromatic- (9-11) and non-aromatic piperazine (12,
13) compounds was seen in good accordance with the piperazine substituent properties. In
contrast, most of the members of group C (14, 15 and 17) demonstrated almost identical
lipophilic character. Taking into consideration the results, lipophilic-dependent trends can be
observed for the ABCB1 modulatory action examined (Table 2). Thus the most lipophilic
hydantoin derivatives (6-7) displayed significantly stronger action than either piperazine (9-
13) or triazine (14-17) ones. Furthermore, compound 6, the outstanding one in terms of
cytotoxicity and antiproliferative properties in the cancer cells, was also the most lipophilic
within the series.
7
6
5
RM0
4
3
2
1
0
5
6
7
8
9
10 11 12 13 14 15 16 17
Compounds
Fig. 7. Lipophilicity data for compounds 5-17. * Lipophilicity tested for the basic form (8), unlike the
hydrochloric salts of the rest of group B (9-17). This relatively overstated lipophilicity of 8, as not
comparable, was excluded from the discussion on results.
2.4.2. Metabolic stability in vitro
The metabolic stability of selenoorganic compounds 5-7 was investigated using mouse
liver microsomes (MLMs). The UPLC spectra of the reaction mixtures after 120 min
incubation with MLMs showed that all examined compounds were metabolically unstable
18

(Fig. 8).
Fig. 8. UPLC spectra of the reaction mixtures after 120 min incubation of selenoethers 5 (A), 6(B) and
7 (C) with MLMs. The peak at the retention time 6.26 min was identified as the contamination.
19

Compound 5 was metabolized completely into four metabolites M1-M4, as no parent
compound was observed at UPLC spectra (Fig. 8A). Only a slight amount (4.5%) of
compound 6 remained in the reaction mixture. This compound was biotransformed mainly
into metabolite M1 and small amounts of three more metabolites M2-M4 (Fig. 8B).
Compound 7 was metabolized mainly into metabolite M1 and four more metabolites M2-M5
(Fig. 8C).
According to UPLC spectra, compound 7 was determined as the most stable among
the tested series, as around 30% of this compound remained in the reaction mixture. The
performed next MS and precise ion fragment analyses of the parent compounds and their
metabolites allowed for a determination of the metabolic pathways and the most probable
structures of metabolites among tested series. Generally, the most common metabolic
pathways of 5 and 6 were ester hydrolysis and hydroxylation (Fig. S1A-C and S2A-D,
Supplementary information). Compound 7 was mostly hydroxylated and decomposed at Se
atom (Fig. S3A-E, Supplementary information). A similar to 7 decomposition pattern for Se
was also observed for compound 5 (Fig. S1D and S3B, Supplementary information). All
metabolic pathways of the tested selenoethers were summarized in Table 4.
Table 4 The molecular masses and metabolic pathways of compounds 5-7.
Molecular
Molecular Number of
mass of the
Substrate
mass
identified
Metabolic pathway
metabolite
(m/z)
(m/z)
metabolites
539.13 (M1)
555.08 (M2)
523.11 (M3)
379.21 (M4)
551.22 (M1)
567.18 (M2)
568.44 (M3)
ester bond hydrolysis, hydroxylation
ester bond hydrolysis, double hydroxylation
ester bond hydrolysis
5
537.14
565.18
4
4
decomposition at the Se atom
ester bond hydrolysis,
ester bond hydrolysis, hydroxylation
ester bond hydrolysis, ketone reduction,
hydroxylation
6
7
568.37 (M4)
ester bond hydrolysis, ketone reduction,
hydroxylation
493.13 (M1)
319.20 (M2)
493.13 (M3)
337.21 (M4)
509.15 (M5)
hydroxylation
decomposition at the Se atom
hydroxylation
477.11
5
decomposition at the Se atom, hydroxylation
double hydroxylation
20

2.4.3. Drug-drug interactions prediction
The potential drug-drug interactions (DDI) of selenoethers 5-7 were predicted by in vitro
assessment of their effect on CYP3A4 isoform being the most involved in drug metabolism.
The compounds 5-7 were screened at 10 µM, and the results were compared to the reference
selective CYP3A4 inhibitor ketoconazole. All examined compounds in a statistically
significant manner (****p <0.0001) reduced CYP3A4 activity (Fig. 9).
Fig. 9. The influence of selenoethers 5-7, and the reference inhibitor ketoconazole (KE) on CYP3A4 activity.
The statistical significance was evaluated by a one-way ANOVA, followed by Bonferroni’s Multiple
Comparison Test (∗∗∗∗p < 0.0001 compared with untreated control).
The weakest inhibition was shown for compound 6, which reduced CYP3A4 activity up to
~53% of untreated control, whereas 5 and 7 decreased its activity up to 28 and 20%,
respectively. However, the observed effects of 5-7 were much weaker than that for the
reference drug ketoconazole, which reduced almost entirely CYP3A4 activity at 1 µM (Fig.
9).
2.5. General and SAR discussion
In order to find new anticancer hybrids able to overcome MDR mechanisms, we explored
the chemical space of moieties that were useful to join the phenylselenoether fragment as a
fixed scaffold, including selenoether linkers. Our rational design of suitable moieties based on
results coming from recent literature evidence [16-18] or our previous experiences in
searching anticancer and anti-MDR agents [10-15]. In this context, our present modifications
divided the chemical space of the phenylselenoether derivatives into three main groups, i.e.,
5-arylhydantoin derived compounds (5-7, group A), aryl- (8-11), alkyl- (12) or ester- (13)
piperazine derivatives (group B) and 1,3,5-triazine derivatives with different alkyl branches
within the Se-ether linker (14-17, group C). The comprehensive biological screening,
21

including cancer efflux pump inhibitory-, cytotoxic and antiproliferative properties for whole
the series (5-17) on the one hand, and ADMET in vitro studies for selected compounds 5-7,
on the other hand, has provided exciting and even unexpected qualitative structure-activity
relationship (Fig. 10).
Fig. 10. SAR overview of compounds
5-17. The common phenylselenoether (red) moiety is probably
responsible for the trend of more potent antiproliferative activity in the resistant T-lymphoma (MDR), than that
in sensitive (PAR) cells in all three series A-C. Advantage of these new-discovered hydantoin selenoethers (red),
referring to previous Se-esters, is the lack of any unpleasant smell and their higher chemical stability
Among the three considered groups, hydantoin derivatives (group A, 5-7) were found to be
the most promising structural moiety, being up to 2.6-fold more effective MDR EPI (at a 10-
fold lower concentration) than the reference ABCB1-inhibitor verapamil. These compunds
possessed also significant ABCB1 substrate potency in Pgp ATPase test, significantly higher
than that of reference verapamil. Both assays indicated the realtively strongest effect for the
compound with the rigid spirofluorene moiety (7), if comparing to 5 and 6 containing rotable
5,5-diphenyl moiety (marked in green, Fig. 10).Furthermore, all three members (5-7)
22

displayed significant cytotoxic and antiproliferative properties in the MDR cancer cells of T-
lymphoma. The length of the linker seems to be of considerable importance, as 5,5-
diphenylhydantoin compound 6 (IC50 <1.5 μM in both cases, Table 3) with the longest (C6)
linker was the best one of the present study. Taking into account our previous results, both
5,5-diphenylhydantoin and 5-spirofluorenehydantoin moieties, appeared in the structures of
highly potent arylpiperazine ABCB1-inhibitors [14]. Their combination with a
phenylpiperazine moiety gave even stronger inhibitory effects than those observed in this
study, more than 20-fold stronger than verapamil [32]. However, those compounds displayed
neither cytotoxic nor antiproliferative actions on cancer cells (T-lymphoma).
Furthermore, a too potent ABCB1-inhibitory property can be considered as risky for host
organisms, taking into consideration the role of ABC-transporters in healthy human cells,
which protect them from toxic substances or drugs. From a therapeutic point of view, the
identified pharmacological properties of seleno-diphenylhydantoin 6 seem to be much more
promising, and thus provides a new therapeutic direction for structural derivatives of
anticonvulsant drug phenytoin (5,5-diphenylhydantoin). It should be noted that the hydantoin
scaffold is a common motive in other anticancer drugs, e.g., the 5,5-dimethylhydantoin
derivative, nilutamide. However, our previous studies on the series of 5,5-dimethylhydantoin
derivatives indicated a weaker (usually negligible) ABCB1-inhibitory properties in T-
lymphoma in comparison to their 5,5-diphenyl analogues [13, 14, 32].
Comparing the piperazine phenylselenoethers (8-13, group B) to hydantoins (5-7), the
phenylpiperazine subgroup demonstrated generally lower pharmacological effects but with
notable mutual differences, almost regular and highly corresponding to their structural traits.
The structural difference of compounds 8-13 concerns substituents at N-piperazine, including
aromatic, alkyl and ester moieties, which influence the hydrophobic properties of the
subseries, in the following order: 3,4-diClPh (11) > 4-FPh (10)> Ph (8) > 2-MeOPh (9)>Me
(12)>EtOCO (13). Almost an identical order can be observed for both ABCB1-inhibitory and
anticancer properties, including cytotoxic and antiproliferative actions (Tables 2 and 3). The
biggest and most hydrophobic 3,4-dichlorophenyl substituted compound (11) was outstanding
in the field of the efflux pump modulatory action (FAR=2.27 at 2 μM), also exceeding the rest
of group B in cytotoxic (IC50=3.50 μM) and antiproliferative action (IC50=3.66 μM). The 4-
fluorophenyl derivative (10), the next in the hydrophobicity order, displayed some EPI
properties at the high concentration (FAR=4.25 at 20 μM), but was especially potent in terms
of antiproliferative action in the MDR T-lymphoma (IC50=3.50 μM). It is worth to underline
23

that both, 11 and 10, displayed corresponding or even slightly stronger antiproliferative
properties than those of hydantoin derivatives 5 and 7, respectively (Table 3).
The last subseries of the triazine phenylselenoethers (14-17, group C) was surprisingly
inactive, taking into consideration increasing literature evidence indicating a beneficial role of
the 1,3,5-triazine scaffold in various cancers [16-18]. This small subseries differs only in the
field of the C1-linker branches, which undoubtedly influence slightly hydrophobicity and the
steric hindrance/flexibility due to the variety of the spacer. In this group, only the dimethyl-
branched derivative 17 displayed notable antiproliferative property in MDR T-lymphoma
(IC50 =8.28 μM). It should be noted that the antiproliferative action of compound 17 is almost
4-fold more potent in the MDR- than in the PAR cells.
The trends of more potent antiproliferative activity in the resistant T-lymphoma (MDR),
than that in sensitive (PAR) cells, was noted for all three series A-C (Table 3). It suggests that
the phenylselenoether moiety, as the characteristic common feature, is responsible for this
favourable behaviour (marked in red, Fig. 10), while changeable moieties could modulate the
intensity of this effect. The strongest result was observed for the 3,4-dichlorophenylpiperazine
derivative 11 and the aforementioned triazine derivative 17.
Comparing the lack of anticancer action in previous studies for the hydantoin-
phenylpiperazines [14, 32] to the significant effects found in this study (groups A and B), it is
not hard to recognize that the selenoether linker is the most probable structural factor,
responsible for an introduction of both, the cytotoxic and antiproliferative, properties into the
considered chemical families of hydantoin and phenylpiperazines. Likewise, the length of the
linker seems to be crucial. It catches the eye that the most active hydantoin derivative 6,
distinctly predominant in the whole series is only one that contains the longest C6-linker,
while the anticancer action is decreasing with C4-linker (5 and 7) and C3-linker of
phenylpiperazine derivatives (8-13, group B), to be the weakest in the case of short and rather
stiff C1-linkers of 1,3,5-triazine derivatives 14-17 (group C). This is especially noticeable in
the results of cytotoxic properties in both MDR and PAR T-lymphoma cells (Table 3).
Analysing the present results for the new Se-ether compounds (5-17) in comparison to
those for the previous selenoesters (4) and selenoanhydride (3, Fig. 1) [15], corresponding
efflux pump inhibitory and cytotoxic effects in MDR T-lymphoma can be observed. In more
detail, the ABCB1-inhibitory properties of the most active hydantoin-selenoethers (5-7) were
in the range of 1.38-2.56 of the action of verapamil (at a 10-fold higher concentration), while
24

they were 1.57 and 3.43, tested in the same conditions for the selenoanhydride 3 and
selenoester 4, respectively. In the case of cytotoxic action in MDR T-lymphoma, the IC50
values were 4.65 μM (3) and 1.03 μM (4) vs 0.90-4.21 μM for the best selenoethers found in
this study. Interestingly the most active compounds (5-7) were also active in neuroblastoma
SH-SY5Y cells.
On the other hand, an undeniable advantage of these new-discovered hydantoin
selenoethers, referring to previous Se-esters, is the lack of any unpleasant smell and their
higher chemical stability observed in the test conditions. Furthermore, the results of the assays
using an in vitro CYP3A4 model for most active compounds (5-7) demonstrated their low
DDI risk in comparison to ketoconazole (Fig. 9). However, their metabolic stability tested in
mouse microsomes was rather low; the highest stability was observed in the case of the
spirofluorene derivative (7). These results require further scientific considerations, i.e., more
comprehensive studies on biotransformation of 5-7 and their potential metabolites, also using
human metabolism models, as well as an estimation of the intrinsic anticancer/ MDR EPI
properties of the most probable metabolites. On the other hand, further rational
pharmacomodulations to improve metabolic stability and general “drugability” within this
interesting new group of hydantoin-selenoethers is needed.
This study also provided an insight into potential molecular mechanisms of actions for the
hydantoin-selenoether compounds. In order to explore the biological effects of the best
compound determined from the mouse T-lymphoma assays, the longest-linker hydantoin
derivative 6, was evaluated in JURKAT human lymphoblastoid cells since these cells
represent a good system to study drug resistance, cell death and proliferation in response to
drugs treatment [38]. It has been reported in colon cancer cells that selenium shows
chemotherapeutic potential against cancer cells by inactivating AKT and leading to
suppression of cyclin D1 while triggering apoptosis [39]. Notably, methylselenol exposure
changed the expression of genes related to the regulation of cell cycle and apoptosis, among
which the tumour suppressor gene p53 [40]. Thus, we focused on the regulation of
proliferation rate and on cyclinD1 and p53 expression. We were pleased to observe that the
selenium-containing compound 6 inhibited cell proliferation through the negative regulation
of cyclin D1, while enhanced the expression of the p53 gene. Furthermore, the co-treatment of
JURKAT cells with doxorubicin and our compound enhances the chemotherapeutic effect of
this drug, suggesting a synergistic activity warranting further studies. This effect is evident
and statistically significant especially after 72 hours of treatment and when compound 6 is
25

used at the higher dose tested (2 μM). At the present this data indicate the effectiveness of
strategies based on different compound combinations to enhance the activity of commonly
used antineoplastic drugs.
3. Conclusions
In the present work, we have discussed a new chemical family of potent selenium-
containing compounds as hybrids of phenylselenoethers with either arylhydantoin or
phenylpiperazine moieties. Comprehensive studies, i.e., design, synthesis and both, biological
and ADMET-screening in vitro, including an insight into cellular mechanisms of anticancer
action and followed by in-depth SAR analysis, have been performed, yielding promising
results.
Among the new hybrids (5-17), the hydantoin derivatives (5, 6, and 7) were significantly
more effective than the reference inhibitor verapamil (up to 2.6-fold at a 10-fold lower
concentration) in order to inhibit the crucial tumour MDR mechanism of ABCB1-efflux
pump. The very high stimulation of Pgp efflux pump (>200% of Pgp basal activity) at the
presence of 5-7 confirmed their substrate potency and the probable competitive action with
123 rhodamine to the binding site in ABCB1-efflux modulating assay. Next, the cytotoxic and
anti-proliferative action in both, sensitive (PAR) and resistant (MDR), mouse T-lymphoma
cell lines revealed that both hydantoin- (5-7) and phenylpiperazine-selenoethers (10 and 11)
possessed a good activity profile, being the 5,5-diphenylhydantoin compound (6) the best one
out of the complete series. The promising antiproliferative action of the hydantoin compounds
6 and 7 were confirmed additionally in non-lymphocytic cancer cells, i.e. neuroblastoma,
suggesting a therapeutically-promising broader spectrum of anticancer potency for this
chemical family, also with an accent on the longer-linker compound (6). The mechanistic
study results indicate that compound 6 inhibits cell cycle progression through the reduction of
the expression of cyclin D1 and cell proliferation by inducing p53 expression, thus giving a
first insight into the mechanism of action. Furthermore, the SAR analysis proved that the
lipophilic properties, together with a longer selenoether spacer, are the likely factors affecting
all, cancer EPI-, antiproliferative- and cytotoxic effects, in the tested cancer models for the
new family of selenocompounds.
Overall, this study discovered a new chemical space of highly active selenium-containing
anticancer agents, worthy of being studied more in-depth in order to improve drug-likeness
and to reveal the precise mode of action. In this context, compound 6, as the most active agent
26

found in this study, will serve as a new lead structure for further pharmacomodulations and
extended mechanistic studies as well as a medicinal chemistry optimization.
4. Experimental
4.1. Chemistry
Reagents were bought from Alfa Aesar (Karlsruhe, Germany) or Sigma Aldrich (Darmstadt,
Germany). Solvents were dried over calcium hydrochloride (toluene) or calcium oxide
(methanol). Reaction progress was verified using Thin Layer Chromatography (TLC), which
was carried out on 0.2 mm Merck silica gel 60 F254 plates. Spots were visualized by UV light
or treatment with Dragendorff reagent. Melting points (mp) were determined using a MEL-
TEMP II apparatus and are uncorrected. The 1H NMR and 13C NMR spectra were obtained on
77
a Varian Mercury-VX 300 MHz spectrometer and for the Se NMR on a Bruker Variance
500MHz spectrometer in DMSO-d6, CDCl3-d, MeOD-d4, or Acetone-d6. Chemical shifts in
the relative NMR spectra were reported in parts per million (ppm) on the δ scale using the
solvent signal as an internal standard. Data are reported as follows: chemical shift,
multiplicity (s, singlet; br. s, broad singlet; d, doublet; t, triplet; dd, doublet of doublet, q,
quintet, td triplet of doublet, m, multiplet),), coupling constant J in Hertz (Hz), number of
protons, proton’s position (Ph-phenyl, Flou-Flourine, Pp-piperazine). LC-MS were carried out
on a system consisting of a Waters Acquity UPLC, coupled to a Waters TQD mass
spectrometer. Retention times (tR) are given in minutes. The UPLC/MS purity of all final
compounds was determined (%). The intermediates 18-20 are described in our previous
works. [14, 21, 22] While the intermediates 21, 23, 25-30 were obtained according to the
procedure described earlier [21] and are known in the literature [24, 26-29, 44, 45] (for details
see Supporting Information).
4.1.1. General procedure for the Se-alkylation (5-14)
As part of this general procedure, the appropriate diphenyl diselenide (9.5 mmol) was
dissolved in a 1:1 mixture of water and THF (50 mL) under protective nitrogen gas. Then,
sodium borohydride (47.5 mmol) was added, and the mixture was stirred for approximately 35
minutes, with a decolourization of the solution observed already after 1-3 minutes. Next, a
solution of hydantoin substitutions or piperazine derivatives (19.37 mmol) in THF or DCM (5
mL) was slowly added, and the reaction mixture was stirred at room temperature and
monitored via TLC until the complete consumption of the starting material which occurred
27

after 24-48 hours. Upon completion, the reaction was quenched with 50 mL of a saturated
aqueous solution of ammonium chloride and extracted with diethyl ether. The combined
organic layers were dried over sodium sulphate and evaporated under reduced pressure to
yield the desired products 5-14.
4.1.1.1. Methyl 2-(2,5-dioxo-4,4-diphenyl-3-(4-(phenylselanyl)butyl)imidazolidin-1-yl)acetate
(5)
Compound 18 (10 mmol) was used. White solid, mp 128˚C. Yield 23.59%. LC/MS+/: purity:
100 %, tR=8.97, (ESI) m/z [M+H]+ 537.33. C28H28O4N2Se (MW 536.12). 1H NMR (DMSO) δ
7.47–7.43 (m, 5H-Ph), 7.29 (s, 10H-2Ph), 4.34 (s, 2H-CH2), 3.71 (s, 3H-CH3), 3.33–3.28 (m,
2H-CH2), 2.62 (t, J = 7.3 Hz, 2H-CH2), 1.37–1.17 (m, 2H-CH2), 0.88 (dt, J = 15.3, 7.7 Hz,
2H-CH2). 13C NMR (DMSO) δ 172.80, 167.95, 136.69, 131.32, 129.15, 128.93, 128.79,
128.15, 128.07, 126.44, 74.51, 73.31, 52.55, 27.61, 26.47, 26.01. 77Se NMR (CDCL3) δ
294.61.
4.1.1.2. Methyl 2-(2,5-dioxo-4,4-diphenyl-3-(6-(phenylselanyl)hexyl)imidazolidin-1-yl)acetate
(6)
Compound 19, Cas Number (1350707-01-5) (10 mmol) was used. Yellowish solid, mp 80˚C.
Yield 23.20%. C30H32O4N2Se (MW 564.15). LC/MS+/: purity: 92.76%, tR=9.65, (ESI) m/z
1
[M+H]+ 565.25. H NMR (CDCl3) δ 7.31–7.27 (m, 2H-Ph), 7.26–7.18 (m, 10H-2Ph), 7.12–
7.04 (m, 3H-Ph), 4.17 (s, 2H-CH2), 3.64 (s, 3H-CH3), 3.20–3.14 (m, 2H-CH2), 2.62 (t, J = 7.5
Hz, 2H-CH2), 1.44 (s, 2H-CH2), 1.36–1.28 (m, 2H-CH2), 0.97 (dt, J = 14.9, 7.5 Hz, 2H-CH2),
0.83 (dt, J = 14.6, 7.3 Hz, 2H-CH2), 0.72 (dt, J = 15.0, 7.5 Hz, 2H-CH2). 13C NMR (CDCl3) δ
173.57, 167.70, 154.88, 136.93, 132.30, 130.56, 128.99, 128.96, 128.79, 128.46, 126.59,
77.26, 77.01, 76.75, 75.25, 52.70, 42.13, 39.84, 33.59, 32.25, 29.69, 28.96, 27.60, 27.57,
26.03. 77Se NMR (CDCL3) δ 290.63.
4.1.1.3. 1'-Methyl-3'-(4-(phenylselanyl)butyl)spiro[fluorene-9,4'-imidazolidine]-2',5'-dione
(7)
Compound 20 (10 mmol) was used. White solid, mp 132˚C. Yield 15.20%. C26H24O2N2Se
1
(MW 476.10). LC/MS+/: purity: 95.87%, tR=8.56, (ESI) m/z [M+H]+ 477.18. H NMR
28

(CDCl3) δ 7.72 (d, J = 7.6 Hz, 2H-Flu), 7.45 (td, J = 7.5, 1.1 Hz, 2H- Flu), 7.36–7.31 (m, 2H-
Flou), 7.28 (td, J = 7.5, 1.0 Hz, 2H- Flu), 7.21 (d, J = 7.5 Hz, 2H- Ph), 7.20–7.17 (m, 3H-Ph),
3.17 (s, 3H-CH3), 2.96 (t, J = 7.4 Hz, 2H-CH2), 2.66–2.55 (m, 2H-CH2), 1.44 (dt, J = 15.1,
13
7.5 Hz, 2H-CH2), 1.20 (dt, J = 15.0, 7.5 Hz, 2H-CH2). C NMR (CDCl3) δ 171.89, 157.27,
141.52, 140.17, 132.64, 130.31, 129.85, 128.83, 128.24, 126.71, 123.57, 120.89, 77.16, 76.91,
76.65, 75.01, 40.63, 28.83, 26.87, 26.83, 25.61. 77Se NMR (CDCL3) δ 292.63.
4.1.1.4. 1-Phenyl-4-(3-(phenylselanyl)propyl)piperazine (8)
Compound 21 (10 mmol), CAS Number (10599-17-4), was used. White solid, mp 248˚C.
Yield 20.11%. C19H24N2Se (MW 360.11). LC/MS+/: purity: 100%, tR=4.82, (ESI) m/z
[M+H]+ 361.07. 1H NMR (MeOD) δ 7.61–7.58 (m, 2H-Ph), 7.34–7.28 (m, 5H-Ph), 7.05–7.02
(m, 2H-Ph), 6.94 (ddd, J = 8.3, 2.0, 1.0 Hz, 1H-Ph), 3.79–3.37 (m, 8H-Pp-2,6-H+ Pp-3,5-H),
3.36 (s, 2H-CH2), 3.05 (t, J = 7.1 Hz, 2H-CH2), 2.24–2.16 (m, 2H-CH2). 13C NMR (DMSO) δ
149.57, 131.56, 129.57, 129.32, 129.07, 126.75, 119.88, 115.87, 55.10, 50.65, 45.21, 42.46,
23.74, 23.32. 77Se NMR (DMSO): 283.99.
4.1.1.5. 1-(2-Methoxyphenyl)-4-(3-(phenylselanyl)propyl)piperazine hydrochloride(9)
Commercial 1-(3-chloropropyl)-4-(2-methoxyphenyl)piperazine (10 mmol), CAS Number
(21279-77-6), was used. White solid, mp 287˚C. Yield 8.6%. Basic form C20H26ON2Se (MW
390.12), C20H27ClON2Se (MW 426.10). LC/MS+/: purity: 100%, tR=4.97, (ESI) m/z [M+H]+
1
391.17. H NMR (DMSO) δ 11.36 (s, 1H, NH+), 7.23–7.15 (m, 6H-Ph), 6.91 (d, J = 8.4 Hz,
2H-Ph), 6.78 (t, J = 7.3 Hz, 1H-Ph), 3.69 (dd, J = 11.7, 5.3 Hz, 2H-CH2), 3.43 (d, J = 10.3 Hz,
2H-CH2), 3.16–2.96 (m, 8H-Pp-2,6-H+ Pp-3,5-H), 2.10–2.02 (m, 2H-CH2). 13C NMR (126
MHz, DMSO) δ 131.58, 129.59, 129.41, 129.31, 129.08, 126.76, 115.86, 50.58, 45.19, 23.74,
23.31, 0.10. 77Se NMR (DMSO): 481.64.
4.1.1.6. 1-(4-Fluorophenyl)-4-(3-(phenylselanyl)propyl)piperazine hydrochloride (10):
Compound 21 (10 mmol), was used. White solid, mp 204˚C. Yield 37.68%. Basic form
C19H23FN2Se (MW 378.10), C19H24ClFN2Se (MW 414.08). LC/MS+/: purity: 100%, tR=4.83,
1
(ESI) m/z [M+H]+ 379.14. H NMR (DMSO) δ 11.10 (s, 1H, NH+), 7.50–7.46 (m, 2H-Ph),
7.31–7.21 (m, 3H-Ph), 7.08–7.02 (m, 2H-Ph), 6.99-6.94 (m, 2H-Ph), 3.65 (d, J = 10.3 Hz,2H-
CH2 ), 3.47 (d, J = 9.8 Hz, 2H-CH2), 3.20–2.96 (m, 8H-Pp-2,6-H+ Pp-3,5-H), 2.08 (dt, J =
29