Synthesis and leishmanicidal activity of novel urea, thiourea, and selenourea derivatives of diselenides

Article abstract of DOI:10.1128/AAC.02200-18

A novel series of thirty-one N-substituted urea, thiourea, and selenourea derivatives containing diphenyldiselenide entities were synthesized, fully characterized by spectroscopic and analytical methods, and screened for their in vitro leishmanicidal activities. The cytotoxic activity of these derivatives was tested against Leishmania infantum axenic amastigotes, and selectivity was assessed in human THP-1 cells. Thirteen of the synthesized compounds showed a significant antileishmanial activity, with 50% effective concentration (EC₅₀) values lower than that for the reference drug miltefosine (EC₅₀, 2.84 M). In addition, the derivatives 9, 11, 42, and 47, with EC₅₀ between 1.1 and 1.95 M, also displayed excellent selectivity (selectivity index ranged from 12.4 to 22.7) and were tested against infected macrophages. Compound 11, a derivative with a cyclohexyl chain, exhibited the highest activity against intracellular amastigotes, with EC₅₀ values similar to those observed for the standard drug edelfosine. Structure-activity relationship analyses revealed that N-aliphatic substitution in urea and selenourea is recommended for the leishmanicidal activity of these analogs. Preliminary studies of the mechanism of action for the hit compounds was carried out by measuring their ability to inhibit trypanothione reductase. Even though the obtained results suggest that this enzyme is not the target for most of these derivatives, their activity comparable to that of the standards and lack of toxicity in THP-1 cells highlight the potential of these compounds to be optimized for leishmaniasis treatment.

Full text of DOI:10.1128/AAC.02200-18

AAC Accepted Manuscript Posted Online 19 February 2019
Antimicrob. Agents Chemother. doi:10.1128/AAC.02200-18
Copyright © 2019 American Society for Microbiology. All Rights Reserved.
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NOVEL UREA, THIOUREA AND SELENOUREA DERIVATIVES
OF DISELENIDES: SYNTHESIS AND LEISHMANICIDAL
ACTIVITY
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Marta Díaz,a,b Héctor de Lucio,c Esther Moreno,a,b,d Socorro
Espuelas,a,b,d Carlos Aydillo,a,b,d Antonio Jiménez-Ruiz,c Miguel
Ángel Toro,c Killian Jesús Gutiérrez,c Victor Martínez-Merino,e
Alfonso Cornejo,e Juan Antonio Palop,a,b,d Carmen Sanmartín,a,b,d,*
Daniel Planoa,b,d
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Departamento de Tecnología y Química Farmacéuticasa and Instituto de Salud
Tropical,b University of Navarra, Pamplona, Spain; Departamento de Biología
de Sistemas, Universidad de Alcalá, Madrid, Spain;c
Instituto de
Investigaciones Sanitarias de Navarra (IdiSNA), Pamplona, Spain;d
Departamento de Química Aplicada, Universidad Pública de Navarra,
Pamplona, Spaine
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* Prof. Carmen Sanmartín
Department of Organic and Pharmaceutical Chemistry
University of Navarra
Irunlarrea, 1, E-31008 Pamplona
SPAIN
+34 948 425 600 (Telephone)
+34 948 425 649 (Fax)
e-mail: sanmartin@unav.es

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Abstract
A novel series of thirty-one N-substituted urea, thiourea and selenourea
derivatives containing diphenyldiselenide entity were synthesized, fully
characterized by spectroscopic and analytical methods, and screened for their
in vitro leishmanicidal activities. The cytotoxic activity of these derivatives was
tested against Leishmania infantum axenic amastigotes, and selectivity was
assessed in human THP-1 cells. Thirteen of the synthesized compounds
showed a significant antileishmanial activity with EC50 values lower than the
reference drug miltefosine (EC50 = 2.84 µM). In addition, the derivatives 9, 11,
42 and 47 with EC50 between 1.1 and 1.95 µM also displayed an excellent
selectivity (SI ranged from 12.4 to 22.7) and were also tested against infected
macrophages. Compound 11, a derivative with a cyclohexyl chain, exhibited the
highest activity against intracellular amastigotes with EC50 values similar to
those observed for the standard drug edelfosine. SAR analyses revealed that
N-aliphatic substitution in urea and selenourea is recommended for the
leishmanicidal activity of these analogs. Preliminary studies of the mechanism
of action for the hit compounds was carried out by measuring their ability to
inhibit trypanothione reductase (TryR). Even though the obtained results
suggest that this enzyme is not the target for most of these derivatives, their
comparable activity with the standards and lack of toxicity in THP-1 cells
highlight the potential of these compounds to be optimized for leishmaniasis
treatment.
Keywords: selenium, selenourea, thiourea, trypanothione reductase, urea
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Leishmaniasis comprises a group of mammalian diseases caused by
diphasic protozoans of the genus Leishmania spp. It is endemic in 98 countries
and approximately 15 million of new cases are diagnosed every year, leading to
high rates of morbidity and mortality. Leishmania spp presents three different
clinical manifestations: cutaneous, mucocutaneous and visceral. Among these
forms, cutaneous is the most common whereas visceral is the most severe form
(1-2). Treatment options are limited and far from being satisfactory. Most of
available front-line agents were developed 50 years ago and include
chemotherapeutic drugs such as injectable pentavalent antimonials and sodium
stibogluconate and meglumine antimoniate. Second-line treatment relies in
highly toxic drugs such as amphotericin B or pentamidine. In this context, the
development of more effective and less toxic drugs represents an urgent need
(3). In this regard, miltefosine, an alkylphosphocholine drug, and the
aminoglycoside antibiotic paromomycin have proven to be effective drugs for
the treatment of leishmaniasis. Newly developed liposomal amphotericin B is a
preferred treatment in developing countries because it efficiently targets
Leishmania spp parasites with low toxic side effects. Moreover, promising
combination therapies are under intense investigation (4-5).
The trace element selenium is a micronutrient element with broad
functions in biological systems. Selenium derivatives have been recognized by
antioxidant, cancer preventing, and antiviral activities. Selenoproteins interfere
with kinetoplastid biochemistry and have anti-parasite activities (6). Similarly,
increased selenium concentration in plasma has been proposed as a new
defensive strategy against Leishmania spp infection (7). In recent years, our
research group and others have been engaged in the design, synthesis and
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biological evaluation of new selenium compounds with potent in vitro
antitrypanosomatic activity (8), mainly against L. infantum. Our data revealed
that some of these compounds possess a potent activity with higher selectivity
than the reference drugs miltefosine and edelfosine. Additionally, leishmanicidal
activity in infected macrophages (THP-1 cells) was comparable to edelfosine (9-
16).
Among
the
different
selenium
entities
tested,
4,4’-
diaminodiphenyldiselenide showed one of the most promising leishmanicidal
activities. This derivative contains as essential pharmacophore the diselenide
group within the framework of molecular symmetry that, in our opinion, appears
as a key factor for leishmanicidal activity. Herein, we designed several
modifications on the side chain of the diselenide core in order to develop
compounds with improved leishmanicidal activity and ADMET properties. For
this purpose, the hit 4,4’-diaminodiphenyldiselenide was modulated by two
strategies: i) the amine group was derivatized to urea, thiourea and selenourea
in order to adjust polarity, solubility and ability to interact and form hydrogen
bonds; ii) introduction of various aromatic systems or a cyclic or linear aliphatic
chain of variable length and flexibility into the pendent amino groups of the
ureidic function. Urea moiety is commonly found in various potent
leishmanicidal compounds (17-18). On the other hand, the thiourea scaffold has
been described for treating parasitic disorders by itself (19-20) or combined with
metals (21). Finally, we further expanded the scope of the reaction to the
synthesis of selenoureas in order to assess the importance of the number of
selenium atoms in the leishmanicidal activity. Regarding the modulation in the
pendent amino groups, various substituents were introduced to the terminal
phenyl ring with the purpose of exploring their influence on activity by regulating
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the electronic and steric features (22). Moreover, both cyclic and acyclic
aliphatic chains have been validated as attractive scaffolds for the development
of new leishmanicidal drugs, given the structural analogy with leishmanicidal
derivatives containing aminoalkylchains previously reported in the literature (23-
24). Figure 1 shows the general structure of the new designed compounds.
Based in previous studies, herein we present the synthesis and leishmanicidal
activity against the amastigote form of L. infantum of thirty-one new derivatives
related to Figure 1. The cytotoxicity of these newly synthesized molecules was
also assessed on one different complementary human cell line (THP-1) in order
to select those compounds with high selectivity. Moreover, leishmanicidal
activity of the most active compounds was evaluated in infected macrophages.
Finally, in order to elucidate the underlying molecular mechanisms, the
inhibitory activity against trypanothione reductase (TryR) was determined.
MATERIALS AND METHODS
Chemistry. Melting points were determined with a Mettler FP82+FP80
apparatus (Greifense, Switzerland) and are not corrected. The 1H NMR and 13
C
NMR spectra were recorded on a Bruker 400 UltrashieldTM and Bruker Avance
Neo spectrometers (Rheinstetten, Germany) using TMS as the internal
standard. The IR spectra were obtained on a Thermo Nicolet FT-IR Nexus
spectrophotometer with KBr pellets. Mass spectrometry was carried out on a
MS-DIP, system MSD/DS 5973N (G2577A) Agilent. Elemental microanalyses
were carried out on vacuum-dried samples using a LECO CHN-900 Elemental
Analyzer. Silica gel 60 (0.040−0.063 mm) 1.09385.2500 (Merck KGaA, 64271
Darmstadt, Germany) was used for Column Chromatography and Alugram SIL
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G/UV254 (Layer: 0.2 mm) (Macherey-Nagel GmbH & Co. KG. Postfach 101352,
D-52313 Düren, Germany) was used for Thin Layer Chromatography.
Chemicals were purchased from E. Merck (Darmstadt, Germany), Scharlau
(F.E.R.O.S.A., Barcelona, Spain), Panreac Química S.A. (MontcadaiReixac,
Barcelona, Spain), Sigma-Aldrich Química, S.A. (Alcobendas, Madrid, Spain),
Acros Organics (Janssen Pharmaceuticalaan 3a, 2440 Geel, België) and
Lancaster (Bischheim-Strasbourg, France).
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4,4’-Diaminodiphenyldiselenide. The synthesis of this compound has
been previously described by Plano et al. [11].
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General procedure for the synthesis of ureas 1-11. To a solution of
4,4’-diaminodiphenyldiselenide (1.17 mmol) in dioxane (25 mL) the
corresponding isocyanate was added (2.34 mmol, 1:2 molar ratio), and the
mixture was kept at room temperature from 24 h to 120 h. The solvent was
removed under vacuum by rotatory evaporation and the residue was treated
with ethyl ether (50 mL) and washed with water (100 mL).
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis(1-phenylurea) (1). From
phenyl isocyanate after 24 h gave 1 as a yellow powder. Yield: 58%; mp
1
277−278 ºC; IR ʋmax (KBr): 3294 (N−H), 1637 (C=O) cm-1; H NMR (400 MHz,
DMSO-d6, δ): 6.98 (t, 2H, J4-3 = J4-5 = 7.0 Hz, B+B’, H4), 7.28 (t, 4H, J3-2 = J5-6
8.0 Hz, B+B’, H3+H5), 7.43-7.46 (m, 8H, A+A’+B+B’, H2+H6), 7.52 (d, 4H, J3-2
=
=
J5-6 = 8.5 Hz, A+A’, H3+H5), 8.74 (bs, 2H, 2NH), 8.85 (bs, 2H, 2NH); 13C NMR
(100 MHz, DMSO-d6, δ): 118.7 (A+A', C3+C5), 119.4 (B+B', C2+C6), 122.5
(A+A', C1), 130 (B+B', C3+C4+C5), 134.1 (A+A', C2+C6), 140.0 (A+A', C4), 140.7
(B+B', C1), 152.8 (C=O); MS (m/z % abundance): 368 (59), 191 (100), 135 (24),
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57 (43); Anal. Calcd for C26H22N4O2Se2 (%): C: 53.8, H: 3.8, N: 9.6. Found: C:
54.1, H: 4.1, N: 9.1.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-nitrophenyl)urea] (2).
From 4-nitrophenyl isocyanate after 72 h gave 2 as a yellow powder. Yield:
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66%; mp 245−247 ºC; IR ʋmax (KBr): 3363 (N−H), 1614 (C=O) cm-1; H NMR
(400 MHz, DMSO-d6, δ): 7.47 (d, 4H, J2-3 = J6-5 = 8.5 Hz, A+A’, H2+H6), 7.56 (d,
4H, J3-2 = J5-6 = 8.5 Hz, A+A’, H3+ H5), 7.69 (d, 4H, J2-3 = J6-5 = 9.1 Hz, B+B’,
H2+H6), 8.19 (d, 4H, J3-2 = J5-6 = 9.1 Hz, B+B’, H3+ H5), 9.10 (bs, 2H, 2NH), 9.50
(bs, 2H, 2NH); 13C NMR (100 MHz, DMSO-d6, δ): 118.3 (B+B', C2+C6), 120.5
(A+A', C3+C5), 122.8 (B+B', C3+C5), 126.1 (A+A', C1), 133.3 (A+A', C2+C6),
139.7 (A+A', C4), 142.0 (B+B', C4), 147.1 (B+B', C1), 152.2 (C=O); MS (m/z %
abundance): 588 (29), 368 (15), 99 (46), 83 (50), 57 (100); Anal. Calcd for
C26H20N6O6Se2 (%): C: 46.6, H: 3.0, N: 12.5. Found: C: 46.5, H: 3.1, N: 12.6.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-methylphenyl)urea]
(3). From 4-methylphenyl isocyanate after 120 h gave 3 as a yellow powder.
Yield: 28%; mp 283−284 ºC; IR ʋmax (KBr): 3315 (N−H), 1643 (C=O) cm-1;1H
NMR (400 MHz, DMSO-d6, δ): 2.24 (s, 6H, 2CH3), 7.09 (d, 4H, J3-2 = J5-6 = 8.1
Hz, B+B’, H3+ H5), 7.33 (d, 4H, J2-3 = J6-5 = 8.1 Hz, B+B’, H2+H6), 7.43 (d, 4H, J2-
3 = J6-5 = 8.5 Hz, A+A’, H2+H6), 7.51 (d, 4H, J3-2 = J5-6 = 8.5 Hz, A+A’, H3+ H5),
8.61 (s, 2H, 2NH), 8.79 (s, 2H, 2NH); 13C NMR (100 MHz, DMSO-d6, δ): 32.0
(CH3), 118.1 (B+B', C2+C6), 118.8 (A+A', C3+C5), 122.4 (A+A', C1), 127.2 (B+B',
C3+C5), 130.7 (A+A', C2+C6), 132.6 (B+B', C1), 137.1 (B+B', C4), 140.8 (A+A',
C4), 154.2 (C=O); MS (m/z % abundance): 240 (24), 107 (80), 83 (58), 57 (100);
Anal. Calcd for C28H26N4O2Se2 .1/2 H2O (%): C: 54.4, H: 4.2, N: 9.0. Found: C:
54.6, H: 4.3, N: 8.8.
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N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-chlorophenyl)urea]
(4). From 4-chlorophenyl isocyanate after 48 h gave 4 as a yellow powder.
Yield: 59%; mp>300 ºC; IR ʋmax (KBr): 3289 (N−H), 1635 (C=O) cm-1, 1H NMR
(400 MHz, DMSO-d6, δ): 7.33 (d, 4H, J2-3 = J6-5 = 8.3 Hz, A+A’, H2+H6), 7.45-
7.54 (m, 12H, B+B’, H2+H3+H5+H6, A+A’, H3+H5), 8.86 (bs, 4H, 4NH); 13C NMR
(100 MHz, DMSO-d6, δ): 119.5 (A+A', C3+C5), 120.3 (B+B', C2+C6), 122.7
(A+A', C1), 126.0 (B+B’, C4), 129.1 (B+B', C3+C5), 134.1 (A+A', C2+C6), 139.0
(B+B', C1), 140.5 (A+A', C4), 152.7 (C=O); MS (m/z % abundance): 338 (29),
143 (85), 87 (54), 57 (100); Anal. Calcd for C26H20Cl2N4O2Se2 (%): C: 48.1, H:
3.1, N: 8.6. Found: C: 48.0, H: 3.1, N: 8.4.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-cyanophenyl)urea]
(5). From 4-cyanophenyl isocyanate after 96 h gave 5 as a yellow powder.
Yield: 70%; mp 185−186 ºC; IR ʋmax (KBr): 3367 (N−H), 2221 (CN), 1689 (C=O)
1
cm-1; H NMR (400 MHz, DMSO-d6, δ): 7.46 (d, 4H, J2-3 = J6-5 = 7.5 Hz, A+A’,
H2+H6), 7.55 (d, 4H, J3-2 = J5-6 = 7.5 Hz, A+A’, H3+ H5), 7.64 (d, 4H, J3-2 = J5-6
=
8.0 Hz, B+B’, H3+H5), 7.73 (d, 4H, J2-3 = J6-5 = 8.0 Hz, B+B’, H2+H6), 9.02 (s, 2H,
2NH), 9.24 (s, 2H, 2NH); 13C NMR (100 MHz, DMSO-d6, δ): 103.9 (B+B', C4),
118.6 (CN), 119.7 (B+B', C2+C6), 123.2 (A+A', C1), 133.8 (A+A', C2+C6), 134.0
(B+B', C3+C5), 140.1 (A+A', C4), 144.5 (B+B', C1), 152.4 (C=O); MS (m/z %
abundance): 156 (27), 92 (21), 83 (28), 71 (45), 57 (100); Anal. Calcd for
C28H20N6O2Se2 (%): C: 53.3, H: 3.2, N: 13.3. Found: C: 53.1, H: 3.5, N: 13.2.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-methoxyphenyl)urea]
(6). From 4-methoxyphenyl isocyanate after 72 h gave 6 as a yellow powder.
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Yield: 65%; mp 274−275 ºC; IR ʋmax (KBr): 3288 (N−H), 1644 (C=O) cm-1; H
NMR (400 MHz, DMSO-d6, δ): 3.74 (s, 6H, 2OCH3), 6.56 (d, 2H, J3-2 = 7.5 Hz,
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B+B’, H3), 6.94 (d, 2H, J5-6’ = 7.5 Hz, B+B’, H5), 7.19 (s, 4H, B+B’, H2+H6), 7.44
(d, 4H, J2-3 = J6-5 = 6.9 Hz, A+A’, H2+H6), 7.53 (d, 4H, A+A’, H3+H5), 8.73 (s, 2H,
2NH), 8.82 (s, 2H, 2NH);13C NMR (100 MHz, DMSO-d6, δ): 55.4 (CH3), 111.1
(B+B', C3+C5), 119.4 (B+B', C2+C6), 122.6 (A+A', C3+C5), 130.0 (A+A', C1),
134.1 (A+A', C2+C6), 140.6 (B+B', C1), 141.2 (A+A', C4), 152.7 (C=O), 160.2
(B+B', C4); Anal. Calcd for C28H26N4O4Se2 (%): C: 52.5, H: 4.0, N: 8.7. Found:
C: 52.3, H: 3.9, N: 8.5.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis(1-benzylurea) (7). From
benzyl isocyanate after 96 h gave 7 as a yellow powder. Yield: 43%; mp
1
213−215 ºC; IR ʋmax (KBr): 3335 (N−H), 1647 (C=O) cm-1; H NMR (400 MHz,
DMSO-d6, δ): 4.31 (d, 4H, JCH2-NH = 5.6 Hz, 2CH2), 6.68 (t, 2H, JNH-CH2 = 5.6 Hz,
NH-CH2), 7.24−7.34 (m, 10H, B+B’, H2+H3+H4+H5+H6), 7.39 (d, 4H, J3-2 = J5-6
=
8.6 Hz, A+A’, H3+H5), 7.45 (d, 4H, J2-3 = J6-5 = 8.4 Hz, A+A’, H2+H6), 8.74 (s, 2H,
2NH-C6H4); 13C NMR (100 MHz, DMSO-d6, δ): 43 (CH2), 118.8 (A+A', C3+C5),
121.7 (A+A', C1), 127.2 (B+B', C4), 127.6 (B+B', C2+C6), 128.8 (B+B', C3+C5),
134.3 (A+A', C2+C6), 140.7 (B+B', C1), 141.5 (A+A', C4), 155.5 (C=O); Anal.
Calcd for C28H26N4O2Se2 .1/2 H2O (%): C: 54.5, H: 4.4, N: 9.1. Found: C: 54.6,
H: 4.5, N: 9.3.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-methoxybenzyl)urea]
(8). From 4-methoxybenzyl isocyanate after 48 h gave 8 as a yellow powder.
1
Yield: 31%; mp 222−224 ºC; IR ʋmax (KBr): 3305 (N−H), 1630 (C=O) cm-1; H
NMR (400 MHz, DMSO-d6, δ): 3.73 (s, 6H, 2OCH3), 4.23 (d, 4H, JCH2-NH = 5.5
Hz, 2CH2), 6.60 (t, 2H, JNH-CH2 = 5.5 Hz, 2NH−CH2), 6.90 (d, 4H, J2-3 = J6-5 = 8.5
Hz, B+B’, H2+H6), 7.23 (d, 4H, J3-2 = J5-6 = 8.5 Hz, B+B’, H3+H5), 7.39 (d, 4H, J2-3
= J6-5 = 8.5 Hz, A+A’, H2+H6), 7.45 (d, 4H, J3-2 = J5-6 = 8.5 Hz, A+A’, H3+ H5),
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8.69 (s, 2H, 2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ): 42.9 (CH2), 56.0
(CH3), 114.4 (B+B', C3+C5), 119.0 (A+A', C3+C5), 122.1 (A+A', C1), 129.2 (B+B',
C2+C6), 133.6 (B+B', C1), 134.2 (A+A', C2+C6), 142.3 (A+A', C4), 158.5 (C=O),
159.4 (B+B', C4); MS (m/z % abundance): 368 (7), 215 (26), 83 (44), 71 (53), 57
(100); Anal. Calcd for C30H30N4O4Se2.1/2 H2O (%): C: 53.2, H: 4.4, N: 8.3.
Found: C: 53.1, H: 4.4, N: 8.2.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(n-butyl)urea] (9). From
butyl isocyanate after 72 h gave 9 as a yellow powder. Yield: 42%; mp 250−251
1
ºC; IR ʋmax (KBr): 3309 (N−H), 2958−2864 (C−H), 1630 (C=O) cm-1; H NMR
(400 MHz, DMSO-d6, δ): 0.89 (t, 6H, JCH3-CH2 = 7.2 Hz, 2CH3), 1.25−1.45 (m,
8H, 2(-CH2−CH2−CH3)), 2.96−3.17 (m, 4H, 2(-NH−CH2), 6.18 (t, 2H, JNH2 CH2 =
-
5.3 Hz, 2NH−CH2), 7.30−7.48 (m, 8H, A+A’, H2+H3+H5+H6), 8.57 (s, 2H,
2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ): 14.2 (C4), 19.1 (C3), 32.5 (C2),
39.0 (C1), 119.1 (A+A', C3+C5), 122.8 (A+A', C1), 134.7 (A+A', C2+C6), 142.5
(A+A', C4), 156.1 (C=O); MS (m/z % abundance): 588 (12), 211 (100), 183 (26),
91 (34), 43 (26); Anal. Calcd for C22H30N4O2Se2.H2O (%): C: 47.3, H: 5.7, N:
10.0. Found: C: 47.4, H: 5.5, N: 9.9.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(n-hexyl)urea]
(10).
From hexyl isocyanate after 72 h gave 10 as a yellow powder. Yield: 25%; mp
180−181 ºC; IR ʋmax (KBr): 3313 (N−H), 2956−2856 (C−H), 1627 (C=O) cm-1;
1H NMR (400 MHz, DMSO-d6, δ): 0.87 (bs, 6H, 2CH3), 1.27 (bs, 12H, 2(-
(CH2)2−(CH2)3−CH3)), 1.41 (bs, 4H, 2(-CH2−CH2−(CH2)3−CH3)), 3.06 (bs, 4H,
2(-CH2−(CH2)4−CH3)), 6.17 (bs, 2H, 2NH−CH2), 7.35 (d, 4H, J2-3 = J6-5 = 8.0 Hz,
A+A’, H2+H6), 7.43 (d, 4H, J3-2 = J5-6 = 8.0 Hz, A+A’, H3+H5), 8.57 (bs, 2H,
2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ): 14.4 (C6), 22.5 (C5), 26.5 (C3),
10

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30.1 (C2), 31.5 (C1+C4), 118.7 (A+A', C3+C5), 121.4 (A+A', C1), 134.4 (A+A',
C2+C6), 141.7 (A+A', C4), 155.4 (C=O); MS (m/z % abundance): 172 (25), 149
(56), 123 (100), 91 (55), 56 (74), Anal. Calcd for C26H38N4O2Se2.1/2 H2O (%):
C: 51.6, H: 6.2, N: 9.2. Found: C: 51.6, H: 6.0, N: 9.1.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-cyclohexylurea]
(11).
From cyclohexyl isocyanate after 120 h gave 11 as a yellow powder. Yield:
59%; mp 255−257 ºC; IR ʋmax (KBr): 3306 (N−H), 2927−2850 (C−H), 1645
(C=O) cm-1; 1H NMR (400 MHz, DMSO-d6, δ): 1.15−1.30 (m, 12H, B+B',
2H3+2H4+2H5), 1.53−1.58 (m, 2H, B+B', H1), 1.65 (d, 4H, J2-3 = J2-1 = 13.0 Hz,
B+B', 2H2), 1.79 (d, 4H, J6-1 = J6-5 = 14.1 Hz, B+B', 2H6), 6.13 (d, 2H, JNH CH =
-
7.8 Hz, 2NH−CH), 7.34 (d, 4H, J2-3 = J6-5 = 8.6 Hz, A+A', H2+H6), 7.43 (d, 4H,
A+A', H3+H5), 8.46 (s, 2H, 2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ)
24.8+25.7 (B+B', C3+C5), 33.3+33.8 (B+B', C2+C4+C6), 48.0 (B+B', C1), 118.6
(A+A', C3+C5), 121.5 (A+A', C1), 127.7 (A+A', C2+C6), 134.4 (A+A', C4), 154.6
(C=O); MS (m/z % abundance): 368 (34), 224 (71), 191 (76), 56 (100), 41 (27);
Anal. Calcd for C26H34N4O2Se2 (%): C: 52.7, H: 5.7, N: 9.4. Found: C: 52.3, H:
5.5, N: 9.8.
General procedure for the synthesis of thioureas 12-22. To a solution
of diselenide (1.17 mmol) in dioxane (25 mL) the corresponding isothiocyanate
(2.34 mmol, 1:2 molar ratio) was added and the mixture was kept at room
temperature from 48 h to 144 h. The solvent was removed under vacuum by
rotatory evaporation and the residue was treated with ethyl ether (50 mL) and
washed with water (100 mL).
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis(1-phenylthiourea)
(12).
From phenyl isothiocyanate after 144 h gave 12 as a yellow powder. Yield:
11

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45%; mp 142−143 ºC; IR ʋmax (KBr): 3193 (N−H), 1588 (C=S) cm-1; H NMR
(400 MHz, DMSO-d6, δ): 7.12-7.15 (m, 2H, B+B', H4), 7.33 (t, 4H, J3-2 = J5-6
7.5 Hz, B+B', H3+H5), 7.46-7.49 (m, 8H, A+A', B+B’, H2+H6), 7.59 (d, 4H, J3-2
=
=
J5-6 = 7.5 Hz, A+A', H3+H5), 9.88 (bs, 4H, 4NH); 13C NMR (100 MHz, DMSO-d6,
δ): 124.1 (A+A', C1), 124.5 (A+A', C3+C5), 125.0 (B+B', C2+C6), 125.4 (B+B',
C4), 128.9 (B+B', C3+C5), 132.5 (A+A', C2+C6), 139.8 (A+A', C4), 140.2 (B+B',
C1), 179.9 (C=S); MS (m/z % abundance): 428 (33), 386 (34), 214 (69), 172
(86), 135 (100), 93 (74), 80 (35); Anal. Calcd for C26H22N4S2Se2 (%): C: 50.9, H:
3.6, N: 9.1. Found: C: 50.6, H: 3.8, N: 8.7.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-nitrophenyl)thiourea]
(13). From 4-nitrophenyl isothiocyanate after 48 h gave 13 as a yellow powder.
Yield: 58%; mp 175−176 ºC; IR ʋmax (KBr): 3345 (N−H), 1570 (C=S) cm-1; H
1
NMR (400 MHz, DMSO-d6, δ): 7.49 (bs, 4H, A+A', H2+H6), 7.62 (bs, 4H, A+A',
H3+H5), 7.81 (bs, 4H, B+B', H2+H6), 8.20 (bs, 4H, B+B', H3+H5), 10.41 (bs, 4H,
4NH); 13C NMR (100 MHz, DMSO-d6, δ): 122.2 (A+A', C1), 124.9 (B+B', C3+C5),
126.2 (B+B', C2+C6), 132.4 (A+A', C3+C5), 139.5 (A+A', C2+C6), 142.9 (A+A',
C4), 146.6 (B+B', C1+C4), 179.7 (C=S); MS (m/z % abundance): 426 (5), 386
(13), 344 (15), 180 (100), 172 (53), 150 (26), 134 (34), 90 (25); Anal. Calcd for
C26H20N6O4S2Se2 .H2O (%): C: 43.3, H: 2.8, N: 11.6. Found: C: 43.6, H: 2.9, N:
11.3.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-
methylphenyl)thiourea] (14). From 4-methylphenyl isothiocyanate after 96 h
gave 14 as a yellow powder. Yield: 63%; mp 151−153 ºC; IR ʋmax (KBr): 3203
(N−H), 1583 (C=S) cm-1; 1H RMN (400 MHz, DMSO-d6, δ): 2.28 (s, 6H, 2CH3),
7.14 (d, 4H, J3-2 = J5-6 = 8.0 Hz, B+B', H3+H5), 7.32 (d, 4H, B+B', H2+H6), 7.48
12

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(d, 4H, J2-3 = J6-5 = 8.2 Hz, A+A', H2+H6), 7.58 (d, 4H, A+A', H3+H5), 9.81 (bs,
4H, 4NH); 13C NMR (100 MHz, DMSO-d6, δ): 21.3 (CH3), 123.0 (A+A', C1),
125.2 (A+A', C3+C5), 127.9 (B+B', C2+C6), 129.0 (B+B', C3+C5), 131.5 (A+A',
C2+C6), 133.2 (B+B', C1), 137.1 (B+B', C4), 140.3 (A+A', C4), 179.8 (C=S); MS
(m/z % abundance): 428 (33), 386 (37), 214 (65), 172 (100), 149 (86), 106 (90),
91 (52); Anal. Calcd for C28H26N4S2Se2 (%): C: 52.5, H: 4.1, N: 8.7. Found: C:
52.1, H: 4.3, N: 8.4.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-
chlorophenyl)thiourea] (15). From 4-chlorophenyl isothiocyanate after 48 h
gave 15 as a yellow powder. Yield: 68%; mp 170−171 ºC; IR ʋmax (KBr): 3210
(N−H), 1583 (C=S) cm-1; 1H NMR (400 MHz, DMSO-d6, δ): 7.44 (d, 4H, J2-3 = J6-
5 = 8.8 Hz, A+A', H2+H6), 7.55 (dd, 8H, J3-2 = J5-6 = 8.8 Hz, A+A', B+B', H3+H5),
7.65 (d, 4H, B+B', H2+H6), 10.01 (bs, 4H, 4NH);13C NMR (100 MHz, DMSO-d6,
δ): 124.6 (A+A', C3+C5), 125.6 (B+B', C2+C6), 125.7 (A+A', C1), 128.8 (B+B',
C4), 128.8 (B+B', C3+C5), 132.5 (A+A', C2+C6), 138.8 (B+B', C1), 139.9 (A+A',
C4), 180.0 (C=S); MS (m/z % abundance): 428 (14), 386 (25), 214 (28), 169
(100), 127 (54), 111 (23); Anal. Calcd for C26H20Cl2N4S2Se2 (%): C: 45.8, H: 2.9,
N: 8.2. Found: C: 45.5, H: 3.0, N: 7.9.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-
cyanophenyl)thiourea] (16). From 4-cyanophenyl isothiocyanate after 144 h
gave 16 as a yellow powder. Yield: 44%; mp 123−124 ºC; IR ʋmax (KBr): 3166
(N−H), 2224 (CN), 1584 (C=S) cm-1; 1H NMR (400 MHz, DMSO-d6, δ): 7.48 (d,
4H, J2-3 = J6-5 = 8.4 Hz, A+A', H2+H6), 7.62 (d, 4H, A+A', H3+H5), 7.75 (d, 4H, J3-5
= J5-6 = 8.8 Hz, B+B', H3+H5), 7.78 (d, 4H, B+B', H2+H6), 10.25 (bs, 2H, 2NH),
10.28 (bs, 2H, 2NH); 13C NMR (100 MHz, DMSO-d6, δ): 104.9 (CN), 114.7
13

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(B+B', C4), 124.2 (A+A', C1), 124.7 (A+A', C3+C5), 125.3 (B+B', C2+C6), 129.8
(A+A', C2+C6), 132.6 (B+B', C3+C5), 139.0 (A+A', C4), 144.2 (B+B', C1), 179.8
(C=S); MS (m/z % abundance): 428 (6), 386 (22), 344 (23), 172 (96), 160 (100),
118 (30), 80 (20); Anal. Calcd for C28H20N6S2Se2.1/2 H2O (%): C: 50.1, H: 3.0,
N: 12.5. Found: C: 49.8, H: 3.3, N: 12.4.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-
methoxyphenyl)thiourea] (17). From 4-methoxyphenyl isothiocyanate after 72
1
h gave 17 as a yellow powder. Yield: 65%; mp 142−144 ºC; IR ʋmax (KBr): H
NMR (400 MHz, DMSO-d6, δ): 3.75 (s, 6H, 2OCH3), 6.91 (d, 4H, J2-3 = J6-5 = 8.9
Hz, B+B', H2+H6), 7.32 (d, 4H, B+B', H3+H5), 7.48 (d, 4H, J3-2 = J5-6 = 8.6 Hz,
A+A', H3+H5), 7.58 (d, 4H, A+A', H2+H6), 9.72 (bs, 4H, 4NH); 13C NMR (100
MHz, DMSO-d6, δ): 56.0 (CH3), 114.2 (B+B', C3+C5), 115.3 (A+A', C3+C5),
126.0 (B+B', C1), 125.4 (A+A', C1), 132.1 (B+B’, C2+C6), 133.6 (A+A’, C2+C6),
139.7 (A+A', C4), 159.2 (B+B', C4), 180.1 (C=S); MS (m/z % abundance): 428
(21), 386 (32), 213 (53), 172 (100), 166 (84), 150 (54), 108 (57), 80 (41); Anal.
Calcd for C28H26N4O2S2Se2 .1/2 H2O (%): C: 48.7, H: 4.1, N: 8.1. Found: C:
49.1, H 3.9, N: 7.8.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis(1-benzylthiourea)
(18).
From benzyl isothiocyanate after 144 h gave 18 as a yellow powder. Yield:
1
71%; mp 146−147 ºC; IR ʋmax (KBr): 3238 (N−H), 1533 (C=S) cm-1; H NMR
(400 MHz, DMSO-d6, δ): 4.77 (d, JCH2-NH = 5.3 Hz, 4H, 2CH2), 7.22−7.28 (m,
2H, B+B', H4), 7.32-7.38 (m, 8H, B+B', H2+H6, H3+H5), 7.45 (d, 4H, J2-3 = J6-5
=
8.6 Hz, A+A', H2+H6), 7.57 (d, 4H, A+A', H3+H5), 8.27 (s, 2H, 2NH−CH2), 9.71
(s, 2H, 2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ): 47.6 (CH2), 124.1 (A+A',
C1), 125.1 (A+A', C3+C5), 127.4 (B+B', C4), 127.9 (B+B', C2+C6), 128.8 (B+B',
14

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C3+C5), 132.7 (A+A', C2+C6), 139.3 (B+B', C1), 140.0 (A+A', C4), 181.1 (C=S);
MS (m/z % abundance): 368 (42), 191 (100), 172 (67), 57 (54); Anal. Calcd for
C28H26N4S2Se2 .1/2 H2O (%): C: 51.8, H: 4.2, N: 8.6. Found: C: 51.9, H: 4.6, N:
8.7.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-
methoxybenzyl)thiourea] (19). From 4-methoxybenzyl isothiocyanate after
120 h gave 19 as a yellow powder. Yield: 49%; mp 174−175 ºC; IR ʋmax (KBr):
1
3220 (N−H), 1583 (C=S) cm-1; H NMR (400 MHz, DMSO-d6, δ): 3.73 (s, 6H,
2OCH3), 4.64 (bs, 4H, 2CH2), 6.90 (d, 4H, J3-2 = J5-6 = 8.8 Hz, B+B', H3+H5),
7.27 (d, 4H, B+B', H2+H6,), 7.44 (d, 4H, J2-3 = J6-5 = 7.8 Hz, A+A', H2+H6), 7.56
(d, 4H, A+A', H3+H5), 8.20 (bs, 2H, 2NH−CH2), 9.66 (bs, 2H, 2NH−C6H4); 13C
NMR (100 MHz, DMSO-d6, δ): 53.9 (CH2), 57.1 (CH3), 112.8 (B+B', C3+C5),
115.0 (A+A', C1), 123.2 (A+A', C3+C5), 124.1 (B+B', C1), 128.8 (B+B', C2+C6),
132.6 (A+A', C2+C6), 140.2 (A+A', C4), 159.1 (B+B', C4), 180.7 (C=S); MS (m/z
% abundance): 428 (55), 214 (100), 172 (44), 136 (96), 121 (82), 106 (36);
Anal. Calcd for C30H30N4O2S2Se2 .1/2 H2O (%): C: 50.7, H: 4.2, N: 7.9. Found:
C: 50.4, H: 4.2, N: 7.7.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(n-butyl)thiourea] (20).
From 4-methoxybenzyl isothiocyanate after 120 h gave 20 as a yellow powder.
1
Yield: 49%; mp 174−175 ºC; IR ʋmax (KBr): 3220 (N−H), 1583 (C=S) cm-1; H
NMR (400 MHz, DMSO-d6, δ): 3.73 (s, 6H, 2OCH3), 4.64 (bs, 4H, 2CH2), 6.90
(d, 4H, J3-2 = J5-6 = 8.8 Hz, B+B', H3+H5), 7.27 (d, 4H, B+B', H2+H6,), 7.44 (d, 4H,
J2-3 = J6-5 = 7.8 Hz, A+A', H2+H6), 7.56 (d, 4H, A+A', H3+H5), 8.20 (bs, 2H,
2NH−CH2), 9.66 (bs, 2H, 2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ): 14.2
(CH3), 20.1 (CH2), 30.1 (CH2), 44.0 (CH2), 123.7 (A+A', C1), 124.7 (A+A',
15

373 C3+C5), 132.7 (A+A', C2+C6), 140.2 (A+A', C4), 180.6 (C=S); MS (m/z %
374 abundance): 428 (55), 214 (100), 172 (44), 136 (96), 121 (82), 106 (36); Anal.
375 Calcd for C30H30N4O2S2Se2 .1/2 H2O (%): C: 50.7, H: 4.2, N: 7.9. Found: C:
376 50.4, H: 4.2, N: 7.7.
377
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(n-hexyl)thiourea] (21).
378 From hexyl isothiocyanate after 120 h gave 21 as a yellow powder. Yield: 65%;
379 mp 132−134 ºC; IR ʋmax (KBr): 3223 (N−H), 2925−2854 (C−H), 1540 (C=S) cm-
1
380 1; H NMR (400 MHz, DMSO-d6, δ): 0.87 (bs, 6H, 2CH3), 1.28 (bs, 12H, 2(-
381 (CH2)2−(CH2)3−CH3)), 1.51 (bs, 4H, 2(-CH2−CH2−(CH2)3−CH3)), 3.44 (bs, 4H,
382 2(-CH2−(CH2)4−CH3)), 7.42 (d, 4H, J2-3 = J6-5 = 8.4 Hz, A+A', H2+H6), 7.55 (d,
383 4H, A+A', H3+H5), 7.85 (bs, 2H, 2NH−CH2), 9.55 (bs, 2H, 2NH−C6H4); 13C NMR
384 (100 MHz, DMSO-d6, δ): 14.4 (CH3), 22.5 (CH2), 26.6 (CH2), 28.8 (CH2), 31.5
385 (CH2), 44.3 (CH2), 123.7 (A+A', C1), 132.7 (A+A', C2+C3+C5+C6), 140.2 (A+A',
386 C4), 180.6 (C=S); MS (m/z % abundance): 428 (14), 386 (6), 214 (32), 172 (45),
387 135 (73), 115 (92), 72 (47), 57 (56), 43 (100); Anal. Calcd for
388 C26H38N4S2Se2.1/2 H2O (%): C: 49.0, H: 6.0, N: 8.8. Found: C: 48.9, H: 6.0, N:
389 8.8.
390
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-cyclohexylthiourea]
391 (22). From cyclohexylisothiocyanate after 96 h gave 22 as a yellow powder.
392 Yield: 62%; mp 143−144 ºC; IR ʋmax (KBr): 3321 (N−H), 2926−2851 (C−H),
1
393 1587 (C=S) cm-1; H NMR (400 MHz, DMSO-d6, δ): 1.10−1.35 (m, 12H, B+B',
394 2H3+2H4+2H5)), 1.56 (bs, 2H, B+B', 2H1), 1.62 (bs, 4H, B+B’, 2H2) 1.80 (bs, 4H,
395 B+B', 2H6), 7.47 (d, 4H, J3-2 = J5-6 = 8.6 Hz, A+A', H3+H5), 7.54 (d, 4H, A+A',
396 H3+H5), 7.81 (d, 2H, JNH CH C
= 8.1 Hz, 2NH−CH), 9.49 (s, 2H, 2NH−C6H4); 13
-
397 NMR (100 MHz, DMSO-d6, δ) 25 (B+B', C3+C5), 26 (B+B', C4), 32 (B+B',
16

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416
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422
C2+C6), 53 (B+B', C1), 124 (A+A', C1), 131 (A+A', C2+C3+C5+C6), 140 (A+A',
C4), 179 (C=S); MS (m/z % abundance): 368 (12), 214 (7), 191 (24), 83 (21), 56
(100), 41 (33); Anal. Calcd for C26H34N4S2Se2 .1/2 H2O (%): C: 49.4, H: 5.2, N:
8.8. Found: C: 49.2, H: 5.3, N: 8.5.
Preparation of formamides 23-30.
N-phenylformamide (23). To a stirred solution of aniline (9.2 mmol) was
added dropwise ethyl formate (9.6 mmol). The reaction mixture was stirred at
150 ºC for 12 h. The reaction mixture was cooled to room temperature and the
precipitate was collected by filtration, dried and washed with ethyl ether (100
mL) to give 23 as a white powder. Yield: 77%; IR ʋmax (KBr): 3364 (N−H), 1634
(C=O) cm-1.
N-(4-methylphenyl)formamide (24). A mixture of 4-methylaniline (5
mmol) and anhydrous ammonium formate (7.5 mmol) in dry acetonitrile (15 mL)
was heated at 100°C for 24 h. Acetonitrile was removed under reduced
pressure. The residue was diluted with ethyl acetate (25 mL) and washed with
water (4 x 15 mL). The organic layer was dried over anhydrous Na2SO4. After
filtration and evaporation of the solvent, 24 was acquired as a white powder.
Yield: 66%; IR ʋmax (KBr): 3117 (N−H), 1637 (C=O) cm-1.
N-(4-chlorophenyl)formamide (25). To a mixture of 4-chloroaniline (10
mmol), formic acid (30 mmol) and zinc dust pre-treated with HCl (1 mmol) was
added and stirred at 70 ºC for 8 h−12 h. The mixture was diluted with CH2Cl2
(50 mL), and filters through celite. Then the filtrate was washed with saturated
NaHCO3 (4 x 30 mL) and brine (2x20 mL), and was dried over anhydrous
Na2SO4. After filtration and evaporation of the solvent, 25 was acquired as a
white powder. Yield: 69%; IR ʋmax (KBr): 3258 (N−H), 1670 (C=O) cm-1.
17

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447
N-(4-cyanophenyl)formamide (26). To a mixture of 4-aminobenzonitrile
(10 mmol), formic acid (30 mmol) and zinc dust pre-treated with HCl (1 mmol)
was added and stirred at 70 ºC for 8 h−12 h. The mixture was diluted with
CH2Cl2 (50 mL), and filters through celite. Then the filtrate was washed with
saturated NaHCO3 (3 x 30 mL) and brine (3 x 30 mL), and was dried over
anhydrous Na2SO4. After filtration and evaporation of the solvent, 26 was
acquired as a white powder. Yield: 82%; IR ʋmax (KBr): 3357 (N−H), 2216 (CN),
1637 (C=O) cm-1.
N-(4-methoxyphenyl)formamide (27). To a mixture of 4-methoxyaniline
(11.25 mmol) and HCOOH (33.75 mmol), PEG-400 (16 g) was added. The
mixture was stirred at room temperature for 24 h and after completion was
diluted with water (50 mL) and extracted with ethyl acetate (5 x 15 mL). Then
the organic layer was dried over anhydrous Na2SO4 and concentrated. The
residue was subjected to column chromatography ethyl (acetate/ hexane 70/30)
to obtain the pure 27 as a white powder. Yield: 68.6%; IR ʋmax (KBr): 3245
(N−H), 1656 (C=O) cm-1.
N-butylformamide (28). To a stirred solution of butan-1-amine (25
mmol) was added dropwise ethyl formate (20.16 mmol). The reaction mixture
was stirred at 150 ºC for 12 h. The reaction mixture was cooled to room
temperature and the precipitate was collected by filtration, dried and washed
with ethyl ether (100 mL) to give 28 as a white powder. Yield: 74.7%; IR ʋmax
(KBr): 3291 (N−H), 2960−2869 (C−H), 1665 (C=O) cm-1.
N-hexylformamide (29). To a stirred solution of hexan-1-amine (15
mmol) was added dropwise ethyl formate (12.10 mmol). The reaction mixture
was stirred at 150 ºC for 12 h. The reaction mixture was cooled to room
18

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470
471
temperature and the precipitate was collected by filtration, dried and washed
with ethyl ether (100 mL) to give 29 as a white powder. Yield: 92%; IR ʋmax
(KBr): 3361 (N−H), 2978−2868 (C−H), 1630 (C=O) cm-1.
N-ciclohexylformamide (30). To a mixture of cyclohexylamine (25
mmol), formic acid (75 mmol) and zinc dust pre-treated with HCl (5 mmol) was
added and stirred at 70 ºC for 8 h−12 h. The mixture was diluted with CH2Cl2
(50 mL), and filters through celite. Then the filtrate was washed with saturated
NaHCO3 (4 x 25 mL) and brine (2 x 25 mL), and was dried over anhydrous
Na2SO4. After filtration and evaporation of the solvent, 30 was acquired as a
white powder. Yield: 16%; IR ʋmax (KBr): 3412 (N−H), 2933−2858 (C−H), 1661
(C=O) cm-1.
General procedure for the synthesis of isoselenocyanates 31-34. To
a mixture of formamide (6.29 mmol) and N,N-diethyletanamine (26.8 mmol) in
dry toluene (50 mL) was added dropwise a solution of phosgene (3.35 mmol) in
dry toluene (10 mL), under N2 atmosphere, on ice over a period of 30 min. Then
black selenium powder (12.58 mmol) was added and the resulting mixture was
refluxed for 24 h in the darkness. After filtered, solvents were removed under
vacuum and the residue was washed with dichloromethane (30 mL). Column
chromatography using ethyl acetate/hexane (70/30) as eluent afforded
isoselenocyanate.
Phenylisoselenocyanate (31). From N-phenylformamide 23 gave 31 as
a brown syrup: Yield 2.66%; IR ʋmax (KBr): 2978−2873 (C−H), 2114 (N=C=Se)
cm-1; 1H NMR (400 MHz, DMSO-d6, δ): 6.92 (t, 2H, J2-3 = J6-5 = 6.3 Hz, H2+H6),
7.28 (t, 1H, H4), 7.62 (d, 2H, J3-2 = J5-6 = 8.4 Hz, H3+H5).
19

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478
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480
481
482
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484
485
486
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490
491
492
493
494
495
4-Methylphenylisoselenocyanate
(32).
From
N-(4-
methylphenyl)formamide 24 gave 32 as a brown syrup: Yield 7.48%; IR ʋmax
(KBr): 2921−2866 (C−H), 2153 (N=C=Se) cm-1; 1H NMR (400 MHz, DMSO-d6,
δ): 2.34 (s, 3H, CH3), 7.21 (d, 2H, J2-3 = J6-5 = 7.9 Hz, H2+H6), 7.26 (d, 2H,
H3+H5).
4-Methoxyphenylisoselenocyanate
(33).
From
N-(4-
methoxyphenyl)formamide 27 gave 33 as an orange syrup: Yield 6.79%; IR ʋmax
(KBr): 2944−2740 (C−H), 2121 (N=C=Se) cm-1; 1H NMR (400 MHz, DMSO-d6,
δ): 3.80 (s, 3H, OCH3), 7.02 (d, 2H, J3-2 = J5-6 = 8.0 Hz, H3+H5), 7.45 (d, 2H,
H2+H6).
Benzylisoselenocyanate (34). From N-benzylformamide gave 34 as a
brown syrup: Yield 11.75 %; IR ʋmax (KBr): 2958−2871 (C−H), 2143 (N=C=Se)
cm-1; 1H NMR (400 MHz, DMSO-d6, δ): 5.15 (s, 2H, CH2), 7.41−7.54 (m, 5H,
H2+H3+H4+H5+H6).
General procedure for the synthesis of isoselenocyanates 35-39. To
a refluxing mixture of formamide (7.2 mmol) and N,N-diethyletanamine (30.5
mmol) in dry dichloromethane (25 mL) was added dropwise a solution of
triphosgene (3.85 mmol) in dry dichloromethane (5 mL), under N2 atmosphere,
over a period of 45 min. After the addition, the resulting mixture was refluxed
for 2.5 h and then black selenium powder (14.4 mmol) was added and refluxed
for 12 h in the darkness. After filtered, solvents were removed under vacuum
and the residue was washed with dichloromethane (30 mL). Column
chromatography using ethyl acetate/hexane (70/30) as eluent afforded
isoselenocyanate.
20

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519
520
4-Chlorophenyl
isoselenocyanate
(35).
From
N-(4-
chlorophenyl)formamide 25 gave 35 as a brown syrup: Yield 55.76%; IR ʋmax
(KBr): 2924−2854 (C−H), 2151 (N=C=Se) cm-1; 1H NMR (400 MHz, DMSO-d6,
δ): 7.02 (d, 2H, J3-2 = J5-6 = 7.6 Hz, H3+H5), 7.49 (d, 2H, H2+H6).
4-Cyanophenyl
isoselenocyanate
(36).
From
N-(4-
cyanophenyl)formamide 26 gave 36 as a brown syrup: Yield 85 %; IR ʋmax
(KBr): 2927−2856 (C−H), 2225 (CN), 2146 (N=C=Se) cm-1; 1H NMR (400 MHz,
DMSO-d6, δ): 7.77 (d, 2H, J3-2 = J5-6 = 8.4 Hz, H3+H5), 7.79 (d, 2H, H2+H6).
Butyl isoselenocyanate (37). From N-butylformamide 28 gave 37 as a
dark syrup: Yield 43.5%; IR ʋmax (KBr): 2923−2876 (C−H), 2144 (N=C=Se) cm-1;
1H NMR (400 MHz, DMSO-d6, δ): 0.86−1.11 (m, 3H, CH3), 1.21−1.32 (m, 4H,
CH2−CH2−CH3), 1.35 (m, 2H, CH2-NCSe).
Hexyl isoselenocyanate (38). From N-hexylformamide 29 gave 38 as a
brown syrup: Yield 62%; IR ʋmax (KBr): 2931−2861 (C−H), 2144 (N=C=Se) cm-1;
1H NMR (400 MHz, DMSO-d6, δ): 0.85−0.91 (m, 3H, CH3), 1.05 (t, 2H, J = 6.99
Hz, (CH2)2−(CH2)2−CH2−CH3), 1.18−1.24 (m, 4H, (CH2)2−(CH2)2−CH2−CH3),
1.28−1.35 (m, 2H,CH2−CH2− (CH2)3−CH3), 1.41−1.49 (m, 2H, CH2-NCSe).
Cyclohexylisoselenocyanate (39). From N-ciclohexylformamide 30
gave 39 as a dark syrup: Yield 92%; IR ʋmax (KBr): 2933−2883 (C−H), 2137
(N=C=Se) cm-1; 1H NMR (400 MHz, DMSO-d6, δ): 1.07 (s, H, H1), 1.28-1.37 (m,
2H, H4), 1.56−1.62 (m, 4H, H3+H5), 1.85−1.90 (m, 4H, H2+H6).
General procedure for the synthesis of selenoureas 40-48. To a
solution of the corresponding isoselenocyanate (2.33 mmol) in dry dioxane (40
mL), at room temperature under nitrogen atmosphere, was added a diselenide
solution (1.17 mmol) in dry dioxane (10 mL). The reaction was kept in the
21

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533
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535
536
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538
539
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541
542
543
544
545
darkness for 145 h. To afford the desired selenourea we proceed accordingly
two different work-up: i) Work-up method A: After stirring the precipitate was
filtered off, washed with dichloromethane (100 mL) and dried in order to obtain
the selenoureas 41 and 44; ii) Work-up method B: After stirring the solvent was
evaporated to yield the solid product, which was washed with dichloromethane
(100 mL) and dried in order to obtain the selenoureas 40, 42-43 and 45-48.
Optimal purification method for compounds 40-44 was the formation of
the corresponding salts by reaction with hydrochloric acid in ethyl ether.
N´,N´´´-(Diselanediyldibenzene-4,1-diyl)bis(1-phenylselenourea) (40).
From phenyl isoselenocyanate 31. The salt formation with hydrochloric ether
gave 40 as a yellow powder. Yield: 2.6%; mp 189−191 ºC; IR ʋmax (KBr): 3427
1
(N−H), 1582 (C=Se) cm-1; H NMR (400 MHz, DMSO-d6, δ): 6.43 (d, 4H, J2-3
=
J6-5 = 7.2 Hz, B+B', H2+H6), 6.63 (d, 4H, J3-2 = J5-6 = 8.8 Hz, A+A', H3+H5), 6.81
(t, 2H, J4-3 = J4-5 = 7.3 Hz, B+B', H4), 7.20 (t, 4H, J3-2 = J5-6 = 8.7 Hz, B+B',
H3+H5), 7.54 (d, 4H, A+A', H2+H6), 9.36 (s, 2H, 2NH−C6H4), 9.56 (s, 2H,
2NH−C6H4Se); 13C NMR (100 MHz, DMSO-d6, δ): 119.2 (A+A', C1), 116.4
(A+A', C3+C5), 117.9 (B+B', C2+C6), 122.0 (B+B', C4), 129.3 (B+B', C3+C5),
132.8 (A+A', C2+C6), 144.3 (A+A', C4), 146.1 (B+B', C1), 179.4 (C=Se); Anal.
Calcd for C26H22N4Se4.3HCl (%): C, 37.4 H, 3.2, N, 6.7. Found: C, 37.3, H, 3.0,
N, 6.7.
N´,N´´´-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-
methylphenyl)selenourea] (41). From 4-methylphenyl isoselenocyanate 32.
The salt formation with hydrochloric ether gave 41 as a yellow powder. Yield:
1
7.5%; mp 212−213 ºC; IR ʋmax (KBr): 3161 (N−H), 1577 (C=Se) cm-1; H NMR
(400 MHz, DMSO-d6, δ): 2.73 (s, 6H, 2CH3), 7.30 (d, 4H, J2-3 = J6-5 = 8.8 Hz,
22

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558
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560
561
562
563
564
565
566
567
568
569
570
B+B', H2+H6), 7.47 (d, 4H, J3-2 = J5-6 = 8.6 Hz, A+A', H3+H5), 7.54 (d, 4H, J3-2 =
J5-6 = 8.8 Hz, B+B', H3+H5), 7.66 (d, 4H, J2-3 = J6-2 = 8.6 Hz, A+A', H2+H6), 8.20
(s, 2H, 2NH−C6H4CH3), 9.66 (s, 2H, 2NH−C6H4Se); 13C NMR (100 MHz,
DMSO-d6, δ): 20.1 (CH3), 104.3 (B+B', C2+C6), 118.8 (A+A', C3+C5), 123.3
(A+A', C1), 129.0 (B+B', C3+C5), 134.2 (B+B', C4), 140.7 (A+A', C2+C6), 162.5
(B+B', C1), 173.0 (A+A', C4), 181.8 (C=Se); MS (m/z % abundance): 222 (99),
197 (36), 91 (100), 65 (35); Anal. Calcd for C28H26N4Se4.3HCl (%): C, 39.8, H,
3.4, N, 6.6. Found: C, 39.5, H, 3.2, N, 6.6.
N´,N´´´-(diselanediyldibenzene-4,1-diyl)bis[1-(4-
chlorophenyl)selenourea] (42). From 4-chlorophenyl isoselenocyanate 35.
The salt formation with hydrochloric ether gave 42 as a yellow powder. Yield:
4.75%; IR ʋmax (KBr): 3367 (N−H), 1625 (C=Se) cm-1; 1H NMR (400 MHz,
DMSO-d6, δ): 7.06−7.11 (m, 4H, A+A', H3+H5), 7.20−7.25 (m, 4H, B+B', H2+H6),
7.29 (d, 4H, J2-3 = J6-5 = 8.6 Hz, A+A', H2+H6), 7.45 (d, 4H, J3-2 = J5-6 = 8.8 Hz,
B+B', H3+H5), 9.33 (s, 2H, 2NH−C6H4CN), 9.59 (s, 2H, 2NH−C6H4Se); 13C NMR
(100 MHz, DMSO-d6, δ): 116.3 (A+A', C3+C5), 119.1 (A+A', C1), 121.3 (B+B',
C2+C6), 128.0 (B+B', C4), 130.2 (B+B', C3+C5), 132.5 (A+A', C2+C6), 142.4
(B+B', C1), 144.1 (A+A', C4), 180.0 (C=Se); Anal. Calcd for
C26H20Cl2N4Se4.4HCl (%): C, 33.9, H, 2.6, N, 6.1. Found: C, 34.1, H, 2.2, N, 5.7.
N´,N´´´-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-
cyanophenyl)selenourea] (43). From 4-cyanophenyl isoselenocyanate 36.
The salt formation with hydrochloric ether gave 43 as a yellow powder. Yield:
7.3%; mp 165−167 ºC; IR ʋmax (KBr): 3077 (N−H), 2223 (CN), 1607 (C=Se) cm-
1
1; H NMR (400 MHz, DMSO-d6, δ): 6.94-7.06 (m, 4H, A+A', H3+H5), 7.12-7.33
(m, 4H, B+B', H2+H6), 7.35-7.53 (m, 4H, A+A', H2+H6), 7.56-7.74 (m, 4H, B+B',
23

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575
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579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
H3+H5), 10.19 (s, 4H, 4NH); 13C NMR (100 MHz, DMSO-d6, δ): 103.8 (B+B', C4),
116.4 (A+A', C3+C5), 118.1 (A+A', C1), 119.3 (CN), 120.4 (B+B', C2+C6), 132.2
(A+A', C2+C6), 133.0 (B+B', C3+C5), 144.1 (A+A', C4), 149.2 (B+B', C1), 180.0
(C=Se); MS (m/z % abundance): 446 (20), 243 (47), 189 (100), 95 (46), 56 (39
); Anal. Calcd for C28H20N6Se4.4HCl (%): C, 44.4, H, 2.6, N, 11.1. Found: C,
44.5, H, 2.6, N, 11.3.
N´,N´´´-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-
methoxyphenyl)selenourea] (44). From 4-methoxyphenyl isoselenocyanate
33. The salt formation with hydrochloric ether gave 44 as a orange powder.
Yield: 8.5%; mp 201−202 ºC; IR ʋmax (KBr): 3310 (N−H), 1553 (C=Se) cm-1; 1H
NMR (400 MHz, DMSO-d6, δ): 3.74 (s, 6H, 2OCH3), 6.90 (d, 4H, J2-3 = J6-5 = 8.4
Hz, B+B', H2+H6), 7.28 (d, 4H, J3-2 = J5-6 = 7.7 Hz, A+A', H3+H5), 7.43 (d, 4H,
B+B', H3+H5), 7.58 (d, 4H, A+A', H2+H6), 10.19 (bs, 4H, 4NH); 13C NMR (100
MHz, DMSO-d6, δ): 55.2 (OCH3), 113.7 (B+B', C3+C5), 115.1 (A+A', C3+C5),
115.5 (B+B', C2+C6), 116.0 (A+A', C1), 126.2 (A+A', C2+C6), 133.1 (B+B', C1),
147.8 (A+A', C4), 157.4 (B+B', C4), 179.2 (C=Se); MS (m/z % abundance): 254
(29), 213 (100), 197 (64), 108 (49), 63 (31); Anal. Calcd for
C28H26N4O2Se4.2HCl (%): C, 33.9, H, 2.6, N, 6.1. Found: C, 34.1, H, 2.2, N, 5.7.
1,1’-(4,4’-Diselanediylbis(4,1-phenylene))bis(3-benzylselenourea)
(45). From benzyl isoselenocyanate 34. Yellow powder. Yield: 11.75%; mp
145−146 ºC; IR ʋmax (KBr): 3120 (N−H), 1570 (C=Se) cm-1; 1H NMR (400 MHz,
DMSO-d6, δ): 4.85 (bs, 4H, 2CH2), 7.27 (bs, 4H, B+B', H2+H6), 7.34 (bs, 10H,
A+A’, H2+H6, B+B’, H3+H4+H5), 7.61 (bs, 4H, A+A’, H3+H5), 8.66 (bs, 2H,
2NH−CH2), 10.07 (bs, 2H, 2NH-C6H4); 13C NMR (100 MHz, DMSO-d6, δ): 50.6
(CH2), 125.3 (A+A', C3+C5), 126.6 (A+A', C1), 127.5 (B+B', C4), 127.9 (B+B',
24

596
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598
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618
619
C2+C6), 128.7 (B+B', C3+C5), 132.7 (A+A', C2+C6), 139.0 (B+B', C1), 139.3
(A+A', C4), 180.3 (C=Se); MS (m/z % abundance): 197 (10), 107 (27), 91 (100),
65 (19); Anal. Calcd for C28H26N4Se4 (%): C, 45.7, H, 3.5, N, 7.6. Found: C,
45.6, H, 3.6, N, 7.4.
1,1’-(4,4’-Diselanediylbis(4,1-phenylene))bis(3-butylselenourea) (46).
From butyl isoselenocyanate 37. Yellow powder. Yield: 15.7%; mp 114−116 ºC;
IR ʋmax (KBr): 3257 (N−H), 2957−2865 (C−H), 1620 (C=Se) cm-1; 1H NMR (400
MHz, DMSO-d6, δ): 0.90 (t, 6H, JCH3-CH2 = 6.8 Hz, 2CH3), 1.30-1.34 (m, 6H, B, -
CH2-CH2−CH2−CH3), 1.53−1.56 (m, 6H, B’ -CH2-CH2−CH2−CH3), 7.33 (d, 4H,
J3-2 = J5-6 = 8.1 Hz, A+A', H3+H5), 7.60 (d, 4H, A+A', H2+H6), 8.27 (bs, 2H,
2NH−CH2), 9.92 (bs, 2H, 2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ): 14.2
(CH3), 20.0 (CH2), 31.0 (CH2), 47.0 (CH2), 124.8 (A+A', C1), 132.9 (A+A',
C2+C3+C5+C6), 139.6 (A+A', C4), 179.2 (C=Se); Anal. Calcd for
C22H30N4Se4.H2O (%): C, 38.6, H, 4.7, N, 8.2. Found: C, 38.3, H, 4.3, N, 8.0.
1,1’-(4,4’-Diselanediylbis(4,1-phenylene))bis(3-hexylselenourea) (47).
From hexyl isoselenocyanate 38. Yellow powder. Yield: 12.2%; mp 115−117 ºC;
IR ʋmax (KBr): 3195 (N−H), 2923−2854 (C−H), 1542 (C=Se) cm-1; 1H NMR (400
MHz, DMSO-d6, δ): 0.88 (bs, 6H, 2CH3), 1.28 (bs, 12H, 2(-CH2)2−(CH2)3−CH3),
1.55 (bs, 4H, 2(-CH2−CH2−(CH2)3−CH3), 3.53 (bs, 4H, 2(-CH2−(CH2)4−CH3),
7.33 (bs, 4H, A+A', H3+H5), 7.59 (bs, 4H, A+A', H2+H6), 8.23 (bs, 2H,
2NH−CH2), 9.88 (s, 2H, 2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ): 14.4
(CH3), 22.5 (CH2), 26.5 (CH2), 28.8 (CH2), 31.4 (CH2), 47.3 (CH2), 124.8 (A+A',
C3+C5), 126.2 (A+A', C1), 133.0 (A+A', C2+C6), 139.6 (A+A', C4), 179.2 (C=Se);
MS (m/z % abundance): 368 (5), 191 (23), 69 ( 8), 57 (21), 43 (100); Anal.
25

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623
624
625
626
627
628
629
630
631
632
Calcd for C26H38N4Se4.H2O (%): C, 42.2, H, 5.4, N, 7.6. Found: C, 42.1, H, 5.1,
N, 7.5.
1,1’-(4,4’-Diselanediylbis(4,1-phenylene))bis(3-ciclohexylselenourea)
(48). From cyclohexylisoselenocyanate 39. Yellow powder. Yield: 21%; mp
175−180 ºC; IR ʋmax (KBr): 3398 (N−H), 2968−2931 (C−H), 1655 (C=Se) cm-1;
1H NMR (400 MHz, DMSO-d6, δ): 1.09−1.33 (m, 12H, B+B', 2H3+2H4+2H5);
1.59−1.61 (m, 2H, B+B', H1), 1.62-2.04 (m, 8H, B+B', 2H2+2H6), 7.18 (s, 4H,
A+A', H2+H6), 7.53 (s, 4H, A+A', H3+H5), 8.69 (s, 2H, 2NH−CH), 10.47 (s, 2H,
2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ) 12.2 (Ccy), 33.0 (Ccy), 53.7 (Ccy),
66.3 (Ccy), 115.0 (A+A', C1), 133.5 (A+A', C2+C6), 138.2 (A+A', C3+C5), 142.1
(A+A', C4), 182.8 (C=Se); MS (m/z % abundance): 368 (44), 191 (100), 163
(54), 135 (45), 84 (59), 56 (66), 41 (87); Anal. Calcd for C26H34N4Se4.H2O (%):
C, 42.4, H, 4.9, N, 7.6. Found: C, 42.3, H, 4.7, N, 7.7.
633
634
635
636
637
638
639
640
641
642
643
Biological evaluation. (i) Cells and culture conditions. L. infantum
axenic amastigotes were grown in M199 (Invitrogen, Leiden, The Netherlands)
medium supplemented with 10% heat inactivated FCS, 1 g/L β-alanine, 100
mg/L L-asparagine, 200 mg/L sacarose, 50 mg/L sodium pyruvate, 320 mg/L
malic acid, 40 mg/L fumaric acid, 70 mg/L succinic acid, 200 mg/L α-ketoglutaric
acid, 300 mg/L citric acid, 1.1 g/L sodium bicarbonate, 5 g/L MES, 0.4 mg/L
hemin, 10 mg/L gentamicin pH 5.4 at 37 ºC.THP-1 cells were kindly provided by
Dr. Michel (Université Nice Sophia Antipolis, Nice, France) and were grown in
RPMI-1640 medium (Gibco, Leiden, The Netherlands) supplemented with 10%
heat inactivated FCS, antibiotics, 1 mM HEPES, 2 mM glutamine and 1mM
sodium pyruvate, pH 7.2 at 37 ºC and 5% CO2.
26

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668
(ii) Leishmanicidal activity and cytotoxicity assays. Drug treatment of
amastigotes was performed during the logarithmic growth phase at a
concentration of 2×106 parasites/mL at 26 ºC or 1×106 parasites/mL at 37 ºC for
24 h, respectively. Drug treatment of Jurkat and THP-1 cells was performed
during the logarithmic growth phase at a concentration of 4×105 cells/mL at 37
ºC and 5% CO2 for 24 h. The percentage of living cells was evaluated by flow
cytometry by the propidium iodide (PI) exclusion method (25).
(iii) Leishmania infection assay. THP-1 cells were seeded at 120,000
cells/mL in 24 multidishes plates (Nunc, Roskilde, Denmark) and differentiated
to macrophages for 24 hours in 1mL of RPMI-1640 medium containing 10
ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO,
USA). Medium culture was removed and 1.2×106 Leishmania amastigotes in
1mL of THP-1 medium were added to each well. 4 hours later all medium with
non-infecting amastigotes was removed, washed 3 times with 1X phosphate
buffered saline (1X PBS) and replaced with new THP-1 medium and
corresponding treatment. After 48 hours treatment, medium was removed; THP-
1 cells were washed 3 times with 1X PBS and detached with TrypLETM Express
(Invitrogen, Leiden, The Netherlands) according to the manufacturer’s
indications. Infection was evaluated by flow cytometry.
(iv) Trypanothione reductase assay. Oxidoreductase activity was
determined according to the method described by Toro et al. (26). Briefly,
reactions were carried out at 26º C in 250 µL of 40 mM pH 8.0 HEPES buffer
containing 1 mM EDTA, 150 µM NADPH, 30 µM NADP+, 25 µM DTNB, 1 µM
T[S]2, 0.02% glycerol, 1.5% DMSO and 7 nM of recombinant Li-TryR. Enzyme
activity was monitored by the increase in absorbance at 412 nm for 1 h at 26°C
27

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in a VERSAmax microplate reader (Molecular Devices, California, USA). All the
assays were conducted in triplicate in at least three independent experiments.
Data were analyzed using a non-lineal regression model with the Grafit6
software (Erithacus, Horley, Surrey, UK).
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RESULTS
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Chemistry. The synthesis of the compounds described here was carried
out according to Figures 2−4. 4,4’-diaminodiphenyldiselenide (Figure 2) was
used as starting material to prepare the target compounds. This compound was
synthesized in good yield and purity as previously described by our group (12).
Compounds 1−22 were synthesized according to Figure 2. Diselenide and
commercial available isocyanate or isothiocyanate were mixed in dioxane at a
molar ratio 1:2, respectively, at room temperature for 24−120 hours. After
removing the solvent, the residue was treated with ethyl ether and washed with
water. The compounds were obtained in yields ranging from 25 to 71%.
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To obtain the planned selenoureas, the synthesis of the corresponding
isoselenocyanates (31−39), that were prepared in two steps, was necessary
(Figure 3). The first step involved formylation of amines to yield formamides
23−30 followed by the treatment with phosgene (31−34) (27) or triphosgene
(35−39) (28) and selenium powder in the presence of triethylamine under reflux.
Compounds were purified by silica gel column chromatography using n-
hexane/ethyl acetate as eluent. The IR spectra of the isoselenocyanates are
quite informative about the presence of the isoselenocyanate functional group
(–NCSe). The stretching frequency was observed at 2115−2224 cm−1.
Formamides 23−30 were prepared through different methods depending
on the type of primary amine (Figure 3). Ethyl formate was used for compounds
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23, 28 and 29; formic acid in the presence of zinc dust (29) for derivatives 25,
26 and 30 or in presence of PEG−400 for 27 (30). Derivative 24 was prepared
with ammonium formate in acetonitrile (31). After isolation of the product,
formamides were afforded in moderate to good overall yields 16−92 %.
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Reaction of isoselenocyanates with 4,4’-diaminodiphenyldiselenide in a
molar ratio 2:1 respectively, under nitrogen atmosphere, in dried dioxane and in
darkness generated selenoureas 40−48, (Figure 4). However, isolation of
selenoureas from the crude reaction mixture was highly tedious and
contaminations from different impurities remained with the desired derivatives,
thus diminishing final yields. Some of them (41 and 44) precipitated and were
collected by filtration and the other ones (40, 42, 43, 45, 46, 47 and 48) were
obtained after the solvent was concentrated to dryness. In both cases the
residue was washed with different solvents or solvent mixtures (ethyl ether,
hexane, ethanol...) generating the target compounds for derivatives 45−48,
exclusively. Optimal purification method for compounds 40−44 was the
formation of the corresponding salts by reaction with hydrochloric acid in ethyl
ether.
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The structures and purity of final compounds as well as all intermediates
were confirmed by spectroscopic data (IR, 1H NMR, 13C NMR), MS and
elemental analyses.
IR spectra of urea, thiourea and selenourea compounds revealed
characteristic strong intensity bands between 3427 and 3120 cm− 1 as a broad
signal due to the presence of hydrogen bonding for the introduction of four N-H
groups. Just above 3000 cm−1 Ar−H stretch was evident and carbonyl group for
ureas appeared as an intense band about 1644 cm−1. IR spectra of selenourea
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compounds revealed selenoyl group band at lower values, ranging from 1542 to
1655 cm−1.
1
In H NMR spectra, the characteristic singlets for N-H protons located
between C=X and phenyl moieties are more shielded and appear at downfield
shifted as singlet in a relatively wide range of 8.20 to 10.47 ppm. The typical
differences for aliphatic amino groups were also noted. Thus, for example, in
case of ureas 8−10 the signals of NHCH2 protons are observed between 6.17
and 6.60 as singlets or triplets. The aromatic rings provide their signals between
6.90 and 7.93 ppm.
In 13C NMR, maximum downfield carbon is the carbon attached to
selenium, appearing in the range of 179−183 ppm, whereas carbonyl carbon
appears at 152−156 ppm. Aromatic carbons provide their signals between 160
and 114 ppm. As a representative example of related structures, the close
range of 13C NMR shifts of C=S (179) for derivative 22 and C=Se (183) for
selenourea derivative 48 indicates their chemical similarity compared with the
C=O (156) of urea derivative 11. Most of the compounds proved to be unstable
towards the harsh conditions of MS and therefore the nominal mass was not
observed.
Biological evaluation. (i) In vitro antileishmanial activity and
cytotoxicity. The synthesized diselenides (1−22 and 40−48) were initially
tested against L. infantum axenic amastigotes according to a previously
described procedure [9]. All the analyses were carried out with a minimum of
three independent experiments. In these assays miltefosine and edelfosine
were used as reference drugs. EC50 values are collected in Table 1. In order to
assess their selectivity, these compounds were tested against leukemia cells
30