Reaction of bis[(2-chlorocarbonyl)phenyl] Diselenide with Phenols, Aminophenols, and Other Amines towards Diphenyl Diselenides with Antimicrobial and Antiviral Properties †

A reaction of bis[(2-chlorocarbonyl)phenyl] diselenide with various mono and bisnucleophiles such as aminophenols, phenols, and amines have been studied as a convenient general route to a series of new antimicrobial and antiviral diphenyl diselenides. The compounds, particularly bis[2-(hydroxyphenylcarbamoyl)]phenyl diselenides and reference benzisoselenazol-3(2H)-ones, exhibited high antimicrobial activity against Gram-positive bacterial species (Enterococcus spp., Staphylococcus spp.), and some compounds were also active against Gram-negative E. coli and fungi (Candida spp., A. niger). The majority of compounds demonstrated high activity against human herpes virus type 1 (HHV-1) and moderate activity against encephalomyocarditis virus (EMCV), while they were generally inactive against vesicular stomatitis virus (VSV).

Inspired by the encouraging findings in the area of antimicrobial and antiviral properties of organoselenium compounds and as a continuation of our interest in the chemistry and biology of this class of compounds, we aimed to develop a series of new diphenyl diselenides and investigate their activity against various microbial strains and viruses, along with selected cyclic analogues. It seemed interesting to focus our attention on a group of 2,2'-dicarbamoylphenyl diselenides 4 as the most reduced analogues of ebselen, with a phenyl ring containing hydroxy and/or methoxy moieties. However, the rationale behind studying these compounds was based not only on the fact that the structural modifications in the diphenyl diselenide scaffold seemed to be interesting from the medicinal chemistry perspective; we also aimed to investigated a synthetic route to reduced forms of ebselenols (2-(hydroxyphenyl)-1,2-benzisoselenazol-3(2H)-ones) and its derivatives via the reaction of bis[(2-chlorocarbonyl)phenyl] diselenide 3 with various mono and bisnucleophiles such as aminophenols, phenols, and amines.
Up to the present time, several methods have been applied for the preparation of bis(2-arylcarbamoyl)phenyl diselenides 4. One path is the reductive benzisoselenazolone ring opening of ebselenols and its derivatives [53], which can be obtained from 2-(chloroseleno)benzoyl chloride with moderate to excellent yields [54][55][56]. An alternative route to ebselenols requires metalation-selenenylation of appropriate benzanilides while protecting the hydroxyl group in the first step [57,58]. Although the reductive ring opening reaction has been successfully applied for the reduction of ebselen and its various alkyl analogues [59], in the case of ebselenols, yields were less satisfactory [53]. Only bis [2-(4-hydroxyphenylcarbamoyl)]phenyl diselenide was prepared in 46% yield while bis [2-(2-hydroxyphenylcarbamoyl)]phenyl diselenide and 2-(3-hydroxyphenylcarbamoyl)]phenyl diselenide were obtained in very low yields of 8.3% and 12%, respectively [53]. Another, general route to ebselenol-derived diselenides which we present in this study is the reaction of bis[(2-chlorocarbonyl)phenyl)] diselenide 3 with the corresponding anilines. In the past, this reaction was successfully applied to obtain various 2,2'-dicarbamoyldiphenyl diselenides [60]. However, to our best knowledge, there is only one example of a reaction with an electron-rich arene system bearing a hydroxyl group [26] but with no synthetic details. Thus, we aimed to investigate the behaviour of bis[(2-chlorocarbonyl)phenyl)] diselenide 3 in the reaction with N and O nucleophiles, such as aminophenols, phenols, and amines. The aminophenols were of particular interest as the ones containing two nucleophilic centers in the molecule, with both amine nitrogen and hydroxide oxygen potentially susceptible toward acylation. The reaction of various O and N nucleophiles with another selenium-containing chloride, 2-(chloroseleno)benzoyl chloride 9, showed the preference of a primary amino group toward selenenylation-acylation [55]. As chloride 9 contains both hard and soft electrophilic centers localized on the carbonyl carbon and selenium atoms, respectively, the results of the reaction with O-nucleophiles strongly depended on the nucleophile structure. However, in the absence of the soft electrophilic center, no side reactions should occur. Thus, we expected that the reaction of bis[(2-chlorocarbonyl)phenyl] diselenide 3 with aminophenols could be considered as a convenient alternative for low-efficient reductive ring opening of ebselenols [53] and the preparation from 2,2'-dicarboxydiphenyl diselenide using active ester coupling agents [23,61], where the protection of the hydroxyl group would be recommended.

Synthesis
Herein we present our investigation of synthetic route to diselenides 4-8 via a reaction of (bis[(2-chlorocarbonyl)phenyl] diselenide), hereafter called chloride 3, with N and O nucleophiles, such as a series of various aminophenols and their derivatives as well as other specific reactants such as morpholine, benzohydrazide, 2-hydroxyacetanilide, and antimycin A (Scheme 1, Table 1). The key chloride 3 was prepared in a three-step synthesis starting from anthranilic acid 1, which was diazotized and reacted with disodium diselenide in a methanolic alkaline medium to give, after acidification, 2,2'-diselenobisbenzoic acid 2 which was further reacted with thionyl chloride [59,60,62].
i -iii xi x R: mixture of C 3 H 7 , C 4 H 9 , C 5 H 11 , C 6 H 13 Scheme 1. Synthesis of bis(2-carbamoy)diphenyl diselenides 4a-p, 5 and 6, phenolic esters 7-8, and benzisoselenazol-3(2H)-ones 10a-g. In a first step, reaction of chloride 3 was tested with various free and protected aminophenols, such as monohydroxyanilines, monomethoxyanilines, and their derivatives containing additional substituents in the benzene ring, such as electron-donating methyl and methoxy groups and electron-withdrawing chloro and carboxymethyl groups. As in the case of 2-(chloroseleno)benzoyl chloride 9, which was intensively studied with various bisnucleophiles [55], the reaction occurred on the amine group of both protected (4k-p) and free phenols (4a-j). In the IR spectra, there were observed signals from primary amide bonds at ca. 3300-3500 cm −1 (N-H), carbonyl groups at ca. 1600-1650 cm −1 (C=O), amide groups at ca. 1500-1550 cm −1 (CONH), and in 1 H-NMR there were present protons of N-H group at ca. 9.6-10.5 ppm (see the Supplementary Materials for details). All compounds were prepared with very good to excellent yields, significantly higher than those achieved by the reductive ring opening method [53]. The simplest three isomeric aminophenols reacted with 73-92% yield towards 4a-c. Reaction with 2-aminophenols containing electron-withdrawing substituents (Cl and COOMe) at the vicinity carbon atom generally resulted in slightly lower yields (4g-i; 57-63%), when compared to those containing electron-donating ones (4d-f; 60-89%). As expected [35], methoxy derivatives 4j-p were also prepared with very good yields (72-99%). As a result of our studies, seven new open-chain analogues of ebselenols 4d-j and two new methoxy derivatives 4n and 4p were developed. Reaction of benzoyl chloride 3 with secondary amines such as morpholine also resulted in secondary amide bond formation to produce compound 5 with a very good yield of 85%. A similar compound containing piperazine moiety was prepared with a comparable yield [64].
Another highly nucleophilic benzohydrazide reacted quantitatively with chloride 3 on its terminal nitrogen atom to form bishydrazide 6. Encouraged by this result, we continued our studies with other N-nucleophiles, focusing on hydrazine, ethylene diamine, tri(hydroxymethyl)aminomethane, and isoniazyde (isonicotinic hydrazide). The resulting oligomeric products were, however, difficult to identify.
Further efforts were directed into the investigation of the behavior of aminophenol derivatives containing blocked amine groups such a 2-hydroxyacetanilide and natural antimycin A. In this case, reaction occurred on the free hydroxyl group only, resulting in phenolic ester derivatives 7 and 8, however, highly sterically hindered antimycin A required an extremely long reaction time. In the IR spectrum of compound 7, a characteristic phenolic ester bond was observed at 1760 cm −1 . Antimycin A is a mixture of at least four closely related compounds, containing alkyl or acyl substituents of different lengths or degrees of branching (usually C 3 -C 6 ) in the dilactone portion of the molecule [65]. Thus, the spectroscopic analysis is complicated and only diagnostic signals could be reported in 13 C-NMR spectra. Fortunately, in HRMS spectra, seven diagnostic molecular species (M + Na + ) were observed with R=C 5 H 11 being a predominant one, which was consistent with the results of the elemental analysis.
Generally, there is a noticeable tendency that the compounds tested have been more active against Gram-positive bacterial strains than against Gram-negative ones. These results are in line with our previous observations [33][34][35] and might indicate the possible mode of action of the whole group. Gram-positive bacteria are more susceptible to plasma membrane-binding agents, absorbing them directly into the plasma membrane, whereas a Gram-negative outer membrane may capture such agents before they penetrate through a cell wall and bind into the inner membrane, which usually results in subsequent cell permeabilization [66]. Using propidium iodide, no significant plasma membrane permeabilization was observed in S. aureus after treating with ebselen [67], however, membrane binding is not always sufficient for its permeabilization [68]. Membrane binding agents alter plasma membrane biophysics, which may be toxic itself.
The antibacterial activity towards Gram-positive species varied in a range of 1 to >128 µg/mL. Particularly interesting is the strong activity towards E. faecalis and E. hirae. These enterococci display highest vulnerability to most of the tested compounds and only compounds 4h-l, 5 and 7 (as well as 4m in the case of E. hirae) exhibit no activity in the tested range of concentrations. Enterococci seem to be significantly more susceptible towards organoselenium compounds than reference antibiotics, with the exception of 4o and 10d, displaying same MIC as ampicillin towards E. faecalis. This makes organoselenium compounds promising for the treatment of enterococci infections, which although being low virulent bacteria, often cause infections in immunocompromised patients [69]. In the case of staphylococci, it can be noticed that compounds 4a-c and 10a-b, containing hydroxyl substituent in the phenyl ring, were the most active against both S. aureus and S. epidermidis. Benzisoselenazol-3(2H)-ones 10a and 10b were slightly more active than their open-chain analogues 4a-c. Only two compounds, 4e and 10a, showed inhibitory effect against E. coli, similar to commonly known antibiotics. None of the compounds showed activity towards P. aeruginosa in the screening range. However, it is worth pointing out that this microorganism was also not susceptible toward the majority of reference antibiotics in the tested range.
Contrary to their cyclic analogues, diselenides appeared to be practically inactive towards both Candida spp., with the exception of compound 4e, which also displayed a high inhibitory effect against Gram-positive bacteria and moderate effects against Gram-negative E. coli ( Table 1). The probable lack of inhibitory effect of the majority of compounds tested might be due to the fact that the antifungal activity of diphenyl diselenides is dependent on the presence of functional groups [40]. However, diphenyl diselenides may still display high activity in reducing germ tube formation, one of the crucial virulent factors of C. albicans [38]. Azoles (ketoconazole and itraconazole), commonly used in antifungal therapy, also did not show activity under the test conditions. However, these two compounds are considered static drugs [70], therefore it is difficult to obtain MIC 90 values for them. Thus, it is possible that organoselenium compounds which lack inhibitory effects may still display synergistic effects. Benzisoselenazol-3(2H)-ones 10a, 10c, 10f-g, and 10e (in the case of C. albicans only) inhibited fungal growth in a range of 16-64 µg/mL (Table 2), comparable to nourseothricin. Amphotericin B exhibited a much higher activity than all the benzizsoelenazol-3(2H)-ones tested, however, this highly cytotoxic compound is considered as a drug of choice for the most severe fungal infections, when the patients are close to death [71]. The majority of diselenides have shown no activity against A. niger in the tested range. Only compound 4e showed weak activity against this filamentous fungus. Benzisoselenazol-3(2H)-ones 4c and 4f showed moderate activity towards A. niger, comparable to other previously tested cyclic analogues of ebselen [33,34].
The compound 5, prepared by reaction with a secondary amine, as well as phenolic ester 7 were inactive against all tested microorganisms.

Antiviral Activity
The antiviral activity of compounds 4, 5, 7, and 10 has been evaluated in vitro towards HHV-1 (human herpes virus type 1, Herpesviridae, enveloped virus), EMCV (encephalomyocarditis virus, Picornaviridae, non-enveloped virus), and VSV (vesicular stomatitis virus, Rhabdoviridae, enveloped virus) in the human cell line A549. The minimum inhibitory concentrations (MICs) for the abovementioned viruses along with cytotoxicity against the testing cell line are summarized in Table 4.
The compounds exhibited high antiviral activity against HHV-1 and moderate to high activity against EMCV, but in almost all cases were inactive towards VSV (Table 4). A similar tendency was observed in our previous studies [32][33][34][35]. The anti-HHV-1 activity of most diselenides was in a range of 2-40 µg/mL; only the one containing morpholine moiety showed lower activity at 200 µg/mL. The benzisoselenazol-3(2H)-ones also showed high activity against HHV-1, in a range of 2-4 µg/mL, comparable to ebselen. The most promising anti-HHV-1 agents would be compounds 4k, 4n, and 10f with higher chemotherapeutic indices (I), indicating that the concentration active against the virus was lower than that showing a cytotoxic effect. Up to the present time, there were only several organoselenium compounds identified with such high chemotherapeutic indices [33]. Generally, the compounds were less active against EMCV and only compounds 4e, 4o-p, and 10c showed activity below 10 µg/mL.

General Information
Melting points were determined on an Electrothermal IA 91100 digital melting-point apparatus using the standard open capillary method. 1 H-and 13 C-NMR spectra were recorded in CDCl 3 or DMSO-d 6 on a Bruker DRX 300 Spectrometer (Bruker, Rheinstetten, Germany) ( 1 H: 300.1 MHz, 13  Analytical thin-layer chromatography (TLC) was performed on PET foils precoated with silica gel (Merck silica gel, 60 F254) (Sigma-Aldrich, Saint Louis, MO, USA), and visualised using UV light (λ max = 254 nm), or by staining with iodine vapours. Selenium powder (100 mesh) (Sigma-Aldrich, Saint Louis, MO, USA) used for Na 2 Se 2 preparation had a purity of ≥99.5%. Acetonitrile (MeCN) (POCH, Gliwice, Poland) and methylene chloride (CH 2 Cl 2 ) (POCH, Gliwice, Poland) were distilled over P 2 O 5 , and CH 2 Cl 2 was stored over sodium hydride (NaH) pills. Triethylamine (Et 3 N) (POCH, Gliwice, Poland), distilled over NaOH, was stored over NaOH pellets. Methanol (MeOH) (POCH, Gliwice, Poland) was refluxed over magnesium shavings (Mg) in the presence of elemental iodine (I 2 ) and distilled over Mg(OMe) 2 was formed and stored over 3A molecular sieves (Sigma-Aldrich, Saint Louis, MO, USA). Diethyl ether (DEE) (POCH, Gliwice, Poland) was distilled over a CaH 2 and LiAlH 4 mixture from water batch at ca. 45 • C. Morpholine (POCH, Gliwice, Poland) was distilled over NaOH pellets (POCH, Gliwice, Poland) before use. Anhydrous sodium carbonate (Na 2 CO 3 ) (POCH, Gliwice, Poland) was ground in a mortar before use. Antimycin A natural product (Sigma-Aldrich, Saint Louis, MO, USA) was used as a mixture of co-crystalized n-alkylated compounds with R = C 3 -C 6 predominantly. 2-Hydroxy-5-carboxymethylaniline was prepared from 3-amino-4-hydroxybenzoic acid (Sigma-Aldrich, Saint Louis, MO, USA) by direct metoksylation in a hot methanol environment and in the presence of H 2 SO 4 (POCH, Gliwice, Poland), as mentioned below. Ebselen was prepared according to the procedure described earlier [59]. Other reagents and starting materials were directly used as obtained commercially. Purity of the products was confirmed by the comparison of their melting point with data given in literature and by spectroscopic methods. The new compounds were fully characterized.
2-Hydroxy-5-carboxymethylaniline. To 2-amino-4-hydroxybenzoic acid (1.53 g, 10 mmol) in dry MeOH (25 mL) was added H 2 SO 4 (1.0 mL, 1.8 g, 19 mmol) portionwise during stirring, and the solution formed was stirred in solvent reflux (+65 • C) for 20 h. The reaction mixture was cooled and poured into 0.5% NaOH (200 mL) and kept in an ice/water bath, NaHCO 3 (3.6 g, 44 mmol) was added and the mixture was washed with DEE (100 mL) followed by the addition of 3.5% HCl to a pH of 8.0, and was then extracted with degased DEE. The collected DEE solution was washed with water, dried over Na 2 SO 4 , and the solvent was distilled off from a water bath to obtain crude 2-hydroxy-5-carboxymethylaniline. 2,2'-Diselenobisbenzoic acid 2. The compound was prepared from anthranilic acid 1 using disodium diselenide with a modified literature procedure [59,60,62]. The anthranilic acid 1 (20.5 g, 0.15 mol) was dissolved in a solution of 37% aq. HCl (30 mL) in distilled water (90 mL) at ca. 75 • C. The solution was cooled in an ice-water bath (0-5 • C) to form anthranilic acid hydrochloride precipitate. To the resulting thick suspension, a cold solution of NaNO 2 (11.4 g, 0.165 mol) in distilled water (90 mL) at ca. −5 • C was added while stirring for 0.5 h, and the reaction was continued for an additional 15 min. The prepared cold solution of anthranilic acid diazonium chloride was added dropwise for 2.5 h to the freshly prepared methanolic solution of disodium diselenide (NaSeSeNa) at −10-7 • C during stirring with nitrogen evolution accompanied by the formation of red selenium . The reaction mixture was warmed to RT (room temperature) and stored overnight. (NaSeSeNa solution preparation: to the suspension of selenium element (11.2 g, 0.15 mol) in NaOH (18 g, 0.45 mol) solution with MeOH (300 mL) in a round-bottom flask equipped with an oil closure, hydrazine monohydrate (1.9 mL, 0.040 mol) was added and gently stirred at RT for 60 h.) The formed grey selenium was filtered, washed with water, and air-dried to obtain selenium powder (6.20 g, 0.083 mol) with 55% yield, which can be reused. The collected filtrates were acidified with 37% aq. HCl (25 mL), gently heated with mixing and left at RT overnight. The solid was filtered off, washed with hot water (ca. 1 L) (until no odor of salicylic acid in the filtrate was detected), and air-dried. The crude product was recrystallized from hot 1,4-dioxane (400 mL) by slow concentration under ca. 200 mmHg to give acid 2. Yield: (11.1 g, 37%); m.p.: 298-300 • C (300-303 • C [59]); as a pale brick powder (1,4-dioxane).
General synthetic procedure for benzisoselenazol-3(2H)-ones 10a-g. To a stirred solution of substituted aniline (13.0 mmol) in anhydrous CH 2 Cl 2 (5 mL) a solution of (2-chloroseleno)benzoyl chloride 9 (1.02 g, 4.0 mmol) in anhydrous CH 2 Cl 2 (25 mL) was added dropwise. The reaction was continued for 2-24 h. The progress of reaction was controlled by TLC (silica gel, eluent CHCl 3 :EtOAc, 4:1, v/v). After the reaction was finished, the solvent was evaporated, HCl (3.5%, w/w, 40 mL) was added, and the mixture was stirred at RT overnight, then filtered off and left to dry in the air. Crude products were purified by column chromatography on silica gel (70-230 mesh) eluted with CHCl 3 to obtain pure 10a-g.

2-(2-Hydroxyphenyl
The general procedure starting from 2-hydroxyaniline (0.44 g, 4.0 mmol) and anhydrous Et 3 N (1.01 g, 1.4 mL, 10 mmol) at RT was employed, with a 20-h reaction time with separation of the product from the crude reaction mixture by column chromatography using EtOAc as an eluent to obtain 10a. Yield: (0.46 g, 40% calculated on benzoyl  [57]. The 77 Se NMR, and HRMS are in agreement with literature data [26,57].  18 (s, 1H, OH). The 13 C-NMR is in agreement with literature data [55].
The general procedure starting from 2-methoxyaniline (1.60 g, 13 mmol) at RT was employed, with a 24-h reaction time followed by separation by column chromatography to obtain 10a. Yield: (0.86 g, 71% calculated on the benzoyl  Prepared according to a modified procedure [35]. The general procedure starting from 3-methoxyaniline (0.493 g, 4.0 mmol) and dry Et 3 N (1.02 g, 1.4 mL, 10 mmol) in anhydrous MeCN (40 mL) at RT with a 2-h reaction time was employed. Water was added portionwise and the solution was left in the refrigerator for crystallization. Then, the solid was filtered off, washed with water and MeCN, and dried in the air to obtain 10d. Yield: (0.79 g, 65%), m.p: 166-168 • C (165.5-167.5 • C [35]); as yellow prisms in (MeCN:H 2 O, 1:1, v/v). The 1 H-NMR and 13 C-NMR are in agreement with literature data [35].

Conclusions
The reaction of bis[(2-chlorocarbonyl)phenyl] diselenide 3 with various aminophenols and their derivatives is a convenient synthetic route to bis [2-(hydroxyphenylcarbamoyl)]phenyl diselenides 4 (reduced forms of ebselenols) and other compounds containing protected hydroxyl groups. This general approach allows the production of ebselenol-derived diselenides with higher yields than the alternative method based on the reductive benzisoselenazolone ring opening of ebselenols. The secondary amine such as morpholine and benzohydrazide also reacted on their nitrogen atoms to form bis [2-(N-morpholinecarbamoyl)]phenyl and bis [2-(N-benzamidecarbamoyl)]phenyl diselenides, respectively, while reaction with aminophenol derivatives containing a blocked amine group such a 2-hydroxyacetanilide and natural antimycin A led to corresponding phenolic esters of bis(2-carboxy)phenyl diselenide. As a result of our studies, 15 new structures have been developed, including 13 diselenides and two benzisoselenazol-3(2H)-ones.
The investigation of the antimicrobial and antiviral activities of bis [2-(hydroxyphenylcarbamoyl)] phenyl diselenides and their derivatives as well as cyclic analogues have revealed new biologically active structures. The compounds, particularly bis [2-(hydroxyphenylcarbamoyl)]phenyl diselenides and reference benzisoselenazol-3(2H)-ones, exhibited high antimicrobial activity against Gram-positive bacterial species (Enterococcus spp., Staphylococcus spp.), and some compounds were also active against Gram-negative E. coli and fungi (Candida spp., A. niger). The antiviral activity evaluation allowed the identification of substances with high anti-HHV-1 and moderate anti-EMCV activities. The chemotherapeutic indices for some of these compounds were significantly higher (concentration active against virus was lower than the one showing a cytotoxic effect) than for other organoselenium compounds previously reported in the literature.
As novel molecules with the ability to inhibit microbial and viral functions are in high demand, the results of our studies are encouraging for further exploration of organoselenium compounds as anti-infective agents.
Supplementary Materials: Supplementary materials are available online.