Editor's Pick Chemistry; Biosynthesis

Synthesis and Leishmanicidal Activity of Novel Urea, Thiourea, and Selenourea Derivatives of Diselenides

Marta Díaz, Héctor de Lucio, Esther Moreno, Socorro Espuelas, Carlos Aydillo, Antonio Jiménez-Ruiz, Miguel Ángel Toro, Killian Jesús Gutiérrez, Victor Martínez-Merino, Alfonso Cornejo, Juan Antonio Palop, Carmen Sanmartín, Daniel Plano
DOI: 10.1128/AAC.02200-18

ABSTRACT

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 μM). In addition, the derivatives 9, 11, 42, and 47, with EC50 between 1.1 and 1.95 μM, 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.

INTRODUCTION

Leishmaniasis comprises a group of mammalian diseases caused by diphasic protozoans of the genus Leishmania. 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. present 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 available front-line agents were developed 50 years ago and include chemotherapeutic drugs, such as injectable pentavalent antimonials, sodium stibogluconate, and meglumine antimoniate. Second-line treatment relies on 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 antiparasite activities (6). Similarly, increased selenium concentration in plasma has been proposed as a new defensive strategy against Leishmania infection (7). In recent years, our research group and others have been engaged in the design, synthesis, and 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 potent activity with higher selectivity than the reference drugs miltefosine and edelfosine. Additionally, leishmanicidal activity in infected macrophages (THP-1 cells) was comparable to that of 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 of the diselenide group within the framework of molecular symmetry that, in our opinion, is a key factor for leishmanicidal activity. Here, we designed several modifications on the side chain of the diselenide core in order to develop compounds with improved leishmanicidal activity and absorption, distribution, metabolism, excretion, and toxicity (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, and (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 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 aminoalkyl chains previously reported in the literature (23, 24). Figure 1 shows the general structure of the newly designed compounds.

FIG 1
FIG 1

Design and general structure for the proposed compounds.

Based on previous studies, here we present the synthesis and leishmanicidal activity against the amastigote form of L. infantum of thirty-one new derivatives (Fig. 1). The cytotoxicity of these newly synthesized molecules was also assessed on a 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.

RESULTS

Chemistry.The synthesis of the compounds described here was carried out according to Fig. 2 to 4. 4,4′-Diaminodiphenyldiselenide (Fig. 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 to 22 were synthesized according to Fig. 2. Diselenide and commercially available isocyanate or isothiocyanate were mixed in dioxane at a molar ratio of 1:2 at room temperature for 24 to 120 h. 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%.

FIG 2
FIG 2

General procedure of synthesis for compounds 1 to 22. Reagents and conditions: (i) DMSO, 15 min, room temperature; (ii) NaBH4, absolute ethanol, 2 h, room temperature, N2; (iii) dioxane (dry), 24 to 120 h, room temperature, dark, N2.

To obtain the planned selenoureas, the synthesis of the corresponding isoselenocyanates (compounds 31 to 39), which were prepared in two steps, was necessary (Fig. 3). The first step involved formylation of amines to yield formamides 23 to 30, followed by the treatment with phosgene (31 to 34) (25) or triphosgene (35 to 39) (26) and selenium powder in the presence of triethylamine under reflux. Compounds were purified by silica gel column chromatography using n-hexane–ethyl acetate as the eluent. The infrared (IR) spectra of the isoselenocyanates are quite informative about the presence of the isoselenocyanate functional group (–NCSe). The stretching frequency was observed at 2,115 to 2,224 cm−1.

FIG 3
FIG 3

General procedure of synthesis for compounds 23 to 39. Reagents and conditions: (i) HCOOC2H5, 12 h, reflux; (ii) HCOOH, Zn (10%), 12 h, 70°C; (iii) HCOOH, PEG-400, room temperature; (iv) HCO2NH4/CH3CN, 8 to 15 h, reflux; (v) triethylamine, phosgene-toluene, 2.5 h, reflux, Se, 12 h, reflux; (vi) triethylamine, triphosgene-DCM, 30 min, 0°C, Se, 24 h, reflux.

Formamides 23 to 30 were prepared through different methods depending on the type of primary amine (Fig. 3). Ethyl formate was used for compounds 23, 28, and 29 and formic acid in the presence of zinc dust (27) for derivatives 25, 26, and 30 or in the presence of polyethylene glycol 400 (PEG 400) for compound 27 (28). Derivative 24 was prepared with ammonium formate in acetonitrile (29). After isolation of the product, formamides were afforded in moderate to good overall yields of 16% to 92%.

Reaction of isoselenocyanates with 4,4′-diaminodiphenyldiselenide in a molar ratio 2:1 under nitrogen atmosphere, in dried dioxane and in darkness, generated selenoureas 40 to 48 (Fig. 4). However, isolation of selenoureas from the crude reaction mixture was highly tedious, and contaminations from different impurities remained with the desired derivatives, diminishing final yields. Some of them (compounds 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, etc.), generating the target compounds for derivatives 45 to 48 exclusively. The optimal purification method for compounds 40 to 44 was the formation of the corresponding salts by reaction with hydrochloric acid in ethyl ether.

FIG 4
FIG 4

General procedure of synthesis for compounds 40 to 48. Reagents and conditions were dioxane (dry), 24 to 120 h, r.t, dark, N2 (i).

The structures and purity of final compounds as well as all intermediates were confirmed by spectroscopic data (IR, 1H nuclear magnetic resonance [NMR], and 13C NMR), mass spectrometry (MS), and elemental analyses.

IR spectra of urea, thiourea, and selenourea compounds revealed characteristic strong-intensity bands between 3,427 and 3,120 cm−1 as a broad signal due to the presence of hydrogen bonding for the introduction of four N–H groups. Just above 3,000 cm−1, an Ar−H stretch was evident, and carbonyl group for ureas appeared as an intense band at about 1,644 cm−1. IR spectra of selenourea compounds revealed selenoyl group bands at lower values, ranging from 1,542 to 1,655 cm−1.

In 1H NMR spectra, the characteristic singlets for N–H protons located between C=X and phenyl moieties are more shielded and appear downfield, shifted as a 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 the case of ureas 8 to 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 to 183 ppm, whereas carbonyl carbon appears at 152 to 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 to C=O (156) of urea derivative 11. Most of the compounds proved to be unstable under 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 to 22 and 40 to 48) were initially tested against L. infantum axenic amastigotes according to a previously described procedure (9). All of the analyses were carried out with a minimum of three independent experiments. In these assays, miltefosine and edelfosine were used as reference drugs. Fifty percent effective concentration (EC50) values are collected in Table 1. In order to assess their selectivity, these compounds were tested against leukemia cells derived from monocytes (THP-1). EC50 values obtained are summarized in Table 1. The selectivity index (SI) was defined as the ratio of the EC50 values of compounds against THP-1 cells relative to those obtained against L. infantum axenic amastigotes.

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TABLE 1

EC50 values for the compounds on amastigotes and cytotoxic activity in THP-1 cell linesEmbedded Image

The newly synthesized compounds displayed high activity, with thirteen of them (5, 7, 9, 10, 11, 20, 22, 40, 41, 42, 44, 47, and 48) showing EC50 values lower than that of miltefosine (EC50 of 2.84 μM) and one of them (40) being more effective than the standard drug edelfosine (EC50 of 0.82 μM). In light of the results, the following structural considerations could be made. Regarding derivatization of the amine group and, as a general trend, the ureas 1 to 11 and selenoureas 40 to 48, considered as a whole, they have better leishmanicidal activity than the corresponding thiourea analogues (compound 1 [EC50 of 3.1 μM] and compound 40 [EC50 of 0.74 μM] versus compound 12 [EC50 of 11.23 μM] or compound 10 [EC50 of 2.03 μM] and compound 47 [EC50 of 1.95 μM] versus compound 21 [EC50 of 5.69 μM]). Regarding the relevance of the presence of additional selenium atoms, comparison of compounds 43 and 46 with analogues 5 and 9, where the selenium was replaced by oxygen, revealed higher activity in the oxygen-containing molecules, particularly in the case of the urea analogue. This fact revealed that the introduction of two additional atoms of selenium is not crucial for the activity.

Inspection of the data in Table 1 shows that within thiourea compounds, introduction of electron-withdrawing substituents in the para position decreases activity (compound 16, 4-CN, EC50 of 12.09 μM; compound 13, 4-NO2, EC50 of 17.7 μM). The elongation effect of the methylene group as a spacer between the aromatic ring and the functional derivatization in compounds 1, 12, and 40 (n = 0) and 7, 18, and 45 (n = 1) was also evaluated. Thus, this spacer causes a drop in the leishmancidal activity in selenoureas, while in ureas and thioreas it is responsible for a significant increase. With regard to the introduction of alkyl side chains, this modification confers a marked leishmanicidal increase in the three series of compounds (9, 10, 11, 20, 21, 22, 46, 47, and 48), with seven of them being more active than miltefosine. This phenomenon indicates that the activity correlates with an increase in the lipophilicity of the compounds. Lipophilic compounds are more permeable to cellular membranes, which could justify this higher in vitro activity. In addition, cyclization of the aliphatic chain improved the activity for thioureas (compound 21, EC50 of 5.69 μM; corresponding cyclic 22, EC50 of 2.71 μM).

In terms of selectivity, compounds 8, 9, 10, and 11 for ureas, 15, 17 to 20, and 22 for thioureas, and 41, 42, 45, and 47 for selenoureas show SI values in the range of 6.1 to 22.76, comparable to or better than those of reference drugs. These compounds also displayed the best inhibitory activity in the cultured amastigote model for each series. The most selective was N′, N′′′-(diselanediylbenzene-4,1-diyl)bis[1-(n-butyl)urea] (9), with an SI of >22.7, followed by derivatives 11 (SI of >15.2), 47 (SI of >12.82), and 42 (SI of >12.36). In particular, compound 9 was found to be 3.8 and 3.2 times more selective than edelfosine (SI of >22.7 versus SI of 6) and miltefosine (SI of >22.7 versus SI of 7), respectively. These results confirm a low toxicity for these diselenide compounds.

(ii) Leishmanicidal activity in infected macrophages. After the first screening and considering their activity and selectivity, four derivatives (9, 11, 42, and 47) were selected and further tested for their leishmanicidal activity on infected THP-1 macrophages. Again, edelfosine was used as a comparative reference. The 50% effective dose (ED50) for each compound was calculated and summarized in Table 2. These compounds reduced the parasite load of the cells, exhibiting ED50 values of 21.5, 3.4, 14.4, and >25 μM, respectively. Among them, compound 11, with an ED50 of 3.4 μM, presented an effectiveness similar to that of the reference drug.

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TABLE 2

ED50 values for the compounds in intracellular amastigotes

(iii) Inhibition of L. infantum trypanothione reductase activity. Going one step further, we investigated whether the most active compounds could act as trypanothione reductase (TryR) inhibitors. Given the essential role of TryR in the antioxidant defences of trypanosomatids, this enzyme has become one of the main exploited targets in Leishmania spp. (30–32). Different inhibitors have been described in the literature, although so far none of them proceeded to the further step of drug development (33). With this purpose, hit compounds were screened at six different concentrations between 0.1 and 75 μM. Mepacrine, a well-known TryR inhibitor, was used as a positive control (34) and dimethyl sulfoxide (DMSO) as a vehicle. The EC50 values obtained are shown in Table 3.

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TABLE 3

EC50 values for the selected compounds against TryR inhibition

According to the results, compound 47 potently inhibits TryR, presenting an EC50 value of 3.77 μM. It is worth noting that this derivative was 4.5-fold more active than the positive control. This inhibitory effect is also accompanied by a good leishmanicidal activity in axenic amastigotes, which suggests that inhibition of TryR is involved in the mechanism of action of this molecule. Its low activity against intracellular amastigotes suggests that this compound does not enter the parasitophorous vacuole or, alternatively, the compound is altered inside it before entering the parasites.

Compound 11, which was demonstrated to be the most potent against infected macrophages, shows mild inhibitory activity toward TryR, which indicates that this enzyme is not its main target. The other two compounds, 9 and 42, evinced a mild leishmanicidal effect on infected macrophages and on TryR activity. In general, the inhibitory effect of these compounds over TryR is not strong enough to support the notion that TryR is their main target inside the cell. Consequently, additional studies are necessary to elucidate the mechanism of action of the compounds presented here.

DISCUSSION

The present report describes the synthesis of 31 new N-functionalized urea, thiourea, and selenourea derivatives from 4,4′-diaminodiphenyldiselenide along with their in vitro antileishmanial activity against amastigote forms of L. infantum. In order to explore their selectivity, THP-1 cells were used. Fifteen derivatives exhibited EC50 values of <3 μM, with thirteen of them showing higher activity than the reference drug miltefosine, in some cases by more than 3.8 times. Our results demonstrate that the incorporation of urea and selenourea into the central scaffold improves leishmanicidal activity, mainly with aliphatic chains.

Four compounds (9, 11, 42, and 47) showing high activity and selectivity were tested for their activity in infected macrophages and for their ability to inhibit trypanothione reductase, a potential therapeutic target for the treatment of leishmaniasis. Compound 11 showed antiparasitic activity comparable to that of edelfosine. On the other hand, compound 47 showed activity against the targeted enzyme, while the rest of the derivatives do not follow this apparent trend, since they are mild inhibitors of TryR. These results indicate that different mechanisms must be involved in the leishmanicidal activity exerted by these hit compounds. A graphical summary of the conclusions drawn from this work is depicted in Fig. 5.

FIG 5
FIG 5

Schematic illustration of conclusions.

Our results provide a basis for further scaffold optimization and structure-based drug design aimed toward the identification and develop of more active, safe, and cost-effective antileishmanial agents.

MATERIALS AND METHODS

Chemistry.Melting points were determined with a Mettler FP82 + FP80 apparatus (Greifense, Switzerland) and are not corrected. The 1H NMR and 13C NMR spectra were recorded on a Bruker 400 Ultrashield and Bruker Avance Neo spectrometers (Rheinstetten, Germany) using tetramethylsilane 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 an MS direct insertion probe, system MSD/DS 5973 N (G2577A), from Agilent. Elemental microanalyses were carried out on vacuum-dried samples using a LECO CHN-900 elemental analyzer. Silica gel 60 (0.040 to 0.063 mm; 1.09385.2500; Merck KGaA, Darmstadt, Germany) was used for column chromatography, and an Alugram SIL G/UV254 (layer, 0.2 mm) (Macherey-Nagel GmbH & Co., Düren, Germany) was used for thin-layer chromatography. Chemicals were purchased from E. Merck (Darmstadt, Germany), Scharlab (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 Pharmaceuticalaa, Geel, Belgium), and Lancaster (Bischheim-Strasbourg, France).

The synthesis of 4,4′-diaminodiphenyldiselenide compound has been previously described by Plano et al. (12).

General procedure for the synthesis of ureas 1 to 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).

(i) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis(1-phenylurea) (1).After 24 h, phenyl isocyanate gave compound 1 as a yellow powder. Yield: 58%; mp 277 to 278°C; IR ʋmax (KBr): 3,294 (N−H), 1,637 (C=O) cm−1; 1H 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 to 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), 57 (43); analysis calculated for C26H22N4O2Se2 (%): C, 53.8; H, 3.8; N, 9.6. Found: C, 54.1; H, 4.1; N, 9.1.

(ii) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-nitrophenyl)urea] (2).After 72 h, 4-nitrophenyl isocyanate gave compound 2 as a yellow powder. Yield: 66%; mp 245 − 247°C; IR ʋmax (KBr): 3,363 (N−H), 1,614 (C=O) cm−1; 1H 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); analysis calculated for C26H20N6O6Se2 (%): C, 46.6; H: 3.0; N, 12.5. Found: C, 46.5; H: 3.1; N, 12.6.

(iii) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-methylphenyl)urea] (3).After 120 h, 4-methylphenyl isocyanate gave compound 3 as a yellow powder. Yield: 28%; mp 283 to 284°C; IR ʋmax (KBr): 3,315 (N−H), 1,643 (C=O) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 2.24 (s, 6×H, 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); analysis calculated for C28H26N4O2Se2·1/2 H2O (%): C, 54.4; H: 4.2; N, 9.0. Found: C, 54.6; H: 4.3; N, 8.8.

(iv) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-chlorophenyl)urea] (4).After 48 h, 4-chlorophenyl isocyanate gave compound 4 as a yellow powder. Yield: 59%; mp > 300°C; IR ʋmax (KBr): 3,289 (N−H), 1,635 (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 to 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); analysis calculated for C26H20Cl2N4O2Se2 (%): C, 48.1; H: 3.1; N, 8.6. Found: C, 48.0; H: 3.1; N, 8.4.

(v) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-cyanophenyl)urea] (5).After 96 h, 4-cyanophenyl isocyanate gave compound 5 as a yellow powder. Yield: 70%; mp 185 to 186°C; IR ʋmax (KBr): 3,367 (N−H), 2,221 (CN), 1,689 (C=O) cm−1; 1H 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); analysis calculated for C28H20N6O2Se2 (%): C, 53.3; H: 3.2; N, 13.3. Found: C, 53.1; H: 3.5; N, 13.2.

(vi) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-methoxyphenyl)urea] (6).After 72 h, 4-methoxyphenyl isocyanate gave compound 6 as a yellow powder. Yield: 65%; mp 274 to 275°C; IR ʋmax (KBr): 3,288 (N−H), 1,644 (C=O) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 3.74 (s, 6×H, 2OCH3), 6.56 (d, 2H, J3-2 = 7.5 Hz, 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); analysis calculated for C28H26N4O4Se2 (%): C, 52.5; H: 4.0; N, 8.7. Found: C, 52.3; H: 3.9; N, 8.5.

(vii) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis(1-benzylurea) (7).After 96 h, benzyl isocyanate gave compound 7 as a yellow powder. Yield: 43%; mp 213 to 215°C; IR ʋmax (KBr): 3,335 (N−H), 1,647 (C=O) cm−1; 1H 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 to 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); analysis calculated for C28H26N4O2Se2·1/2 H2O (%): C, 54.5; H, 4.4; N, 9.1. Found: C, 54.6; H, 4.5; N, 9.3.

(viii) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-methoxybenzyl)urea] (8).After 48 h, 4-methoxybenzyl isocyanate gave compound 8 as a yellow powder. Yield: 31%; mp 222 to 224°C; IR ʋmax (KBr): 3,305 (N−H), 1,630 (C=O) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 3.73 (s, 6×H, 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), 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); analysis calculated for C30H30N4O4Se2·1/2 H2O (%): C, 53.2; H, 4.4; N, 8.3. Found: C, 53.1; H, 4.4; N, 8.2.

(ix) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(n-butyl)urea] (9).After 72 h, butyl isocyanate gave compound 9 as a yellow powder. Yield: 42%; mp 250 to 251°C; IR ʋmax (KBr): 3,309 (N−H), 2,958 to 2,864 (C−H), 1,630 (C=O) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 0.89 (t, 6×H, JCH3-CH2 = 7.2 Hz, 2CH3), 1.25 − 1.45 (m, 8H, 2(-CH2 −CH2 −CH3)), 2.96 to 3.17 (m, 4H, 2(-NH−CH2 ), 6.18 (t, 2H, JNH2-CH2 = 5.3 Hz, 2NH−CH2), 7.30 to 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); analysis calculated for C22H30N4O2Se2·H2O (%): C, 47.3; H, 5.7; N, 10.0. Found: C, 47.4; H, 5.5; N, 9.9.

(x) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(n-hexyl)urea] (10).After 72 h, hexyl isocyanate gave compound 10 as a yellow powder. Yield: 25%; mp 180 to 181°C; IR ʋmax (KBr): 3,313 (N−H), 2,956 to 2,856 (C−H), 1,627 (C=O) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 0.87 (bs, 6×H, 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), 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); analysis calculated for C26H38N4O2Se2·1/2 H2O (%): C, 51.6; H, 6.2; N, 9.2. Found: C, 51.6; H, 6.0; N, 9.1.

(xi) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-cyclohexylurea] (11).After 120 h, cyclohexyl isocyanate gave compound 11 as a yellow powder. Yield: 59%; mp 255 to 257°C; IR ʋmax (KBr): 3,306 (N−H), 2,927 to 2,850 (C−H), 1,645 (C=O) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 1.15 to 1.30 (m, 12H, B + B′, 2H3 + 2H4 + 2H5), 1.53 to 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); analysis calculated 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 to 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).

(i) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis(1-phenylthiourea) (12).After 144 h, phenyl isothiocyanate gave compound 12 as a yellow powder. Yield: 45%; mp 142 to 143°C; IR ʋmax (KBr): 3,193 (N−H), 1,588 (C=S) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 7.12 to 7.15 (m, 2H, B + B′, H4), 7.33 (t, 4H, J3-2 = J5-6 = 7.5 Hz, B + B′, H3 + H5), 7.46 to 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); analysis calculated for C26H22N4S2Se2 (%): C, 50.9; H, 3.6; N, 9.1. Found: C, 50.6; H, 3.8; N, 8.7.

(ii) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-nitrophenyl)thiourea] (13).After 48 h, 4-nitrophenyl isothiocyanate gave compound 13 as a yellow powder. Yield: 58%; mp 175 to 176°C; IR ʋmax (KBr): 3,345 (N−H), 1570 (C=S) cm−1; 1H 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); analysis calculated for C26H20N6O4S2Se2·H2O (%): C, 43.3; H, 2.8; N, 11.6. Found: C, 43.6; H, 2.9; N, 11.3.

(iii) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-methylphenyl)thiourea] (14).After 96 h, 4-methylphenyl isothiocyanate gave compound 14 as a yellow powder. Yield: 63%; mp 151 to 153°C; IR ʋmax (KBr): 3,203 (N−H), 1,583 (C=S) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 2.28 (s, 6×H, 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 (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); analysis calculated for C28H26N4S2Se2 (%): C, 52.5; H, 4.1; N, 8.7. Found: C, 52.1; H, 4.3; N, 8.4.

(iv) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-chlorophenyl)thiourea] (15).After 48 h, 4-chlorophenyl isothiocyanate gave compound 15 as a yellow powder. Yield: 68%; mp 170 to 171°C; IR ʋmax (KBr): 3,210 (N−H), 1,583 (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); analysis calculated for C26H20Cl2N4S2Se2 (%): C, 45.8; H, 2.9; N, 8.2. Found: C, 45.5; H, 3.0; N, 7.9.

(v) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-cyanophenyl)thiourea] (16).After 144 h, 4-cyanophenyl isothiocyanate gave compound 16 as a yellow powder. Yield: 44%; mp 123 to 124°C; IR ʋmax (KBr): 3,166 (N−H), 2,224 (CN), 1,584 (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 (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 (28), 80 (20); analysis calculated for C28H20N6S2Se2·1/2 H2O (%): C, 50.1; H, 3.0; N, 12.5. Found: C, 49.8; H, 3.3; N, 12.4.

(vi) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-methoxyphenyl)thiourea] (17).After 72 h, 4-methoxyphenyl isothiocyanate gave compound 17 as a yellow powder. Yield: 65%; mp 142 to 144°C; IR ʋmax (KBr): 1H NMR (400 MHz, DMSO-d6, δ): 3.75 (s, 6×H, 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); analysis calculated for C28H26N4O2S2Se2·1/2 H2O (%): C, 48.7; H, 4.1; N, 8.1. Found: C, 49.1, H 3.9; N, 7.8.

(vii) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis(1-benzylthiourea) (18).After 144 h, benzyl isothiocyanate gave 18 as a yellow powder. Yield: 71%; mp 146 to 147°C; IR ʋmax (KBr): 3,238 (N−H), 1,533 (C=S) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 4.77 (d, JCH2-NH = 5.3 Hz, 4H, 2CH2), 7.22 to 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′, 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); analysis calculated for C28H26N4S2Se2·1/2 H2O (%): C, 51.8; H, 4.2; N, 8.6. Found: C, 51.9; H, 4.6; N, 8.7.

(viii) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-methoxybenzyl)thiourea] (19).After 120 h, 4-methoxybenzyl isothiocyanate gave 19 as a yellow powder. Yield: 49%; mp 174 to 175°C; IR ʋmax (KBr): 3,220 (N−H), 1,583 (C=S) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 3.73 (s, 6×H, 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); analysis calculated for C30H30N4O2S2Se2·1/2 H2O (%): C, 50.7; H, 4.2; N, 7.9. Found: C, 50.4; H, 4.2; N, 7.7.

(ix) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(n-butyl)thiourea] (20).After 120 h, 4-methoxybenzyl isothiocyanate gave 20 as a yellow powder. Yield: 49%; mp 174 to 175°C; IR ʋmax (KBr): 3,220 (N−H), 1,583 (C=S) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 3.73 (s, 6×H, 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′, C3 + C5), 132.7 (A + A′, C2 + C6), 140.2 (A + A′, C4), 180.6 (C=S); MS (m/z % abundance): 428 (55), 214 (100), 172 (44), 136 (96), 121 (82), 106 (36); analysis calculated for C30H30N4O2S2Se2·1/2 H2O (%): C, 50.7; H, 4.2; N, 7.9. Found: C, 50.4; H, 4.2; N, 7.7.

(x) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(n-hexyl)thiourea] (21).After 120 h, hexyl isothiocyanate gave 21 as a yellow powder. Yield: 65%; mp 132 to 134°C; IR ʋmax (KBr): 3,223 (N−H), 2,925 to 2,854 (C−H), 1,540 (C=S) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 0.87 (bs, 6×H, 2CH3), 1.28 [bs, 12H, 2(-(CH2)2−(CH2 )3 −CH3)], 1.51 [bs, 4H, 2(-CH2−CH2 −(CH2)3−CH3)], 3.44 [bs, 4H, 2(-CH2 −(CH2)4−CH3)], 7.42 (d, 4H, J2-3 = J6-5 = 8.4 Hz, A + A′, H2 + H6), 7.55 (d, 4H, A + A′, H3 + H5), 7.85 (bs, 2H, 2NH−CH2), 9.55 (bs, 2H, 2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ): 14.4 (CH3), 22.5 (CH2), 26.6 (CH2), 28.8 (CH2), 31.5 (CH2), 44.3 (CH2), 123.7 (A + A′, C1), 132.7 (A + A′, C2 + C3 + C5 + C6), 140.2 (A + A′, C4), 180.6 (C=S); MS (m/z % abundance): 428 (14), 386 (6), 214 (32), 172 (45), 135 (73), 115 (92), 72 (47), 57 (56), 43 (100); analysis calculated for C26H38N4S2Se2·1/2 H2O (%): C, 49.0; H, 6.0; N, 8.8. Found: C, 48.9; H, 6.0; N, 8.8.

(xi) N′, N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-cyclohexylthiourea] (22).After 96 h, cyclohexylisothiocyanate gave 22 as a yellow powder. Yield: 62%; mp 143 to 144°C; IR ʋmax (KBr): 3,321 (N−H), 2,926 to 2,851 (C−H), 1,587 (C=S) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 1.10 to 1.35 (m, 12H, B + B′, 2H3 + 2H4 + 2H5)), 1.56 (bs, 2H, B + B′, 2H1), 1.62 (bs, 4H, B + B′, 2H2) 1.80 (bs, 4H, B + B′, 2H6), 7.47 (d, 4H, J3-2 = J5-6 = 8.6 Hz, A + A′, H3 + H5), 7.54 (d, 4H, A + A′, H3 + H5), 7.81 (d, 2H, JNH-CH = 8.1 Hz, 2NH−CH), 9.49 (s, 2H, 2NH−C6H4); 13C NMR (100 MHz, DMSO-d6, δ) 25 (B + B′, C3 + C5), 26 (B + B′, C4), 32 (B + B′, 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); analysis calculated 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 to 30.(i) N-Phenylformamide (23). To a stirred solution of aniline (9.2 mmol), we added dropwise ethyl formate (9.6 mmol). The reaction mixture was stirred at 150°C 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): 3,364 (N−H), 1,634 (C=O) cm−1.

(ii) 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× with 15 ml each time). 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): 3,117 (N−H), 1,637 (C=O) cm−1.

(iii) N-(4-Chlorophenyl)formamide (25). To a mixture of 4-chloroaniline (10 mmol), formic acid (30 mmol) and zinc dust pretreated with HCl (1 mmol) were added and stirred at 70°C for 8 h to 12 h. The mixture was diluted with CH2Cl2 (50 ml) and filtered through celite. The filtrate then was washed with saturated NaHCO3 (4× with 30 ml each time) and brine (2× with 20 ml each time) 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): 3,258 (N−H), 1,670 (C=O) cm−1.

(iv) N-(4-Cyanophenyl)formamide (26). To a mixture of 4-aminobenzonitrile (10 mmol), formic acid (30 mmol) and zinc dust pretreated with HCl (1 mmol) were added and stirred at 70°C for 8 h to 12 h. The mixture was diluted with CH2Cl2 (50 ml) and filtered through celite. The filtrate then was washed with saturated NaHCO3 (3× with 30 ml each time) and brine (3× with 30 ml each time) 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): 3,357 (N−H), 2,216 (CN), 1,637 (C=O) cm−1.

(v) 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× with 15 ml each time). The organic layer then was dried over anhydrous Na2SO4 and concentrated. The residue was subjected to column chromatography ethyl (70:30 acetate-hexane) to obtain the pure compound 27 as a white powder. Yield: 68.6%; IR ʋmax (KBr): 3,245 (N−H), 1,656 (C=O) cm−1.

(vi) N-Butylformamide (28). To a stirred solution of butan-1-amine (25 mmol), ethyl formate (20.16 mmol) was added dropwise. The reaction mixture was stirred at 150°C 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): 3,291 (N−H), 2,960 to 2,869 (C−H), 1,665 (C=O) cm−1.

(vii) N-Hexylformamide (29). To a stirred solution of hexan-1-amine (15 mmol), ethyl formate (12.10 mmol) was added dropwise. The reaction mixture was stirred at 150°C 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 29 as a white powder. Yield: 92%; IR ʋmax (KBr): 3,361 (N−H), 2,978 to 2,868 (C−H), 1,630 (C=O) cm−1.

(viii) N-Ciclohexylformamide (30). To a mixture of cyclohexylamine (25 mmol), formic acid (75 mmol) and zinc dust pretreated with HCl (5 mmol) were added and stirred at 70°C for 8 h to 12 h. The mixture was diluted with CH2Cl2 (50 ml) and filtered through celite. The filtrate then was washed with saturated NaHCO3 (4× with 25 ml each time) and brine (2× with 25 ml each time) and was dried over anhydrous Na2SO4. After filtration and evaporation of the solvent, compound 30 was acquired as a white powder. Yield: 16%; IR ʋmax (KBr): 3,412 (N−H), 2,933 to 2,858 (C−H), 1,661 (C=O) cm−1.

General procedure for the synthesis of isoselenocyanates 31 to 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. Black selenium powder (12.58 mmol) then was added, and the resulting mixture was refluxed for 24 h in darkness. After being 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.

(i) Phenylisoselenocyanate (31). N-phenylformamide compound 23 gave compound 31 as a brown syrup. Yield: 2.66%; IR ʋmax (KBr): 2,978 to 2,873 (C−H), 2,114 (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).

(ii) 4-Methylphenylisoselenocyanate (32). N-(4-Methylphenyl)formamide compound 24 gave compound 32 as a brown syrup. Yield: 7.48%; IR ʋmax (KBr): 2,921 to 2,866 (C−H), 2,153 (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).

(iii) 4-Methoxyphenylisoselenocyanate (33). N-(4-Methoxyphenyl)formamide compound 27 gave compound 33 as an orange syrup. Yield: 6.79%; IR ʋmax (KBr): 2,944 to 2,740 (C−H), 2,121 (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).

(iv) Benzylisoselenocyanate (34). N-Benzylformamide gave compound 34 as a brown syrup. Yield: 11.75%; IR ʋmax (KBr): 2,958 to 2,871 (C−H), 2,143 (N=C=Se) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 5.15 (s, 2H, CH2), 7.41 to 7.54 (m, 5H, H2 + H3 + H4 + H5 + H6).

General procedure for the synthesis of isoselenocyanates 35 to 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 darkness. After being filtered, solvents were removed under vacuum and the residue was washed with dichloromethane (30 ml). Column chromatography using ethyl acetate-hexane (70:30) as the eluent afforded isoselenocyanate.

(i) 4-Chlorophenyl isoselenocyanate (35). N-(4-Chlorophenyl)formamide compound 25 gave compound 35 as a brown syrup. Yield: 55.76%; IR ʋmax (KBr): 2,924 to 2,854 (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).

(ii) 4-Cyanophenyl isoselenocyanate (36). N-(4-Cyanophenyl)formamide compound 26 gave compound 36 as a brown syrup. Yield: 85%; IR ʋmax (KBr): 2,927 to 2,856 (C−H), 2,225 (CN), 2,146 (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).

(iii) Butyl isoselenocyanate (37). N-Butylformamide compound 28 gave compound 37 as a dark syrup. Yield: 43.5%; IR ʋmax (KBr): 2,923 to 2,876 (C−H), 2,144 (N=C=Se) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 0.86 to 1.11 (m, 3H, CH3), 1.21 to 1.32 (m, 4H, CH2−CH2−CH3), 1.35 (m, 2H, CH2-NCSe).

(iv) Hexyl isoselenocyanate (38). N-Hexylformamide compound 29 gave compound 38 as a brown syrup. Yield: 62%; IR ʋmax (KBr): 2,931 to 2,861 (C−H), 2144 (N=C=Se) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 0.85 to 0.91 (m, 3H, CH3), 1.05 (t, 2H, J=6.99 Hz, (CH2)2−(CH2)2−CH2−CH3), 1.18 to 1.24 (m, 4H, (CH2)2−(CH2)2−CH2−CH3), 1.28 to 1.35 (m, 2H,CH2−CH2− (CH2)3−CH3), 1.41 to 1.49 (m, 2H, CH2-NCSe).

(v) Cyclohexylisoselenocyanate (39). N-Ciclohexylformamide compound 30 gave compound 39 as a dark syrup. Yield: 92%; IR ʋmax (KBr): 2,933 to 2,883 (C−H), 2,137 (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 to 1.62 (m, 4H, H3 + H5), 1.85 to 1.90 (m, 4H, H2 + H6).

General procedure for the synthesis of selenoureas 40 to 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 mixture was kept in darkness for 145 h. To afford the desired selenourea, we used two different work-ups. For 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. For 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 to 48.

Optimal purification method for compounds 40 to 44 was the formation of the corresponding salts by reaction with hydrochloric acid in ethyl ether.

(i) N′,N′′′-(Diselanediyldibenzene-4,1-diyl)bis(1-phenylselenourea) (40). From phenyl isoselenocyanate compound 31, the salt formation with hydrochloric ether gave compound 40 as a yellow powder. Yield: 2.6%; mp 189 to 191°C; IR ʋmax (KBr): 3,427 (N−H), 1,582 (C=Se) cm−1; 1H 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); analysis calculated for C26H22N4Se4·3HCl (%): C, 37.4; H, 3.2; N, 6.7. Found: C, 37.3; H, 3.0; N, 6.7.

(ii) N′,N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-methylphenyl)selenourea] (41). From 4-methylphenyl isoselenocyanate compound 32, the salt formation with hydrochloric ether gave compound 41 as a yellow powder. Yield: 7.5%; mp 212 to 213°C; IR ʋmax (KBr): 3,161 (N−H), 1,577 (C=Se) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 2.73 (s, 6×H, 2CH3), 7.30 (d, 4H, J2-3 = J6-5 = 8.8 Hz, 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); analysis calculated for C28H26N4Se4.3HCl (%): C, 39.8; H, 3.4; N, 6.6. Found: C, 39.5; H, 3.2; N, 6.6.

(iii) N′,N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-chlorophenyl)selenourea] (42). From 4-chlorophenyl isoselenocyanate compound 35, the salt formation with hydrochloric ether gave compound 42 as a yellow powder. Yield: 4.75%; IR ʋmax (KBr): 3,367 (N−H), 1,625 (C=Se) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 7.06 to 7.11 (m, 4H, A + A′, H3 + H5), 7.20 to 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); analysis calculated for C26H20Cl2N4Se4.4HCl (%): C, 33.9; H, 2.6; N, 6.1. Found: C, 34.1; H, 2.2; N, 5.7.

(iv) N′,N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-cyanophenyl)selenourea] (43). From 4-cyanophenyl isoselenocyanate compound 36, the salt formation with hydrochloric ether gave compound 43 as a yellow powder. Yield: 7.3%; mp 165 to 167°C; IR ʋmax (KBr): 3,077 (N−H), 2,223 (CN), 1,607 (C=Se) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 6.94 to 7.06 (m, 4H, A + A′, H3 + H5), 7.12-7.33 (m, 4H, B + B′, H2 + H6), 7.35 to 7.53 (m, 4H, A + A′, H2 + H6), 7.56 to 7.74 (m, 4H, B + B′, 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); analysis calculated for C28H20N6Se4·4HCl (%): C, 44.4; H, 2.6; N, 11.1. Found: C, 44.5; H, 2.6; N, 11.3.

(v) N′,N′′′-(Diselanediyldibenzene-4,1-diyl)bis[1-(4-methoxyphenyl)selenourea] (44). From 4-methoxyphenyl isoselenocyanate compound 33, the salt formation with hydrochloric ether gave compound 44 as an orange powder. Yield: 8.5%; mp 201 to 202°C; IR ʋmax (KBr): 3,310 (N−H), 1,553 (C=Se) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 3.74 (s, 6×H, 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); analysis calculated for C28H26N4O2Se4·2HCl (%): C, 33.9; H, 2.6; N, 6.1. Found: C, 34.1; H, 2.2; N, 5.7.

(vi) 1,1´-(4,4´-Diselanediylbis(4,1-phenylene))bis(3-benzylselenourea) (45). From benzyl isoselenocyanate compound 34 we got compound 45 as a yellow powder. Yield: 11.75%; mp 145 to 146°C; IR ʋmax (KBr): 3,120 (N−H), 1,570 (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′, 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); analysis calculated for C28H26N4Se4 (%): C, 45.7; H, 3.5; N, 7.6. Found: C, 45.6; H, 3.6; N, 7.4.

(vii) 1,1´-(4,4´-Diselanediylbis(4,1-phenylene))bis(3-butylselenourea) (46). From butyl isoselenocyanate compound 37 we got compound 46 as a yellow powder. Yield: 15.7%; mp 114 to 116°C; IR ʋmax (KBr): 3,257 (N−H), 2,957 to 2,865 (C−H), 1,620 (C=Se) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 0.90 (t, 6×H, JCH3-CH2 = 6.8 Hz, 2CH3), 1.30 to 1.34 (m, 6×H, B,-CH2 -CH2 −CH2 −CH3), 1.53 to 1.56 (m, 6×H, 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); analysis calculated for C22H30N4Se4·H2O (%): C, 38.6; H, 4.7; N, 8.2. Found: C, 38.3; H, 4.3; N, 8.0.

(viii) 1,1´-(4,4´-Diselanediylbis(4,1-phenylene))bis(3-hexylselenourea) (47). From hexyl isoselenocyanate compound 38, we got compound 47 as a yellow powder. Yield: 12.2%; mp 115 to 117°C; IR ʋmax (KBr): 3,195 (N−H), 2,923 to 2,854 (C−H), 1,542 (C=Se) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 0.88 (bs, 6×H, 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); analysis calculated for C26H38N4Se4·H2O (%): C, 42.2; H, 5.4; N, 7.6. Found: C, 42.1; H, 5.1; N, 7.5.

(ix) 1,1´-(4,4´-Diselanediylbis(4,1-phenylene))bis(3-ciclohexylselenourea) (48). From cyclohexylisoselenocyanate compound 39, we got compound 48 as a yellow powder. Yield: 21%; mp 175 to 180°C; IR ʋmax (KBr): 3,398 (N−H), 2,968 to 2,931 (C−H), 1,655 (C=Se) cm−1; 1H NMR (400 MHz, DMSO-d6, δ): 1.09 to 1.33 (m, 12H, B + B′, 2H3 + 2H4 + 2H5); 1.59 to 1.61 (m, 2H, B + B′, H1), 1.62 to 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); analysis calculated for C26H34N4Se4·H2O (%): C, 42.4; H, 4.9; N, 7.6. Found: C, 42.3; H, 4.7; N, 7.7.

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 fetal calf serum (FCS), 1 g/liter β-alanine, 100 mg/liter l-asparagine, 200 mg/liter sacarose, 50 mg/liter sodium pyruvate, 320 mg/liter malic acid, 40 mg/liter fumaric acid, 70 mg/liter succinic acid, 200 mg/liter α-ketoglutaric acid, 300 mg/liter citric acid, 1.1 g/liter sodium bicarbonate, 5 g/liter morpholineethanesulfonic acid, 0.4 mg/liter hemin, and 10 mg/liter gentamicin, pH 5.4, at 37°C. THP-1 cells were kindly provided by T. 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 1 mM sodium pyruvate, pH 7.2, at 37°C and 5% CO2.

(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°C or 1 × 106 parasites/ml at 37°C for 24 h. 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°C and 5% CO2 for 24 h. The percentage of living cells was evaluated by flow cytometry by the propidium iodide (PI) exclusion method (35).

(iii) Leishmania infection assay. THP-1 cells were seeded at 120,000 cells/ml in 24-multidish plates (Nunc, Roskilde, Denmark) and differentiated to macrophages for 24 h in 1 ml 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 1 ml of THP-1 medium were added to each well. Four h later all medium with noninfecting amastigotes was removed, washed 3 times with 1× phosphate-buffered saline (1× PBS), and replaced with new THP-1 medium and corresponding treatment. After 48 h of treatment, medium was removed; THP-1 cells were washed 3 times with 1× PBS and detached with TrypLE Express (Invitrogen, Leiden, The Netherlands) according to the manufacturer′s instructions. Infection was evaluated by flow cytometry.

(iv) Trypanothione reductase assay. Oxidoreductase activity was determined according to the method described by Toro et al. (36). Briefly, reactions were carried out at 26°C in 250 μl of 40 mM HEPES buffer, pH 8.0, containing 1 mM EDTA, 150 μM NADPH, 30 μM NADP+, 25 μM 5,5′-dithiobis-(2-nitrobenzic acid), 1 μM tripanothione, 0.02% glycerol, 1.5% DMSO, and 7 nM recombinant Li-TryR. Enzyme activity was monitored by the increase in absorbance at 412 nm for 1 h at 26°C in a VERSAmax microplate reader (Molecular Devices, CA, USA). All assays were conducted in triplicate in at least three independent experiments. Data were analyzed using a nonlineal regression model with Grafit6 software (Erithacus, Horley, Surrey, UK).

ACKNOWLEDGMENTS

We thank the Foundation for Applied Medical Investigation (FIMA), University of Navarra. We also acknowledge the Ministerio de Educación y Ciencia, Spain (grant SAF2015-64629-C2), and Comunidad de Madrid (BIPEDD-2-CM ref. S-2010/BMD-2457) for financial support. We also thank the Caixa Foundation, Roviralta, and Ubesol for supporting this research. This work, including the efforts of M.D., was funded by Foundation for Applied Medical Investigation (ISTUN-API-2011/02). This work, including the efforts of A.J.-R., was funded by Ministerio de Educación y Ciencia, Spain (grant SAF2015-64629-C2), and Comunidad de Madrid (BIPEDD-2-CM ref. S-2010/BMD-2457). We have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

FOOTNOTES

    • Received 18 October 2018.
    • Returned for modification 19 November 2018.
    • Accepted 13 February 2019.
    • Accepted manuscript posted online 19 February 2019.

  • Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02200-18.

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KEYWORDS

selenium
selenourea
thiourea
trypanothione reductase
urea

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