Phenylselanyl group incorporation for "glutathione peroxidase-like" activity modulation

Article abstract of DOI:10.3390/molecules25153354

The ability of organoselenium molecules to mimic the activity of the antioxidant selenoenzyme glutathione peroxidase (GPx) allows for their use as antioxidant or prooxidant modulators in several diseases associated with the disruption of the cell redox homeostasis. Current drug design in the field is partially based on specific modifications of the known Se-therapeutics aimed at achieving more selective bioactivity towards particular drug targets, accompanied by low toxicity as the therapeutic window for organoselenium compounds tends to be very narrow. Herein, we present a new group of Se-based antioxidants, structurally derived from the well-known group of GPx mimics-benzisoselenazol-3(2H)-ones. A series of N-substituted unsymmetrical phenylselenides with an o-amido function has been obtained by a newly developed procedure: a copper-catalyzed nucleophilic substitution by a Se-reagent formed in situ from diphenyl diselenide and sodium borohydride. All derivatives were tested as antioxidants and anticancer agents towards breast (MCF-7) and leukemia (HL-60) cancer cell lines. The highest H2O2-scavenging potential was observed for N-(3-methylbutyl)-2-(phenylselanyl)benzamide. The best antiproliferative activity was found for (-)-N-(1S,2R,4R)-menthyl-2-(phenylselanyl)benzamide (HL-60) and ((-)-N-(1S,2R,3S,6R)-(2-caranyl))benzamide (MCF-7). The structure-activity correlations, including the differences in reactivity of the obtained phenyl selenides and corresponding benzisoselenazol-3(2H)-ones, were performed.

Full text of DOI:10.3390/molecules25153354

molecules
Article
Phenylselanyl Group Incorporation for “Glutathione
Peroxidase-Like” Activity Modulation
Magdalena Obieziurska-Fabisiak 1, Agata J. Pacuła 1, Lucia Capoccia 2,
Joanna Drogosz-Stachowicz 3, Anna Janecka 3, Claudio Santi 2 and Jacek Scianowski
´
1,
*
1
Department of Organic Chemistry, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Street,
87-100 Torun, Poland; magdao@umk.pl (M.O.-F.); pacula@umk.pl (A.J.P.)
Dipartimento di Scienze Farmaceutiche, Universita di Perugia, Via del Liceo 1, 06134 Perugia, Italy;
lucia.capoccia@outlook.it (L.C.); claudio.santi@unipg.it (C.S.)
Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Mazowiecka 6/8,
92-215 Lodz, Poland; joanna.drogosz@stud.umed.lodz.pl (J.D.-S.); anna.janecka@umed.lodz.pl (A.J.)
Correspondence: jsch@umk.pl
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Francesca Marini
Received: 10 July 2020; Accepted: 23 July 2020; Published: 24 July 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The ability of organoselenium molecules to mimic the activity of the antioxidant
selenoenzyme glutathione peroxidase (GPx) allows for their use as antioxidant or prooxidant
modulators in several diseases associated with the disruption of the cell redox homeostasis.
Current drug design in the field is partially based on specific modifications of the known
Se-therapeutics aimed at achieving more selective bioactivity towards particular drug targets,
accompanied by low toxicity as the therapeutic window for organoselenium compounds tends
to be very narrow. Herein, we present a new group of Se-based antioxidants, structurally
derived from the well-known group of GPx mimics—benzisoselenazol-3(2H)-ones. A series of
N-substituted unsymmetrical phenylselenides with an o-amido function has been obtained by a newly
developed procedure: a copper-catalyzed nucleophilic substitution by a Se-reagent formed in situ
from diphenyl diselenide and sodium borohydride. All derivatives were tested as antioxidants
and anticancer agents towards breast (MCF-7) and leukemia (HL-60) cancer cell lines. The highest
H2O2-scavenging potential was observed for N-(3-methylbutyl)-2-(phenylselanyl)benzamide. The best
antiproliferative activity was found for (
−
)-N-(1S,2R,4R)-menthyl-2-(phenylselanyl)benzamide (HL-60)
and (( )-N-(1S,2R,3S,6R)-(2-caranyl))benzamide (MCF-7). The structure–activity correlations, including the
−
differences in reactivity of the obtained phenyl selenides and corresponding benzisoselenazol-3(2H)-ones,
were performed.
Keywords: selenides; antioxidant activity; anticancer activity
1. Introduction
Drug design is a multi-step process, focused on the obtainment of the most specific ligand–receptor
interaction correlated to a suitable structural core that is able to equip the molecule with a potential
biological activity. The first discovered lead compound is subsequently variously functionalized in
order to increase (and maximize) the desired therapeutic activity over the toxicity. The pharmacophore
modeling often includes installation of aromatic or heteroaromatic rings, which are easy to introduce,
can be further manipulated and are often responsible of the activity [1,2].
In the field of organoselenium chemistry, the design of Se-based therapeutics is often connected
with the ability of selenium pharmacophores to mimic the activity of glutathione peroxidase (GPx).
Over the years, the role of organoselenium compounds as redox-modulators was well-established
with numerous examples of biologically active molecules [
3
–
6], including the antioxidant agent
Molecules 2020, 25, 3354; doi:10.3390/molecules25153354
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3354
2 of 13
N-phenylbenzisoselenazol-3(2H)-one (named as Ebselen)
1
, currently in phase II clinical trial for
noise-induced hearing loss [
Se-molecules possess aromatic or heteroaromatic rings as the core of the molecule [
are presented in Scheme 1 and also include other Se-therapeutics currently in clinical trial:
ethaselen , Trx reductase inhibitor, antitumor agent [ ] and 4,4-dimethyl-benziso-2H-selenazine
7
]. Similarly, to ebselen
1
, a significant number of proven bioactive
3]. Examples
2
8
3, anti-inflammatory therapeutic tested in chronic plaque psoriasis [9].
O
O
O
N
Se
N
Se
N
Se
NH
Se
1
Ebselen,
2
Ethaselen,
3
ALT 2074,
Scheme 1. Bioactive organoselenium compounds 1–3 that possess aromatic rings in their structure.
Many research groups continue the study of new strategies and structural modifications to obtain
new Se-antioxidants that have high and selective activity. In our previous work, we explored the
possibility to improve the GPx-like activity of ebselen with the introduction of specific functionalities that
would enable new highly efficient biocatalyst [10
were obtained and easily transformed into the corresponding diselenides
seleninic acids, as well as to their potassium salts (path ) [16]. Determination of the antioxidant and
–
12]. Various N-aromatic and N-aliphatic derivatives
4
5
(path ) [13 15] and
a
–
6
b
antiproliferative potential of all obtained molecules revealed a particular structure–activity relationship.
Besides the observed influence of the N-substituent on their biological potential, it was also recently
highlighted by Santi and co-workers [17], that the form of the Se-moiety is crucial for the specific
catalytic activity of the designed GPx-mimics. To further differentiate the structures and to broaden
the scope of the tested molecules, we introduced a phenylselanyl group as a new benzisoselenazolone
core modification (7b–23b, Scheme 2).
Scheme 2. Possible structural modification of N-substituted benzisozelenazol-3(2H)-ones 4.
This modification allowed us to obtain a large group of GPx mimetics 7b–23b and to determine
whether the introduction of an additional aromatic ring and the exchange of Se-N for Se-Car bond is
justified in order to obtain higher therapeutic potential of the N-substituted ebselen-like antioxidants.
2. Results and Discussion
The first step of the research involved the synthesis of N-substituted o-iodobenzamides 7a
The compounds were obtained through the reaction of the corresponding amines with o-iodobenzoic
acid chloride Benzamides 7a 23a were further transformed to the final N-aliphatic 7b 12b,
–23a.
8.
–
–

Molecules 2020, 25, 3354
3 of 13
N-aromatic 13b
–
17b and chiral N-terpenyl [18] phenylselenides 18b
–
23b by a copper-catalyzed
nucleophilic aromatic substitution. The selenium nucleophile was prepared in situ from diphenyl
diselenide and sodium borohydride. All derivatives were obtained in moderate to good yields
(Scheme 3).
Scheme 3. Synthesis of N-substituted phenylselenides 7b–23b.
Considering the mechanism of the reaction, we assume that the first step includes the
base-promoted formation of (1,10-ph)Cu-SePh complex 25. The oxidative addition to the Car-I
bond of the amide leads to the copper(III) complex 26 in which both the arene and Se-nucleophile are
ligated to the metal. Next, through the reductive elimination pathway, the final selenide is formed
with the regeneration of the (1,10-ph)Cu(I)I catalyst 24 (Scheme 4).
Scheme 4. Putative mechanism for the Cu-catalyzed introduction of phenylselenyl group.
The final goal of the research was to evaluate the obtained phenylselenides 7b–23b as possible
GPx-mimics and anticancer agents. The antioxidant capacity was tested by a conventionally used
NMR-activity assay proposed first by Iwaoka and co-workers [19]. The rate of H2O2 reduction by
the Se-catalyst was indirectly evaluated by the oxidation of dithiothreitol as a mimic of the reducing

Molecules 2020, 25, 3354
4 of 13
thiol cofactor. The conversion of the dithiol DTTred to the disulphide DTTox was observed in 1HNMR
spectra in the specific time intervals. The results for the most active derivatives are presented in
Scheme 5. The results obtained for all compounds are reported in Supporting Information.
Scheme 5. Results of the antioxidant activity measurements.
The highest antioxidant potential was observed for N-butyl 9b, N-3-methylbutyl 10b and N-pinanyl
phenyl selenide 22b. The results of the three selected Se-catalysts were compared to corresponding
benzisoselenazol-3(2H)-ones 27–29. It could be noticed that the bulkiness of the substituent enhances
the H2O2 scavenging activity of benzisoselenazolones (reactivity: 29
>
28
>
27) but decreased it for the
29, the hindrance of
corresponding phenylselenides (reactivity: 10b 22b 9b). For compounds 27–
>
>
the N-substituent facilitated the cleavage of the Se-N bond that accelerated the Se-moiety oxidation by
hydrogen peroxide. On the contrary, the reaction of -SePh group with H2O2 proceeded more efficiently
when the alkyl chain did not hinder the selenium atom.
To investigate the mechanism of the antioxidant activity, we have performed an additional 77Se
NMR experiment of the H2O2-oxidation product of the most reactive N-butyl phenylselenide 9b
(the sample was stored for 12 h before the NMR recording). A signal at 853 ppm indicated the
formation of corresponding selenooxide. Based on these observations, supported by previous literature
reports [20,21], we assume that the possible GPx-like catalytic cycle of the tested phenyl selenides
involves the formation of the selenoxide 30, which is further hydrated to the corresponding hydrated
oxide 31. The final H2O2 reduction and thiol oxidation proceeds through the reversible formation of
the peroxy-hydrated oxide 32 (Scheme 6).

Molecules 2020, 25, 3354
5 of 13
Scheme 6. Plausible mechanism for the H2O2 reduction by the Se-catalysts in the presence of thiols.
Next, all phenylselenides 7b
(3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay against breast (MCF-7) and
leukemia (HL-60) cell lines. The best results were obtained for N-terpene derivatives 18b 19b and
–23b were evaluated as antiproliferative agents by the MTT
,
compared with the data for the corresponding benzisoselenazol-3(2H)-ones 33 and 34 (Table 1). For the
rest of the tested samples 7b–17b, 20b–23b, the IC50 values were above 50 µM.
Table 1. Cytotoxic activity of N-terpene derivatives and the corresponding benzisoselenazol-3(2H)-ones.
MCF-7
IC50 [µM]
HL-60
IC50 [µM]
MCF-7
IC50 [µM]
HL-60
IC50 [µM]
Structure
Structure
36.1 ± 0.6
16.3 ± 0.3
10.7 ± 0.6
16.3 ± 0.2
11.9 ± 0.2
24.3 ± 2.4
62 ± 2.0
18b
19b
33
34
203 ± 2.0
Additionally, it was observed that the antiproliferative potential of analogs increased when the
phenylselanyl moiety was introduced into the structure, showing that an additional aromatic ring can
be beneficial for the compound’s cytotoxicity.
We have previously noticed that the internal 2-methylbutyl carbon chain is a repetitive
element in the structure of the active benzisoselenazol-3(2H)-ones 28, 33 and 35, which indicates
its potential role as a pharmacophore. Additional carbon chains or functional groups attached to
the 2-methylbutyl substituent influenced the inhibitory potential. The antiproliferative activity was
the highest for compounds with the carbon chain expanded to the cyclic menthyl functionality,
benzisoselenazol-3(2H)-one 30 and phenylselenide 18b with IC50 values 11.9
10.7 ± 0.6 µM (HL-60), respectively (Scheme 7).
±
0.2 µM (MCF-7) and

Molecules 2020, 25, 3354
6 of 13
Scheme 7. Repetitive carbon chain in the structure of bio-active derivatives 18b, 28, 33 and 35.
3. Materials and Methods
3.1. General
NMR spectra were recorded on Bruker Avance III/400 or Bruker Avance III/700 (Karlsruhe,
Germany) for 1H and 176.1 MHz or 100.6 MHz for 13C (see Supplementary Material). Chemical shifts
were recorded relative to SiMe4 (δ0.00) or solvent resonance (CDCl3 δ7.26, CD3OD δ3.31). Multiplicities
were given as: s (singlet), d (doublet), dd (double doublet), ddd (double double doublet), t (triplet),
dt (double triplet), and m (multiplet). The 77Se NMR spectra were recorded on Bruker Avance III/400
or Bruker Avance III/700 with diphenyl diselenide as an external standard. NMR spectra were carried
out using ACD/NMR Processor Academic Edition. Melting points were measured with a Büchi Tottoli
SPM-20 heating unit (Büchi Labortechnik AG, Flawil, Switzerland) and were uncorrected. Elemental
analyses were performed on a Vario MACRO CHN analyzer. Optical rotations were measured in
10-mm cells with a polAAr 3000 polarimeter. Column chromatography was performed using Merck
40-63D 60Å silica gel (Merck, Darmstadt, Germany). Commercially available solvents DMF, DCM,
and MeOH (Aldrich, St. Louis, MO, USA) and chemicals were used without further purification.
3.2. Procedures and Analysis Data
3.2.1. Synthesis of N-substituted o-iodobenzamides 7a–23a
2% NaOH (4.4 mL) was added to a solution of an amine (1.0 mmol) in DCM (2 mL). The mixture
was cooled to 0 ◦C and o-iodobenzoic acid chloride (1.1 mmol) dissolved in DCM (3 mL) was added
dropwise. The reaction mixture was stirred at room temperature for 20 h and the product was extracted
with DCM. Combined organic layers were washed with saturated NaHCO3 and dried over magnesium
sulfate. The solvent was removed under reduced pressure and the product was obtained as white solid.
((−
)-N-(1R,2S,5R)-menthyl)-o-iodobenzamide 18a Yield: 98%, mp 146–148 ◦C; [α]D20
= 0.88 (d, J = 7.0 Hz, 3H, CH3), 0.92 (s, 3H, CH3), 0.93 (s, 3H,
=
−
38.93 (c = 5.73,
CHCl3); 1H NMR (700 MHz, CDCl3)
δ
CH3), 0.98–1.03 (m, 1H), 1.11–1.18 (m, 2H), 1.51–1.57 (m, 2H), 1.69–1.75 (m, 2H), 2.08–2.12 (m, 1H),
2.17–2.20 (m, 1H), 3.95–4.00 (m, 1H), 5.41 (d, J = 9.1 Hz, 1H, NH), 7.06–7.09 (m, 1Har), 7.35–7.38 (m,
2Har), 7.84 (dd, J1 = 0.7, J2 = 7.7 Hz, 1Har); 13C NMR (100.6 MHz, CDCl3)
δ = 16.20 (CH3), 21.20 (CH3),
22.15 (CH3), 23.78 (CH2), 26.91 (CH), 31.89 (CH), 34.52 (CH2), 42.86 (CH2), 48.13 (CH), 50.75 (CH),
92.32 (Car), 128.08 (CHar), 128.12 (CHar), 130.86 (CHar), 139.88 (CHar), 142.93 (Car), 168.61 (C=O); IR:
3230, 2951, 2916, 2867, 1636, 1584, 1540, 1462, 1430, 1385, 1367, 1341, 1325, 1307, 1261, 1161, 1147, 1116,
1107, 1059, 1043, 1014 cm−1; Elemental Anal. Calcd for C17H24INO (385.09): C, 53.00; H, 6.28; N, 3.64
Found: C, 53.18; H, 6.34; N, 3.76.
((
−
)-N-(1S,2R,3S,6R)-(2-caranyl))-o-iodobenzamide 19a Yield: 90%; mp 145–147 ◦C; [α]D20
= 21.47
−
(c = 4.77, CHCl3); 1H NMR (700 MHz, CDCl3) 0.64–0.68 (m, 1H), 0.94–1.01 (m, 1H), 1.02 (d, J = 7.7
Hz, 3H, CH3), 1.05 (s, 3H, CH3), 1.11 (s, 3H, CH3), 1.23–1.28 (m, 1H), 1.55–1.60 (m, 2H), 1.71–1.74 (m,
1H), 1.77–1.82 (m, 1H), 3.54–3.58 (m, 1H), 5.72 (d, J = 8.4 Hz, 1H, NH), 7.09 (dt, J1 = 2.1, J2 = 8.4 Hz,
1Har), 7.38 (dt, J1 = 0.7, J2 = 7.7 Hz, 1Har), 7.43 (dd, J1 = 2.1, J2 = 7.7 Hz, 1Har), 7.87 (dd, J1 = 0.7, J2 = 7.7

Molecules 2020, 25, 3354
7 of 13
Hz, 1Har); 13C NMR (100.6 MHz, CDCl3)
δ
= 15.55 (2
×
CH3), 17.59 (C), 19.03 (CH2), 20.28 (CH), 28.71
(CH), 29.25 (CH3), 30.83 (CH2), 34.92 (CH), 50.41 (CH), 92.38 (Car), 128.14 (CHar), 128.35 (CHar), 130.90
(CHar), 139.96 (CHar), 142.81 (C), 168.50 (C=O); IR: 3249, 2915, 2862, 1656, 1630, 1585, 1546, 1459, 1430,
1375, 1330, 1257, 1115, 1015 cm−1; Elemental Anal. Calcd for C17H22INO (383.27): C, 53.27; H, 5.79; N,
3.65 Found: C, 53.05; H, 5.71; N, 3.53.
N-bornyl-o-iodobenzamide 20a Yield: 93%; mp 122–123 ◦C (lit. [14] mp 119–121 ◦C); [α]D20 = +11.01 (c
= 5.54, CHCl3); 1H NMR (700 MHz, CDCl3) 0.90 (s, 3H, CH3), 0.91–0.99 (m, 1H), 0.96 (s, 3H, CH3), 0.99
(s, 3H, CH3), 1.17–1.21 (m, 1H), 1.41–1.46 (m, 1H), 1.56–1.61 (m, 1H), 1.70 (t, J = 9.1 Hz, 1H), 1.76–1.81
(m, 1H), 2.42–2.47 (m, 1H), 4.41–4.45 (m, 1H), 5.78 (d, J = 8.4 Hz, 1H, NH), 7.07–7.10 (m, 1Har), 7.35–7.40
(m, 2Har), 7.85 (dd, J1 = 0.7, J2 = 7.7 Hz, 1Har); 13C NMR (100.6 MHz, CDCl3)
δ = 13.96 (CH3), 18.72
(CH3), 19.81 (CH3), 28.20 (CH2), 28.37 (CH2), 37.44 (CH2), 44.92 (CH), 48.38 (C), 49.70 (C), 54.64 (CH),
92.40 (Car), 128.20 (CHar), 128.42 (CHar), 130.96 (CHar), 139.83 (CHar), 142.84 (Car), 169.45 (C=O); IR:
3319, 2981, 2950, 2877, 1642, 1584, 1561, 1510, 1479, 1459, 1429, 1388, 1374, 1361, 1310, 1290, 1262, 1228,
1205, 1172, 1154, 1115, 1063, 1046, 1012 cm−1; Elemental Anal. Calcd for C17H22INO (383.27): C, 53.27;
H, 5.79; N, 3.65 Found: C, 53.11; H, 5.65; N, 3.47.
(
−
)-N-(1S,2R,5S)-myrtanyl-o-iodobenzamide 21a Yield: 83%; mp 142–144 ◦C; [α]D20
= 8.23 (c = 5.33,
−
1
CHCl3); H NMR (400 MHz, CDCl3) 0.96 (d, J = 9.6 Hz, 1H), 1.11 (s, 3H, CH3), 1.24 (s, 3H, CH3),
1.55–1.64 (m, 1H), 1.88–2.10 (m, 5H), 2.35–2.44 (m, 2H), 3.47–3.51 (m, 2H), 5.79 (bs, 1H, NH), 7.09–7.13
(m, 1Har), 7.37–7.42 (m, 2Har), 7.87 (dd, J1 = 0.8, J2 = 8.4 Hz, 1Har); 13C NMR (100.6 MHz, CDCl3)
δ = 17.56 (CH2), 21.56 (CH3), 26.00 (CH2), 27.99 (CH3), 33.13 (CH2), 38.74 (C), 41.22 (CH), 41.36 (CH),
43.92 (CH), 45.75 (CH2), 92.41 (Car), 128.16 (CHar), 128.31 (CHar), 130.98 (CHar), 139.84 (CHar), 142.67
(Car), 169.33 (C=O); IR: 3239, 2935, 2903, 2889, 2859, 1636, 1584, 1542, 1462, 1430, 1382, 1364, 1316, 1292,
1260, 1218, 1156, 1113, 1054, 1014 cm−1; Elemental Anal. Calcd for C17H22INO (383.27): C, 53.27; H,
5.79; N, 3.65 Found: C, 53.55; H, 5.84; N, 3.74.
(
−
)-N-(1R,2R,3R,5S)-isopinocamphyl-o-iodobenzamide 22a Yield: 78%; mp 130–132 ◦C; [α]D20
= −18.67
(c = 4.88, CHCl3); 1H NMR (400 MHz, CDCl3) 0.91 (d, J = 10.0 Hz, 1H), 1.12 (s, 3H, CH3), 1.26 (s, 3H,
CH3), 1.28 (s, 3H, CH3), 1.73–1.78 (m, 1H), 1.88–1.91 (m, 1H), 1.93–1.97 (m, 1H), 2.01–2.05 (m, 1H),
2.44–2.50 (m, 1H), 2.73–2.80 (m, 1H), 4.48–4.56 (m, 1H), 5.67 (bs, 1H, NH), 7.09–7.14 (m, 1Har), 7.38–7.45
(m, 2Har), 7.88 (dd, J1 = 0.8, J2 = 8.0 Hz, 1Har); 13C NMR (100.6 MHz, CDCl3)
δ = 20.98 (CH3), 23.39
(CH3), 28.00 (CH3), 35.31 (CH2), 36.91 (CH2), 38.49 (C), 41.60 (CH), 46.20 (CH), 47.85 (CH), 48.58 (CH),
92.47 (Car), 128.22 (CHar), 128.37 (CHar), 130.94 (CHar), 139.78 (CHar), 142.68 (Car), 168.78 (C=O); IR:
3242, 2980, 2969, 2900, 2867, 1632, 1584, 1556, 1534, 1458, 1428, 1384, 1372, 1348, 1336, 1319, 1301, 1259,
1227, 1160, 1056, 1016 cm−1; Elemental Anal. Calcd for C17H22INO (383.27): C, 53.27; H, 5.79; N, 3.65
Found: C, 53.49; H, 5.87; N, 3.71.
(+)-N-(1R,2R,3R,5S)-isopinocamphyl-o-iodobenzamide 23a Yield: 79%; mp 142–144 ◦C; [α]D20 = +17.17
(c = 6.00, CHCl3) 1H NMR (400 MHz, CDCl3) 0.90 (d, J = 10.0 Hz, 1H), 1.11 (s, 3H, CH3), 1.25 (s, 3H,
CH3), 1.27 (s, 3H, CH3), 1.72–1.77 (m, 1H), 1.88–1.90 (m, 1H), 1.94–1.96 (m, 1H), 2.02–2.03 (m, 1H),
2.43–2.49 (m, 1H), 2.72–2.79 (m, 1H), 4.49–4.55 (m, 1H), 5.65 (bs, 1H, NH), 7.09–7.13 (m, 1H, 1Har),
7.40–7.44 (m, 2H, 2Har), 7.87 (d, J = 7.2 Hz, 1H, 1Har); 3C NMR (100.6 MHz, CDCl3)
δ = 20.99 (CH3),
23.40 (CH3), 28.00 (CH3), 35.33 (CH2), 36.94 (CH2), 38.50 (C), 41.60 (CH), 46.22 (CH), 47.83 (CH), 48.58
(CH), 92.49 (Car), 128.26 (CHar), 128.41 (CHar), 130.97 (CHar), 139.80 (CHar), 142.68 (Car), 168.80 (C=O);
IR: 3305, 2962, 2922, 2891, 2863, 1632, 1582, 1526, 1450, 1428, 1378, 1347, 1334, 1313, 1294, 1274, 1257,
1229, 1220, 1163, 1015 cm−1; Elemental Anal. Calcd for C17H22INO (383.27): C, 53.27; H, 5.79; N, 3.65
Found: C, 53.59; H, 5.70; N, 3.78.
3.2.2. Synthesis of N-substituted phenylselenides 10–15
To a solution of a diphenyl diselenide (0.5 mmol) in dry toluene (5 mL), sodium borohydride
(1.5 mmol) was added and stirred at room temperature. Next, DMSO was added dropwise until the
solution discolored. Then, respectively, CuI (0.1 mmol), 1,10-phenanthroline (0.2 mmol) and an amide
(1.0 mmol) were added. The mixture was stirred under reflux for 18 h. The solution was cooled to room

Molecules 2020, 25, 3354
8 of 13
temperature and brine (5 mL) was added. The product was extracted with chloroform (2 × 10 mL),
and the combined organic layers were washed with water (2 10 mL), brine (2 10 mL) and dried
×
×
over magnesium sulphate. The solvent was removed under reduced pressure and the obtained crude
product was isolated by column chromatography (silica gel, DCM).
N-ethyl-2-(phenylselanyl)benzamide 7b Yield: 38%; mp 89–91 ◦C (lit. [20] mp 84–86 ◦C); 1H NMR
(400 MHz, CDCl3)
7.11–7.13 (m, 1Har), 7.17–7.22 (m, 2Har), 7.35–7.41 (m, 3Har), 7.51–7.54 (m, 1Har), 7.63–7.65 (m, 2Har);
13C NMR (100.6 MHz, CDCl3)
= 14.81 (CH3), 35.03 (CH2), 125.80 (CHar), 127.36 (CHar), 128.48 (CHar),
129.59 (2 × CHar), 130.01 (Car), 130.90 (CHar), 131.34 (CHar), 134.79 (Car), 134.82 (Car), 135.95 (2 × CHar),
168.19 (C=O); 77Se NMR (76 MHz, CDCl3)
= 435.56 ppm; IR: 3267, 3069, 2971, 2927, 2869, 1621,
1585, 1553, 1462, 1448, 1437, 1377, 1359, 1311, 1286, 1261, 1167, 1145, 1121, 1093, 1063, 1033, 1018 cm−1
δ = 1.27 (t, J = 7.2 Hz, 3H, CH3), 3.49–3.56 (m, 2H, N-CH2), 6.08 (bs, 1H, NH),
δ
δ
;
Elemental Anal. Calcd for C15H15NOSe (305.04): C, 59.22; H, 4.97; N, 4.60 Found: C, 59.15; H, 4.89;
N, 4.53.
N-propyl-2-(phenylselanyl)benzamide 8b Yield: 41%; mp 74–76 ◦C (lit. [21] mp 78–79 ◦C); 1H NMR
(400 MHz, CDCl3)
6.22 (bs, 1H, NH), 7.09–7.11 (m, 1H, 1Har), 7.16–7.20 (m, 2H, 2Har), 7.34–7.40 (m, 3H, 3Har), 7.51–7.54
(m, 1H, 1Har), 7.62–7.65 (m, 2H, 2Har); 13C NMR (100.6 MHz, CDCl3)
= 11.51 (CH3), 22.88 (CH2),
41.87 (CH2), 125.76 (CHar), 127.39 (CHar), 128.51 (CHar), 129.59 (2 CHar), 129.99 (Car), 130.89 (CHar),
131.21 (CHar), 134.79 (Car), 134.89 (Car), 136.03 (2
CHar), 168.35 (C=O); 77Se NMR (76 MHz, CDCl3)
δ = 1.01 (t, J = 7.6 Hz, 3H, CH3), 1.63–1.69 (m, 2H, CH2), 3.19–3.46 (m, 2H, N-CH2),
δ
×
×
δ
= 436.03 ppm; IR: 3277, 3054, 2960, 2922, 2870, 1618, 1585, 1549, 1458, 1436, 1380, 1359, 1313, 1288, 1259,
1145, 1100, 1066, 1033, 1017 cm−1; Elemental Anal. Calcd for C16H17NOSe (319.05): C, 60.38; H, 5.38; N,
4.40 Found: C, 60.55; H, 5.42; N, 4.45.
N-butyl-2-(phenylselanyl)benzamide 9b Yield: 60%; mp 121–125 ◦C; 1H NMR (700 MHz, CDCl3)
δ = 0.96 (t, J = 7.0 Hz, 3H, CH3), 1.40–1.44 (m, 2H, CH2), 1.58–1.61 (m, 2H, CH2), 3.45–3.48 (m, 2H,
N-CH2), 6.09 (bs, 1H, NH), 7.07–7.09 (m, 1Har), 7.16–7.19 (m, 2Har), 7.34–7.38 (m, 3Har), 7.49–7.50 (m,
1Har), 7.61–7.63 (m, 2Har); 13C NMR (100.6 MHz, CDCl3)
39.49 (CH2), 125.38 (CHar), 126.97 (CHar), 128.09 (CHar), 129.18 (2
130.87 (CHar), 134.40 (Car), 134.45 (Car), 135.57 (2
CHar), 167.87 (C=O); 77Se NMR (76 MHz, CDCl3)
δ
= 13.39 (CH3), 19.79 (CH2), 31.24 (CH2),
CHar), 129.59 (Car), 130.48 (CHar),
×
×
δ
= 435.10 ppm; IR: 3277, 3054, 2960, 2922, 2870, 1618, 1585, 1549, 1458, 1436, 1380, 1359, 1313, 1288, 1259,
1145, 1100, 1066, 1033, 1017 cm−1; Elemental Anal. Calcd for C17H19NOSe (333.06): C, 61.45; H, 5.76; N,
4.22 Found: C, 61.29; H, 5.69; N, 4.16.
N-hexyl-2-(phenylselanyl)benzamide 11b Yield: 40%; mp 87–89 ◦C; 1H NMR (400 MHz, CDCl3)
δ
= 0.92(t, J = 7.2 Hz, 3H, CH3), 1.31–1.42 (m, 6H, 3
N-CH2), 6.27 (bs, 1H, NH), 7.08–7.12 (m, 1Har), 7.16–7.19 (m, 2Har), 7.34–7.42 (m, 3Har), 7.52–7.54 (m,
1Har), 7.62–7.65 (m, 2Har); 13C NMR (100.6 MHz, CDCl3)
= 14.05 (CH3), 22.58 (CH2), 26.71 (CH2),
29.56 (CH2), 31.52 (CH2), 40.22 (CH2), 125.76 (CHar), 127.46 (CHar), 128.49 (CHar), 129.58 (2 × CHar),
130.05 (Car), 130.85 (CHar), 131.20 (CHar), 134.84 (Car), 134.85 (Car), 136.00 (2 CHar), 168.33 (C=O);
×
CH2), 1.59–1.66 (m, 2H, CH2), 3.43–3.48 (m, 2H,
δ
×
77Se NMR (76 MHz, CDCl3)
δ = 435.14 ppm; IR: 3317, 2957, 2918, 2927, 2871, 2852, 1617, 1585, 1543, 1462,
1434, 1376, 1331, 1306, 1265, 1200, 1189, 1154, 1032, 1020 cm−1; Elemental Anal. Calcd for C19H23NOSe
(361.09): C, 63.33; H, 6.43; N, 3.89 Found: C, 63.19; H, 6.34; N, 3.70.
N-(3-methylbutyl)-2-(phenylselanyl)benzamide 10b Yield: 54%; mp 76–78 ◦C; 1H NMR (700 MHz,
CDCl3)
(m, 2H, N-CH2), 6.05 (bs, 1H, NH), 7.08–7.09 (m, 1H, 1Har), 7.16–7.19 (m, 2Har), 7.34–7.38 (m, 3Har),
7.48–7.50 (m, 1Har), 7.61–7.62 (m, 2Har); 13C NMR (100.6 MHz, CDCl3)
= 22.49 (CH3), 25.99 (CH),
38.43 (CH2), 38.49 (CH2), 125.83 (CHar), 127.38 (CHar), 128.47 (CHar), 129.59 (2 CHar), 130.04 (Car),
130.88 (CHar), 131.39 (CHar), 134.68 (Car), 134.96 (Car), 135.90 (2
CHar), 168.23 (C=O); 77Se NMR
δ
= 0.95 (d, J = 7.0 Hz, 6H, 2
×
CH3), 1.49–1.52 (m, 2H, CH2), 1.67–1.72 (m, 1H, CH), 3.46–3.49
δ
×
×
(76 MHz, CDCl3) δ = 435.83 ppm; IR: 3315, 2955, 2927, 2870, 1624, 1585, 1564, 1564, 1536, 1462, 1434,
1384, 1364, 1342, 1304, 1284, 1268, 1255, 1226, 1158, 1023 cm−1; Elemental Anal. Calcd for C18H21NOSe
(347.08): C, 62.43; H, 6.11; N, 4.04 Found: C, 62.61; H, 6.19; N, 4.13.

Molecules 2020, 25, 3354
9 of 13
N-cyclohexyl-2-(phenylselanyl)benzamide 12b Yield: 60%; mp 183–185 ◦C (lit. [22] mp 179–181 ◦C);
1H NMR (400 MHz, CDCl3)
= 1.21–1.31 (m, 3H), 1.40–1.50 (m, 2H, CH2), 1.65–1.69 (m, 1H), 1.74–1.80
δ
(m, 2H, CH2), 2.05–2.09 (m, 2H, CH2), 3.97–4.07 (m, 1H, N-CH), 5.32 (bs, 1H, NH), 7.09–7.11 (m, 1Har),
7.17–7.23 (m, 2Har), 7.35–7.41 (m, 3Har), 7.50–7.54 (m, 1Har), 7.60–7.65 (m, 2Har); 13C NMR (100.6 MHz,
CDCl3)
128.42 (CHar), 129.55 (2
135.88 (2
CHar), 167.38 (C=O); 77Se NMR (76 MHz, CDCl3)
δ
= 24.83 (2
×
CH2), 25.55 (CH2), 33.07 (2
CHar), 130.01 (Car), 130.80 (CHar), 131.26 (CHar), 134.60 (Car), 135.04 (Car),
= 434.17 ppm; IR: 3251, 3053, 2924, 2849,
×
CH2), 48.87 (CH), 125.76 (CHar), 127.33 (CHar),
×
×
δ
1618, 1583, 1541, 1459, 1448, 1436, 1377, 1337, 1299, 1283, 1256, 1240, 1191, 1149, 1120, 1081, 1066, 1030,
1018 cm−1; Elemental Anal. Calcd for C19H21NOSe (359.08): C, 63.68; H, 5.91; N, 3.91 Found: C, 63.52;
H, 5.86; N, 3.83.
N-phenyl-2-(phenylselanyl)benzamide 13b Yield: 90%; mp 139–140 ◦C (lit. [23] mp 139–141 ◦C); 1H
NMR (700 MHz, CDCl3) 7.14–7.18 (m, 2Har), 7.22–7.26 (m, 2Har), 7.33–7.39 (m, 5Har), 7.59–7.61 (m,
4Har), 7.65–7.67 (m, 1Har), 7.87 (bs, 1H, NH); 13C NMR (75.5 MHz, CDCl3)
δ
= 120.18 (2
CHar), 129.65 (2 CHar), 129.76
(Car), 131.33 (CHar), 131.86 (CHar), 134.86 (Car), 135.05 (Car), 135.79 (2 CHar), 137.71 (Car), 166.33
= 434.60 ppm; IR: 3328, 3046, 1641, 1596, 1581, 1519, 1494, 1435,
×
CHar),
124.67 (CHar), 126.16 (CHar), 127.76 (CHar), 128.57 (CHar), 129.05 (2
×
×
×
(C=O); 77Se NMR (76 MHz, CDCl3)
δ
1319, 1290, 1271, 1251, 1177, 1152, 1139, 1104, 1074, 1063, 1045, 1025 cm−1; Elemental Anal. Calcd for
C19H15NOSe (353.03): C, 64.78; H, 4.29; N, 3.98 Found: C, 64.92; H, 4.34; N, 4.09.
N-(p-chlorophenyl)-2-(phenylselanyl)benzamide 14b Yield: 44%; mp 167–169 ◦C; 1H NMR (700 MHz,
CDCl3) 7.20–7.21 (m, 1Har), 7.24–7.28 (m, 2Har), 7.32–7.38 (m, 5Har), 7.53–7.55 (m, 2Har), 7.58–7.59
(m, 2Har), 7.66 (dd, J1 = 1.4, J2 = 7.0 Hz, 1Har), 7.84 (bs, 1H, NH); 13C NMR (75.5 MHz, CDCl3)
δ =
121.32 (2
2 × CHar), 131.52 (CHar), 132.11 (CHar), 134.68 (Car), 135.60 (2
77Se NMR (76 MHz, CDCl3)
= 433.36 ppm; IR: 3349, 3053, 1656, 1592, 1579, 1509, 1491, 1457, 1433,
×
CHar), 126.32 (CHar), 127.83 (CHar), 128.61 (CHar), 129.08 (2
×
CHar), 129.65 (2
×
Car), 129.69
(
×
CHar), 136.24 (2
×
Car), 166.21 (C=O);
δ
1394, 1354, 1311, 1289, 1235, 1180, 1140, 1118, 1093, 1074, 1046, 1030, 1012, 1000 cm−1; Elemental Anal.
Calcd for C19H14ClNOSe (386.99): C, 59.01; H, 3.65; N, 3.62 Found: C, 59.32; H, 3.55; N, 3.76.
N-(p-bromophenyl)-2-(phenylselanyl)benzamide 15b Yield: 60%; mp 176–178 ◦C; 1H NMR (400 MHz,
CDCl3) 7.22–7.24 (m, 1Har), 7.27–7.31 (m, 2Har), 7.35–7.41 (m, 3Har), 7.48–7.53 (m, 4Har), 7.60–7.62 (m,
2Har), 7.68–7.70 (m, 1Har), 7.86 (bs, 1H, NH); 13C NMR (75.5 MHz, CDCl3) 121.62 (2
(CHar), 127.86 (CHar), 128.58 (CHar), 129.68 (2 CHar), 129.98 (2
Car), 131.51 (CHar), 132.02 (2 × CHar),
132.22 (CHar), 134.53 (Car), 134.88 (Car), 135.51 (2
CHar), 136.76 (Car), 166.18 (C=O); 77Se NMR (76
MHz, CDCl3)
×
CHar), 126.37
×
×
×
δ
= 433.40 ppm; IR: 3277, 1644, 1589, 1512, 1487, 1457, 1436, 1428, 1390, 1312, 1286, 1250,
1237, 1179, 1164, 1139, 1071, 1030, 1021, 1006 cm−1; Elemental Anal. Calcd for C19H14BrNOSe (430.94):
C, 52.93; H, 3.27; N, 3.25 Found: C, 53.11; H, 3.35; N, 3.40.
N-(p-iodophenyl)-2-(phenylselanyl)benzamide 16b Yield: 22%; mp 179–181 ◦C; 1H NMR (700 MHz,
CDCl3) 7.18–7.20 (m, 1Har), 7.23–7.25 (m, 2Har), 7.33–7.35 (m, 2Har), 7.36–7.38 (m, 3Har), 7.57–7.59 (m,
2Har), 7.62–7.65 (m, 2Har), 7.94 (bs, 1H, NH); 13C NMR (75.5 MHz, CDCl3) 121.87 (2
×
CHar), 126.35
Car), 131.53 (CHar), 132.19 (CHar),
CHar), 166.18 (C=O); 77Se NMR
= 433.36 ppm; IR: 3348, 3049, 2922, 2852, 1651, 1585, 1506, 1486, 1456, 1436, 1429,
(CHar), 127.85 (CHar), 128.60 (CHar), 129.68 (2
134.60 (Car), 134.53 (Car), 135.54 (2 CHar), 137.47 (Car), 137.98 (2
(76 MHz, CDCl3)
×
CHar), 129.98 (2
×
×
×
δ
1387, 1352, 1313, 1288, 1233, 1187, 1166, 1139, 1120, 1063, 1045, 1029, 1019, 1002 cm−1; Elemental Anal.
Calcd for C19H14INOSe (478.93): C, 47.72; H, 2.95; N, 2.93 Found: C, 47.59; H, 3.01; N, 3.10.
N-(p-metoxyphenyl)-2-(phenylselanyl)benzamide 17b Yield: 45%; mp 131–132 ◦C; 1H NMR (700 MHz,
CDCl3) 3.83 (s, 3H, CH3), 6.71 (ddd, J1 = 0.7, J2 = 2.8 Hz, J2 = 8.4 Hz, 1Har), 7.05 (dd, J1 = 1.4, J2 = 8.4 Hz
,
1Har), 7.18 (dd, J1 = 0.7, J2 = 7.7 Hz, 1Har), 7.23–7.28 (m, 3Har), 7.33–7.39 (m, 4Har), 7.60–7.61 (m, 2Har),
7.65 (dd, J1 = 1.4, J2 = 7.7 Hz, 1Har), 7.80 (bs, 1H, NH); 13C NMR (75.5 MHz, CDCl3) 55.37 (CH3), 105.74
(CHar), 110.66 (CHar), 112.22 (CHar), 126.22 (CHar), 127.77 (CHar), 128.54 (CHar), 129.64 (2 × CHar),
129.71 (CHar), 131.34 (CHar), 131.96 (CHar), 134.64 (Car), 135.18 (Car), 136.13 (2
×
CHar), 138.91 (2 × Car),
= 433.96 ppm; IR: 3300, 2922, 1640, 1597,
1581, 1564, 1521, 1489, 1464, 1453, 1427, 1306, 1287, 1273, 1253, 1201, 1172, 1156, 1140, 1038, 1022 cm−1
160.22 (Car), 166.26 (C=O); 77Se NMR (76 MHz, CDCl3)
δ
;

Molecules 2020, 25, 3354
10 of 13
Elemental Anal. Calcd for C20H17NO2Se (383.04): C, 62.83; H, 4.48; N, 3.66 Found: C, 62.69; H, 4.39;
N, 3.50.
(−
)-N-(1R,2S,5R)-menthyl-2-(phenylselanyl)benzamide 18b Yield: 27%, mp 153–155 ◦C; [α]D20
=
−
31.11
(c = 2.61, CHCl3); 1H NMR (400 MHz, CDCl3)
δ = 0.88 (d, J = 6.8 Hz, 3H, CH3), 0.93 (d, J = 6.4 Hz, 3H,
CH3), 0.94 (d, J = 6.8 Hz, 3H, CH3), 1.16–1.20 (m, 2H), 1.51–1.59 (m, 3H), 1.71–1.81 (m, 2H), 2.01–2.09
(m, 1H), 2.12–2.18 (m, 1H), 3.95–4.04 (m, 1H), 5.78 (d, J = 8.8 Hz, 1H, NH), 7.10–7.12 (m, 1Har), 7.17–7.23
(m, 2Har), 7.34–7.40 (m, 3Har), 7.50–7.52 (m, 1Har), 7.62–7.64 (m, 2Har); 13C NMR (100.6 MHz, CDCl3)
δ
= 16.26 (CH3), 21.25 (CH3), 22.17 (CH3), 23.85 (CH2), 26.99 (CH), 31.93 (CH), 34.56 (CH2), 43.02 (CH2),
48.25 (CH), 50.63 (CH), 125.79 (CHar), 127.26 (CHar), 128.41 (CHar), 129.57 (2 CHar), 130.12 (Car),
130.76 (CHar), 131.33 (CHar), 134.51 (Car), 135.42 (Car), 135.86 (2
CHar), 167.60 (C=O); 77Se NMR
×
×
(76 MHz, CDCl3) δ = 434.56 ppm; IR: 3364, 2960, 2936, 2863, 1637, 1582, 1522, 1458, 1435, 1338, 1305,
1255, 1157, 1031, 1020 cm−1; Elemental Anal. Calcd for C23H29NOSe (415.14): C, 66.66; H, 7.05; N, 3.38
Found: C, 66.78; H, 7.13; N, 3.23.
(−
)-N-(1S,2R,3S,6R)-(2-caranyl)-2-(phenylselanyl)benzamide 19b Yield: 53%; mp 115–116 ◦C; [α]D20
1
=
−
15.11 (c = 6.15, CHCl3); H NMR (400 MHz, CDCl3) 0.59–0.62 (m, 1H), 0.65–0.69 (m, 1H), 1.00
(d, J = 6.4 Hz, 3H, CH3), 1.05 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.24–1.32 (m, 1H), 1.55–1.62 (m, 1H),
1.72–1.88 (m, 3H), 3.59–3.65 (m, 1H), 6.15 (d, J = 8.8 Hz, 1H, NH), 7.10–7.13 (m, 1Har), 7.17–7.24 (m,
2Har), 7.35–7.42 (m, 3Har), 7.57–7.59 (m, 1Har), 7.64–7.67 (m, 2Har); 13C NMR (100.6 MHz, CDCl3)
δ =
15.55 (CH3), 17.58 (C), 18.88 (CH3), 19.09 (CH2), 20.25 (CH), 28.88 (CH), 29.30 (CH3), 30.80 (CH2), 35.32
(CH), 50.21 (CH), 125.77 (CHar), 127.38 (CHar), 128.43 (CHar), 129.58 (2 CHar), 130.10 (Car), 130.79
(CHar), 131.32 (CHar), 134.72 (Car), 135.25 (Car), 135.94 (2
CHar), 167.41 (C=O); 77Se NMR (76 MHz,
×
×
CDCl3) δ = 435.56 ppm; IR: 3233, 2920, 2863, 1623, 1582, 1540, 1477, 1459, 1437, 1375, 1331, 1284, 1258,
1243, 1161, 1114, 1086, 1055, 1021 cm−1; Elemental Anal. Calcd for C23H27NOSe (413.13): C, 66.98; H,
6.60; N, 3.40 Found: C, 67.12; H, 6.69; N, 3.51.
N-borynyl-2-(phenylselanyl)benzamide 20b Yield: 46%; mp 118–120 C; [α]D20
= 32.10 (c = 4.71,
−
◦
CHCl3); 1H NMR (400 MHz, CDCl3) 0.93 (s, 3H, CH3), 0.94 (s, 3H, CH3), 1.02 (s, 3H, CH3), 1.17–1.24
(m, 1H), 1.42–1.51 (m, 1H), 1.55–1.62 (m, 2H), 1.72–1.74 (m, 1H), 1.76–1.86 (m, 1H), 2.44–2.52 (m, 1H),
4.46–4.52 (m, 1H), 5.32 (d, J = 8.8 Hz, 1H, NH), 7.12–7.14 (m, 1Har), 7.19–7.26 (m, 2Har), 7.34–7.40 (m,
3Har), 7.56–7.58 (m, 1Har), 7.61–7.63 (m, 2Har); 13C NMR (100.6 MHz, CDCl3)
δ
= 13.81 (CH3), 18.71
(CH3), 19.84 (CH3), 28.27 (CH2), 28.43 (CH2), 37.72 (CH2), 44.97 (CH), 48.26 (C), 49.72 (C), 54.48 (CH),
126.00 (CHar), 127.51 (CHar), 128.39 (CHar), 129.60 (2 CHar), 130.09 (Car), 130.87 (CHar), 131.64 (CHar),
134.09 (Car), 135.57 (Car), 135.62 (2 = 432.53 ppm;
CHar), 168.36 (C=O); 77Se NMR (76 MHz, CDCl3)
×
×
δ
IR: 3359, 2950, 2885, 1629, 1582, 1561, 1516, 1476, 1456, 1435, 1389, 1374, 1363, 1309, 1277, 1256, 1223,
1204, 1173, 1152, 1109, 1065, 1030, 1021 cm−1; Elemental Anal. Calcd for C23H27NOSe (413.13): C, 66.98;
H, 6.60; N, 3.40 Found: C, 66.79; H, 6.54; N, 3.32.
(
−
)-N-(1S,2R,5S)-myrtanyl-2-(phenylselanyl)benzamide 21b Yield: 55%; mp 115–117 ◦C; [α]D20
= −8.00
(c = 2.70, CHCl3); 1H NMR (400 MHz, CDCl3) 0.94 (d, J = 9.6 Hz, 1H), 1.11 (s, 3H, CH3), 1.23 (s, 3H,
CH3), 1.53–1.63 (m, 2H), 1.87–2.06 (m, 4H), 2.31–2.43 (m, 2H), 3.47–3.51 (m, 2H), 6.13 (bs, 1H, NH),
7.10–7.13 (m, 1Har), 7.17–7.23 (m, 2Har), 7.35–7.42 (m, 3Har), 7.51–7.53 (m, 1Har), 7.62–7.64 (m, 2Har);
13C NMR (100.6 MHz, CDCl3)
δ
= 19.94 (CH2), 23.25 (CH3), 26.02 (CH2), 27.99 (CH3), 33.24 (CH2),
38.75 (C), 41.39 (CH), 41.41 (CH), 43.91 (CH), 45.73 (CH2), 125.86 (CHar), 127.41 (CHar), 128.43 (CHar),
129.58 (2 CHar), 130.06 (Car), 130.86 (CHar), 131.44 (CHar), 134.52 (Car), 135.16 (Car), 135.83 (2 CHar),
168.27 (C=O); 77Se NMR (76 MHz, CDCl3)
= 434.53 ppm; IR: 3333, 2980, 2905, 1631, 1584, 1560, 1530,
×
×
δ
1462, 1432, 1383, 1364, 1314, 1284, 1254, 1219, 1154, 1058, 1032, 1019, 1001 cm−1; Elemental Anal. Calcd
for C23H27NOSe (413.13): C, 66.98; H, 6.60; N, 3.40 Found: C, 67.12; H, 6.69; N, 3.50.
(
)-N-(1R,2R,3R,5S)-isopinocamphyl-2-(phenylselanyl)benzamide 22b Yield: 53%; mp 117–119 ◦C;
−
[
α]2D0
=
14.75 (c = 4.73, CHCl3); 1H NMR (400 MHz, CDCl3) 0.88 (d, J = 10.0 Hz, 1H), 1.11 (s, 3H, CH3),
−
1.22 (d, J = 7.2 Hz, 3H, CH3), 1.27 (s, 3H, CH3), 1.63–1.69 (m, 1H), 1.87–1.89 (m, 2H), 2.00–2.03 (m, 1H),
2.43–2.49 (m, 1H), 2.72–2.79 (m, 1H), 4.48–4.56 (m, 1H), 6.01 (d, J = 7.2 Hz, 1H, NH), 7.12–7.14 (m, 1Har),
7.18–7.25 (m, 2Har), 7.35–7.40 (m, 3Har), 7.54–7.56 (m, 1Har), 7.61–7.64 (m, 2Har); 13C NMR (100.6 MHz,

Molecules 2020, 25, 3354
11 of 13
CDCl3)
(CH), 47.85 (CH), 48.53 (CH), 125.97 (CHar), 127.51 (CHar), 128.39 (CHar), 129.61 (2 × CHar), 130.08
(Car), 130.84 (CHar), 131.55 (CHar), 134.10 (Car), 135.44 (Car), 135.63 (2
CHar), 167.72 (C=O); 77Se
δ = 20.88 (CH3), 23.38 (CH3), 28.01 (CH3), 35.34 (CH2), 37.21 (CH2), 38.45 (C), 41.62 (CH), 46.49
×
NMR (76 MHz, CDCl3) δ = 431.80 ppm; IR: 3303, 2953, 2903, 2868, 1617, 1582, 1559, 1528, 1475, 1461,
1434, 1375, 1337, 1318, 1300, 1286, 1255, 1226, 1160, 1062, 1031, 1021 cm−1; Elemental Anal. Calcd for
C23H27NOSe (413.13): C, 66.98; H, 6.60; N, 3.40 Found: C, 66.78; H, 6.52; N, 3.34.
(+)-N-(1R,2R,3R,5S)-isopinocamphyl-2-(phenylselanyl)benzamide 23b Yield: 54%; mp 128–130 ◦C;
α]2D0 = +14.35 (c = 4.43, CHCl3); H NMR (400 MHz, CDCl3) 0.89 (d, J = 10.0 Hz, 1H), 1.11 (s, 3H,
1
[
CH3), 1.21 (d, J = 7.2 Hz, 3H, CH3), 1.26 (s, 3H, CH3), 1.63–1.69 (m, 1H), 1.86–1.93 (m, 2H), 1.99–2.06
(m, 1H), 2.42–2.48 (m, 1H), 2.71–2.78 (m, 1H), 4.48–4.56 (m, 1H), 6.09 (d, J = 7.2 Hz, 1H, NH), 7.09–7.15
(m, 1Har), 7.16–7.23 (m, 2Har), 7.34–7.41 (m 3Har), 7.53–7.57 (m, 1Har), 7.59–7.64 (m, 2Har); 13C NMR
(75.5 MHz, CDCl3)
(CH), 46.21 (CH), 47.81 (CH), 48.48 (CH), 125.82 (CHar), 127.54 (CHar), 128.40 (CHar), 129.58 (2
130.08 (Car), 130.77 (CHar), 131.27 (CHar), 134.33 (Car), 135.19 (Car), 135.73 (2 CHar), 167.74 (C=O);
77Se NMR (76 MHz, CDCl3)
= 431.86 ppm; IR: 3311, 3055, 2958, 2908, 2869, 1623, 1584, 1563, 1531,
1476, 1454, 1434, 1376, 1351, 1335, 1311, 1298, 1285, 1258, 1226, 1163, 1092, 1064, 1031, 1021, 1000 cm−1
δ
= 20.88 (CH3), 23.38 (CH3), 28.02 (CH3), 35.25 (CH2), 37.08 (CH2), 38.45 (C), 41.59
×
CHar),
×
δ
;
Elemental Anal. Calcd for C23H27NOSe (413.13): C, 66.98; H, 6.60; N, 3.40 Found: C, 66.68; H, 6.70;
N, 3.51.
3.3. Antioxidant Activity Assay
To a solution of compounds 7b
–
23b (0.015 mmol) and dithiothreitol DTTred (0.15 mmol) in 1.1 mL
1
of CD3OD, 30% H2O2 (0.15 mmol) was added. H NMR spectra were measured right after the addition
of hydrogen peroxide, and then in specific time intervals. The concentration of the substrate was
determined according to the changes in the integration on the 1H NMR spectra [19].
3.4. MTT Viability Assay
The MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay, which measures
activity of cellular dehydrogenases, was based on the method of Mosmann [24]. Briefly, cells were
seeded into 96-well plates (about 1.5
grow for 24 h. Subsequently, 100 L of the tested compounds in the medium was added to a final
concentration of 0–250 M, for 24 h, followed by the addition of 100 L MTT, 3 mg/mL in PBS, for the
next 3 h. After the incubation, the medium was removed. Remaining insoluble formazan crystals were
dissolved in 100 L DMSO. The absorbance of the blue formazan product was measured at 570 nm in
×
104 cells per well, in 100
µL) and then left to adhere and
µ
µ
µ
µ
the plate reader spectrophotometer Infinite M200 (Tecan, Grödig, Austria) and compared with the
control (untreated cells). All experiments were performed three times in triplicate. The concentration
of tested compounds required to inhibit cell viability by 50% (IC50) was calculated using Microsoft
Excel software for semi-log curve fitting with linear regression analysis.
4. Conclusions
Herein, we have presented the synthesis of a new group of GPx-mimics, unsymmetrical phenyl
selenides, functionalized on one of the phenyl rings with a N-substituted o-amido group. The obtained
compounds were diversified on the nitrogen atom with aromatic and aliphatic groups, including
chiral terpene scaffolds. The molecules were designed as N-substituted benzisoselenazol-3(2H)-ones
(“ebselen-like” therapeutics), whereas the Se-N bond was cleaved and a -SePh group was installed with a
secondary amide moiety -C(O)NHR functionalization. The compounds were further tested as potential
antioxidants and anticancer agents. The highest peroxide scavenging activity, significantly higher than
for ebselen, was found for N-(3-methylbutyl)-2-(phenylselanyl)benzamide. The significant cytotoxicity
was observed for derivatives with terpenyl carane and p-menthane skeletons. The performed in vitro
studies revealed that, however, the antioxidant potential was not improved in most cases in comparison
to the results obtained for corresponding benzisoselenazol-3(2H)-ones; the modification was beneficial

Molecules 2020, 25, 3354
12 of 13
for a higher antiproliferative effect towards MCF-7 and HL-60 cancer cells. It can therefore be concluded
that an additional aromatic ring attached to the selenium atom may have a positive influence on the
enhanced cytotoxity of the selected phenylselenides.
Supplementary Materials: The following are available online, results of the antioxidant activity evaluation;
results of the antiproliferative activity evaluation, 1H- and 13C-NMR spectra of compounds 18a
and 77Se spectra of compounds 7b–23b.
– C
23a, and 1H, 13
´
Author Contributions: Conceptualization, J.S.; Data curation, M.O.-F., L.C. and J.D.-S.; Formal analysis,
´
´
J.D.-S.; Investigation, M.O.-F., A.J.P., L.C. and A.J.; Methodology, J.S.; Writing—original draft, A.J.P. and J.S.;
´
Writing—review & editing, A.J., C.S. and J.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding and the APC was funded by “Excellence Initiative—Research
University” programme at Nicolaus Copernicus University.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Polêto, M.D.; Rusu, V.H.; Grisci, B.I.; Dorn, M.; Lins, R.D.; Verli, H. Aromatic Rings Commonly Used in
Medicinal Chemistry: Force Fields Comparison and Interactions with Water Toward the Design of New
Chemical Entities. Front. Pharmacol. 2018, 9, 395–414. [CrossRef]
2.
3.
4.
Ward, S.E.; Beswick, P. What does the aromatic ring number mean for drug design? Expert Opin. Drug Discov.
2014, 9, 9–18. [CrossRef] [PubMed]
Orian, L.; Toppo, S. Organochalcogen peroxidase mimetics as potential drugs: A long story of a promise still
unfulfilled. Free Rad. Biol. Med. 2014, 66, 65–74. [CrossRef] [PubMed]
´
Pacuła, A.J.; Mangiavacchi, F.; Sancineto, L.; Lenardao, E.J.; Scianowski, J.; Santi, C. An Update on
“Selenium Containing Compounds from Poison to Drug Candidates: A Review on the GPx-like Activity”.
Curr. Chem. Biol. 2015, 9, 97–112.
5.
6.
Lenardão, E.J.; Santi, C.; Sancineto, L. New Frontiers in Organoselenium Compounds; Springer International
Publishing: Cham, Switzerland, 2018.
Singh, F.V.; Wirth, T. Synthesis of Organoselenium Compounds with Potential Biological Activities.
In Organoselenium Compounds in Biology and Medicine: Synthesis, Biological and Therapeutic Treatments; Royal
Society of Chemistry: London, UK, 2017; pp. 77–121.
7.
8.
9.
Kil, J.; Lobarinas, E.; Spankovich, C.; Griffiths, S.K.; Antonelli, P.J.; Lynch, E.D.; Le Prell, C.G. Safety
and efficacy of ebselen for the prevention of noise-induced hearing loss: A randomised, double-blind,
placebo-controlled, phase 2 trial. Lancet 2017, 390, 969–979. [CrossRef]
Ye, S.; Yang, Y.; Wu, L.; Ma, W.; Zeng, H. Ethaselen: A novel organoselenium anticancer agent targeting
thioredoxin reductase 1 reverses cisplatin resistance in drug-resistant K562 cells by inducing apoptosis.
Biomed. Biotechnol. 2017, 18, 373–382. [CrossRef] [PubMed]
Asaf, R.; Blum, S.; Miller-Lotan, R.; Levy, A.P. BXT-51072 and the Prevention of Myocardial
Ischemia-Reperfusion Injury. Lett. Drug Des. Dis. 2007, 4, 160–162. [CrossRef]
´
10. Pacuła, A.J.; Scianowski, J.; Aleksandrzak, K.B. Highly efficient synthesis and antioxidant capacity of
N-substituted benzisoselenazol-3(2H)-ones. RSC Adv. 2014, 4, 48959–48962. [CrossRef]
´
11. Obieziurska, M.; Pacuła, A.J.; Długosz-Pokorska, A.; Krzemin´ski, M.; Janecka, A.; Scianowski, J. Bioselectivity
Induced by Chirality of New Terpenyl Organoselenium Compounds. Materials 2019, 12, 3579. [CrossRef]
´
12. Kaczor-Keller, K.B.; Pawlik, A.; Scianowski, J.; Pacuła, A.; Obieziurska, M.; Marcheggiani, F.; Cirilli, I.;
Tiano, L.; Antosiewicz, J. In Vitro Anti-Prostate Cancer Activity of Two Ebselen Analogues. Pharmaceuticals
2020, 13, 47. [CrossRef]
´
13. Pacuła, A.J.; Kaczor, K.B.; Wojtowicz, A.; Antosiewicz, J.; Janecka, A.; Długosz, A.; Janecki, T.; Scianowski, J.
New glutathione peroxidase mimetics—Insights into antioxidant and cytotoxic activity. Bioorg. Med. Chem.
2017, 25, 126–131. [CrossRef]
´
14. Pacuła, A.J.; Kaczor, K.B.; Antosiewicz, J.; Janecka, A.; Długosz, A.; Janecki, T.; Wojtczak, A.; Scianowski, J.
New Chiral Ebselen Analogues with Antioxidant and Cytotoxic Potential. Molecules 2017, 22, 492. [CrossRef]
[PubMed]

Molecules 2020, 25, 3354
13 of 13
´
15. Pacuła, A.J.; Obieziurska, M.; Scianowski, J.; Kaczor, K.B.; Antosiewicz, J. Synthesis of biologically active
diaryl diselenides under water control. Arkivoc 2018, 3, 144–155.
16. Nascimento, V.; Cordeiro, P.S.; Arca, M.; Marini, F.; Sancineto, L.; Braga, A.L.; Lippolis, V.; Iwaoka, M.; Santi, C.
Fast and easy conversion of ortho amidoaryldiselenides into the corresponding ebselen-like derivatives
driven by theoretical investigations. New J. Chem. 2020, 44, 9444–9451. [CrossRef]
´
17. Obieziurska, M.; Pacuła, A.J.; Laskowska, A.; Długosz-Pokorska, A.; Janecka, A.; Scianowski, J. Seleninic
Acid Potassium Salts as Water-Soluble Biocatalysts with Enhanced Bioavailability. Materials 2020, 13, 661.
[CrossRef]
´
18. Pacuła, A.J.; Scianowski, J. Terpenes as Green Starting Materials for New Organoselenium and
Organotellurium Compounds. Curr. Green Chem. 2016, 3, 36–50.
19. Kumakura, F.; Mishra, B.; Priyadarsini, K.I.; Iwaoka, M. A Water-Soluble Cyclic Selenide with Enhanced
Glutathione Peroxidase-Like Catalytic Activities. Eur. J. Org. Chem. 2010, 3, 440–444. [CrossRef]
20. Detty, M.R.; Friedman, A.E.; Oseroff, A.R. A Mechanism for the Oxidation of Glutathione to Glutathione
Disulfide with Organotellurium(IV) and Organoselenium(IV) Compounds. A Stepwise Process with
Implications for Photodynamic Therapy and Other Oxidative Chemotherapy. J. Org. Chem. 1994, 59,
8245–8250. [CrossRef]
21.
Wójtowicz, H.; Brza˛szcz, M.; Kloc, K.; Młochowski, J. Selective oxidation of aromatic aldehydes to
arenecarboxylic acids using ebselen-tert-butyl hydroperoxide catalytic system. Tetrahedron 2001, 57, 9743–9748.
[CrossRef]
22. Ma, W.; Weng, Z.; Fang, X.; Gu, L.; Song, Y.; Ackermann, L. Ruthenium-Catalyzed C–H Selenylations of
Benzamides. Eur. J. Org. Chem. 2019, 1, 41–45. [CrossRef]
23. Lisiak, R.; Młochowski, J. Selenium–Nitrogen Bond Cleavage in Selenazole Ring System with Grignard
Reagent: A Convenient Synthesis of Unsymmetrically Substituted Selenides. Synth. Commun. 2009, 39,
3141–3155. [CrossRef]
24. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and
cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [CrossRef]
Sample Availability: Not available.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).