Towards New Catalytic Antioxidants : A Simple and Mild Synthesis of Selenenylsulfides

A new methodology for the synthesis of small molecules containing the S-Se bond is reported. Aryland alkyl-selenols react smoothly with N-thiophthalimides to afford the corresponding selenenylsulfides through a clean SN2 path occurring at the sulfur atom. The reaction proceeds under very mild conditions in DMF in absence of catalysts for most of the substrates. The scope of the reaction was found to be broad, allowing a wide series of selenols and N-thiophtalimides to be efficiently employed in this procedure. Owing to the instability of the S-Se bond, selenenylsulfides exhibited a remarkable tendency to disproportionate to the corresponding symmetric diselenides and disulfides. Preliminary evaluation of the catalytic antioxidant properties of novel selenenylsulfides showed their behaviour as GPx mimics.


Introduction
Antioxidants are compounds able to prevent or inhibit the oxidation of other molecules, and can be natural or synthetic [1,2].Natural systems include both low molecular weight derivatives, such as polyphenols, ascorbic acid, lipoic acid and vitamin A, and also several enzymes, namely glutathione peroxidase (GPx), thioredoxine reductase (TrxR), superoxide dismutase (SOD), and catalase.Elements like zinc, selenium, iron, and copper are responsible of the activity of certain enzymes.Also, synthetic antioxidants found an extensive use in medicinal and food chemistry.
The catalytic activity of enzymes and of synthetic antioxidants has been ascribed to the heteroatom present in the active site.Selenium derivatives are well known to behave as mimics of the selenoenzyme GPx [12][13][14].Besides this activity, the positive effect of selenium containing molecules against various diseases has been also attributed to other antioxidant mechanisms, such as ROS (reactive oxygen species) scavenging properties and metal coordination (iron and copper) to prevent DNA damage [15].Organoselenium compounds usually behave as more efficient systems with respect to sulfur analogues in cancer prevention or as radical scavengers.
On the basis of the catalytic cycle proposed for GPx, the selenol moiety of the enzyme (EnzSeH) is oxidized by the peroxide, forming a selenenic acid (EnzSeOH), which reacts with a thiol (typically glutathione, GSH) leading to a selenenylsulfide (EnzSeSG).The mixed selenosulfide reacts with a second equivalent of GSH, to reform the selenol, together with oxidized glutathione GSSG.The selenenylsulfide, containing the Se-S bridge, represents the key intermediate for the activity of the enzyme [7,[16][17][18][19].Taking into account the importance of this process, several synthetic approaches to obtain selenated small molecules as enzyme mimics have been developed.However, to the best of our knowledge, a limited number of methods are reported for the preparation of selenenylsulfides.These methods typically exploited the reactivity of diselenides in thiol-diselenide exchange or foresee the reaction of sulfenyl derivatives with thiols [16,17,[20][21][22].
It is well established that the Se-S bridge is unstable and undergoes disproportionation reaction to afford diselenides and disulfides.Some examples are reported on selenenylsulfides stabilized by sterically bulky groups or by intramolecular Se . . .Het (N, O) interactions [23].
Owing to the interest in these selenium containing compounds, the search for novel procedures to prepare small molecules containing the Se-S unit is of great interest.
Our long-dated interest in the chemistry of silyl chalcogenides allowed to disclose novel procedures to access a plethora of sulfur and selenium containing molecules exploiting the nucleophilic behaviour of the chalcogen atom towards various electrophiles [24][25][26][27][28][29].Recently we reported the reaction of bis(trimethylsilyl)selenide (HMDSS) with N-thiophtalimides as a mild, general metal free procedure for the synthesis of variously substituted disulfides [30].
Indeed, despite the interest in the reactivity of selenols as nucleophilic selenium transfer reagents, their use has been critically limited by their instability.However, HMDSS was efficient in the opening of ring strained heterocycles, namely epoxides, episulfides and aziridines, leading to a wide range of β-functionalized selenols, which were stable enough to react with electrophiles under controlled catalytic conditions [31].Differently substituted stable aryl selenols were also prepared by reduction of the parent diselenides to explore their activity as enzyme inhibitors [32].
On the basis of our findings on the behaviour of selenols we report here a new approach for the preparation of selenenylsulfides using N-thiophtalimides as suitable electrophiles.

Results and Discussion
We began our investigations by studying the reaction of benzeneselenol 1a with 2,4-dimethoxyphenyl N-thiophthalimide 2a (Table 1).The reaction was initially performed at 0 • C for 2 h in the presence of Et 3 N, using CHCl 3 as the solvent (Table 1, entry 1).Interestingly, under these conditions the formation of the desired selenenylsulfide 3a was observed, albeit together with a comparable amount of diselenide 4a and disulfide 5a.Furthermore, ca.30% of the starting N-thiophthalimide 2a remained unreacted.Similar results, both in terms of conversion and 3a:4a:5a ratio, were obtained upon performing the reaction at −78 • C for 4 h (entry 2).We evaluated whether the solvent could have an effect on the reaction outcome.We found that the use of acetonitrile in place of chloroform gave the diselenide 4a and the disulfide 5a as main reaction products, together with a larger amount of unreacted 2a (ca.50%, entry 3).The use of toluene gave slight 3a:4a:5a ratio improvements, although poor conversion was achieved (Table 1, entry 4).Remarkably, when DMF was used as solvent in the presence of Et 3 N or Cs 2 CO 3 /TBAI, complete consumption of 2a was observed within 2 h.Under these conditions a comparable mixture of compounds 3a, 4a, and 5a was observed (entries 5 and 6).Finally, we were delighted to find that the desired selenenylsulfide 3a was smoothly formed as major product simply treating selenol 1a with N-thiophthalimide 2a in DMF, in absence of any catalyst (entry 7).This observation seems to suggest that the formation of selenenylsulfide 3a is the result of a clean S N 2 at sulfur of 2a by the nucleophile 1a, favoured in polar aprotic solvents such as DMF.
In our hands compound 3a proved to be rather unstable, as the selenenylsulfide exhibited a remarkable tendency to form the corresponding symmetrical diselenide 4a and disulfide 5a.Indeed, selenenylsulfide 3a significantly decomposed on silica gel during flash chromatography purification (36% isolated yield).Similar behaviour was observed when using a different stationary phase such as neutral Al 2 O 3 .We also observed that both bases and acids could promote the conversion of 3a into 4a and 5a.Intriguingly, the mild acidity of chloroform proved to be sufficient to induce fast conversion of 3a, which could also be observed while acquiring the 1 H NMR spectrum in CDCl 3 .Having established optimum reaction conditions for the conversion of selenols and N-thiophthalimides into the corresponding selenenylsulfides, we explored the scope and the limitation of the reaction.We initially focused our attention on variations of the N-thiophthalimide structure.We found that, under the optimised reaction conditions, variously substituted N-arylthiophthalimides 2a-f, bearing different substituents, such as alkyl, unprotected hydroxyl, methoxy, and silyloxy moieties at different positions of the aromatic ring, reacted smoothly with benzeneselenol 1a, enabling the formation of aryl(phenylselanyl)sulfanes 3a-f (Scheme 1).Furthermore, the reaction was also applied to the N-arylthio derivative of δ-tocopherol 2g, leading to the formation of compound 3g bearing a vitamin E-like skeleton and the phenylseleno moiety.Intriguingly, the 2-chloro-1-N-cyclohexylthiophthalimide 2h proved to be scarcely reactive under the standard conditions.Nonetheless, 1a and 2h readily reacted in the presence of Cs2CO3 and TBAI to afford alkyl(phenylselanyl)sulfane 3h.However, as previously observed for compound 3a, although selenenylsulfides 3b-h were formed in good yields, a significant decomposition occurred during purification.Whereas pure 3b,c,h could be isolated in moderate yields, aryl(phenylselanyl)sulfane 3d-g gave only the corresponding diselenide 4a and disulfides 5d-g upon flash column chromatography (Scheme 1).Having demonstrated that a variety of aryl-and alkyl-N-thiophthalimides could be successfully employed in this reaction, we next explored the scope of the reaction with respect to differently substituted selenols.Pleasingly, arylselenols 1b-d, bearing methyl and methoxy groups onto different position of the aromatic ring, reacted efficiently with N-arylthiophthalimide 2a and N-alkylthiophthalimide 2h to yield the corresponding substituted selenenylsulfides 3i-m.Despite the instability exhibited by S-Se bonds, compounds 3i-m were obtained in good to moderate isolated yields (Scheme 1).
Finally, in order to evaluate whether this reactivity could also be extended to functionalized alkyl selenols, β-hydroxy selenol 1e was treated with N-thiophthalimides 2a and 2h.Unfortunately, albeit selenenylsulfides 3n and 3o were efficiently formed, complete disproportionation occurred during purification on silica gel, leading exclusively to the isolation of diselenide 4e and disulfides 5a,h (Scheme 1).
Worthy of mention is the complete chemoselectivity observed in the reactions of selenols with N-thiophthalimide 2h.In fact, despite working in the presence of a remarkably strong nucleophilic selenate ion and a potentially very good leaving group, no trace of the attack on the chloro a Determined by 1 H NMR of the crude reaction mixture.b Longer reaction time led to improved conversion but lower 3a:4a:5a ratio.c 29%, 12%, 48%, and 33% of starting N-thiophthalimide 2a remained unreacted, respectively for entries 1, 2, 3, and 4.
Having established optimum reaction conditions for the conversion of selenols and N-thiophthalimides into the corresponding selenenylsulfides, we explored the scope and the limitation of the reaction.We initially focused our attention on variations of the N-thiophthalimide structure.We found that, under the optimised reaction conditions, variously substituted N-arylthiophthalimides 2a-f, bearing different substituents, such as alkyl, unprotected hydroxyl, methoxy, and silyloxy moieties at different positions of the aromatic ring, reacted smoothly with benzeneselenol 1a, enabling the formation of aryl(phenylselanyl)sulfanes 3a-f (Scheme 1).Furthermore, the reaction was also applied to the N-arylthio derivative of δ-tocopherol 2g, leading to the formation of compound 3g bearing a vitamin E-like skeleton and the phenylseleno moiety.Intriguingly, the 2-chloro-1-N-cyclohexylthiophthalimide 2h proved to be scarcely reactive under the standard conditions.Nonetheless, 1a and 2h readily reacted in the presence of Cs 2 CO 3 and TBAI to afford alkyl(phenylselanyl)sulfane 3h.However, as previously observed for compound 3a, although selenenylsulfides 3b-h were formed in good yields, a significant decomposition occurred during purification.Whereas pure 3b,c,h could be isolated in moderate yields, aryl(phenylselanyl)sulfane 3d-g gave only the corresponding diselenide 4a and disulfides 5d-g upon flash column chromatography (Scheme 1).Having demonstrated that a variety of aryl-and alkyl-N-thiophthalimides could be successfully employed in this reaction, we next explored the scope of the reaction with respect to differently substituted selenols.Pleasingly, arylselenols 1b-d, bearing methyl and methoxy groups onto different position of the aromatic ring, reacted efficiently with N-arylthiophthalimide 2a and N-alkylthiophthalimide 2h to yield the corresponding substituted selenenylsulfides 3i-m.Despite the instability exhibited by S-Se bonds, compounds 3i-m were obtained in good to moderate isolated yields (Scheme 1).substituted carbon was observed, being the sulfur atom of 3h the unique electrophilic partner of these reactions.The amount of diselenides 4 and disulfides 5 could not be reduced working under strictly controlled conditions (i.e. using dry-box, in absence of light, fast manipulation).The observed behaviour is in accordance with previous reports on the low stability of this kind of structures which, amongst other factors, depends on the nature of substituents on the sulfur or selenium atoms [19,23].
As stated above, a selenenylsulfide is postulated to be the key intermediate of the GPx catalytic cycle.The selenium atom of a selenocysteine residue (Sec) of the GPx catalytic triad and the thiol group of a molecule of GSH are indeed involved in the formation of the selenenylsulfide which, upon reaction with a second molecule of GSH regenerates the catalytically active selenol moiety of Sec.This biochemical mechanism allows the reduction, in living cells, of harmful hydroperoxides to safe alcohols and water, therefore accounting for the biological relevance of S-Se bonds formation [1,3,4].
Thus, having developed a novel procedure for the synthesis of small molecules containing the S-Se moiety, we sought to preliminary test their GPx-like activity.The thiol peroxidase-like activity of compounds 3h and 3l was studied according to the DTT oxidation test (Figure 1) [33][34][35].Indeed, selenenylsulfides 3h,l behaved as medium effective catalysts with T50 around 70 minutes.[36]Furthermore, in line with previous findings on the antioxidant properties of different organoselenium compounds [37,38], the presence of a methoxy group onto the arylseleno moiety renders selenenylsulfide 3l more efficient than 3h (Figure 1).To get more insight on the GPx activity Scheme 1. Scope of the synthesis of selenenylsulfides from selenols and N-thiophthalimides.
Finally, in order to evaluate whether this reactivity could also be extended to functionalized alkyl selenols, β-hydroxy selenol 1e was treated with N-thiophthalimides 2a and 2h.Unfortunately, albeit selenenylsulfides 3n and 3o were efficiently formed, complete disproportionation occurred during purification on silica gel, leading exclusively to the isolation of diselenide 4e and disulfides 5a,h (Scheme 1).
Worthy of mention is the complete chemoselectivity observed in the reactions of selenols with N-thiophthalimide 2h.In fact, despite working in the presence of a remarkably strong nucleophilic selenate ion and a potentially very good leaving group, no trace of the attack on the chloro substituted carbon was observed, being the sulfur atom of 3h the unique electrophilic partner of these reactions.
The amount of diselenides 4 and disulfides 5 could not be reduced working under strictly controlled conditions (i.e., using dry-box, in absence of light, fast manipulation).The observed behaviour is in accordance with previous reports on the low stability of this kind of structures which, amongst other factors, depends on the nature of substituents on the sulfur or selenium atoms [19,23].
As stated above, a selenenylsulfide is postulated to be the key intermediate of the GPx catalytic cycle.The selenium atom of a selenocysteine residue (Sec) of the GPx catalytic triad and the thiol group of a molecule of GSH are indeed involved in the formation of the selenenylsulfide which, upon reaction with a second molecule of GSH regenerates the catalytically active selenol moiety of Sec.This biochemical mechanism allows the reduction, in living cells, of harmful hydroperoxides to safe alcohols and water, therefore accounting for the biological relevance of S-Se bonds formation [1,3,4].Thus, having developed a novel procedure for the synthesis of small molecules containing the S-Se moiety, we sought to preliminary test their GPx-like activity.The thiol peroxidase-like activity of compounds 3h and 3l was studied according to the DTT oxidation test (Figure 1) [33][34][35].Indeed, selenenylsulfides 3h,l behaved as medium effective catalysts with T 50 around 70 min [36] Furthermore, in line with previous findings on the antioxidant properties of different organoselenium compounds [37,38], the presence of a methoxy group onto the arylseleno moiety renders selenenylsulfide 3l more efficient than 3h (Figure 1).To get more insight on the GPx activity of these new compounds [39][40][41][42], in Figure 1 are reported data obtained for diselenides 4a and 4c and disulfide 5h measured under the same conditions.Thus, while diselenides 4a,c promoted a faster oxidation than selenenylsulfides with T 50 around 20 min, disulfide 5h did not exhibit significant catalytic antioxidant properties (Figure 1). of these new compounds [39][40][41][42], in Figure 1 are reported data obtained for diselenides 4a and 4c and disulfide 5h measured under the same conditions.Thus, while diselenides 4a,c promoted a faster oxidation than selenenylsulfides with T50 around 20 minutes, disulfide 5h did not exhibit significant catalytic antioxidant properties (Figure 1).

Experimental Section
All the reactions were performed under a positive pressure of nitrogen and were monitored by TLC using commercially available precoated plates (silica gel 60 F 254) and compounds were visualised by fluorescence quenching or by staining the plates with acidic p-anisaldehyde solution.Silica gel 60, 230-400 mesh, was used for flash column chromatography.Dry solvents were obtained using a Pure Solv™ Micro system.Commercially available reagents were used as obtained from freshly opened containers without further purification.Aryl selenols 1a-d [32], alkyl selenol 1e [31], and N-thiophthalimides 2 [43][44][45][46][47][48] were prepared according to reported procedures.Spectroscopic data of diselenides 4a-e [26,49] and disulfides 5a-h [30] matched those previously reported in the literature. 1 H and 13 C NMR spectra were recorded in CDCl3 or C6D6 with a Varian Mercury Plus instrument or with a Varian INOVA instrument at 400 and 100 MHz, respectively.The corresponding residual non-deuterated solvent was used as a reference (CDCl3: 7.26 ppm for 1 H and 77.0 ppm for 13 C; C6D6: 7.16 ppm for 1 H and 128.0 ppm for 13 C). 77Se NMR spectra were recorded using Bruker 400 Ultrashield spectrometer (Bruker, Milan, Italy), operating at 76 MHz.Diphenyl diselenide (PhSe)2 was used as an external reference for 77 Se NMR (δ = 461 ppm). 1 H NMR data are reported as follows: chemical shift, integration, multiplicity (s = singlet, bs = broad singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublet, etc.), coupling constant (J) or line separation (ls), and assignment.Mass spectra (MS) were determined by ESI (Thermo Fisher Scientific, Milan, Italy).See Electronic Supplementary Material for details.

General Procedure for the Synthesis of Selenenylsulfides 3
A solution of selenol 1 (0.20 mmol, 1.0 eq.) in dry DMF (0.5 mL) was added to a solution of N-thiophthalimide 2 (0.24 mmol, 1.2 eq.) in dry DMF (1.5 mL) at room temperature under inert atmosphere (N2); the reaction mixture was stirred for 4 h (reaction progress monitored by TLC).Afterwards, the mixture was diluted with Et2O (5 mL) and then H2O (3 mL) was added.The organic

Experimental Section
All the reactions were performed under a positive pressure of nitrogen and were monitored by TLC using commercially available precoated plates (silica gel 60 F 254) and compounds were visualised by fluorescence quenching or by staining the plates with acidic p-anisaldehyde solution.Silica gel 60, 230-400 mesh, was used for flash column chromatography.Dry solvents were obtained using a Pure Solv™ Micro system.Commercially available reagents were used as obtained from freshly opened containers without further purification.Aryl selenols 1a-d [32], alkyl selenol 1e [31], and N-thiophthalimides 2 [43][44][45][46][47][48] were prepared according to reported procedures.Spectroscopic data of diselenides 4a-e [26,49] and disulfides 5a-h [30] matched those previously reported in the literature. 1 H and 13 C NMR spectra were recorded in CDCl 3 or C 6 D 6 with a Varian Mercury Plus instrument or with a Varian INOVA instrument at 400 and 100 MHz, respectively.The corresponding residual non-deuterated solvent was used as a reference (CDCl 3 : 7.26 ppm for 1 H and 77.0 ppm for 13 C; C 6 D 6 : 7.16 ppm for 1 H and 128.0 ppm for 13 C). 77Se NMR spectra were recorded using Bruker 400 Ultrashield spectrometer (Bruker, Milan, Italy), operating at 76 MHz.Diphenyl diselenide (PhSe) 2 was used as an external reference for 77 Se NMR (δ = 461 ppm). 1 H NMR data are reported as follows: chemical shift, integration, multiplicity (s = singlet, bs = broad singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublet, etc.), coupling constant (J) or line separation (ls), and assignment.Mass spectra (MS) were determined by ESI (Thermo Fisher Scientific, Milan, Italy).See Electronic Supplementary Material for details.

General Procedure for the Synthesis of Selenenylsulfides 3
A solution of selenol 1 (0.20 mmol, 1.0 eq.) in dry DMF (0.5 mL) was added to a solution of N-thiophthalimide 2 (0.24 mmol, 1.2 eq.) in dry DMF (1.5 mL) at room temperature under inert atmosphere (N 2 ); the reaction mixture was stirred for 4 h (reaction progress monitored by TLC).Afterwards, the mixture was diluted with Et 2 O (5 mL) and then H 2 O (3 mL) was added.The organic phase was extracted with Et 2 O (5 mL), washed with water (3 × 5 mL) and brine (5 mL), and then dried over Na 2 SO 4 ; filtered and concentrated in vacuo.The crude material was purified by flash chromatography to afford selenenylsulfides 3.

Table 1 .
Optimization of the reaction conditions for the synthesis of

Table 1 .
Optimization of the reaction conditions for the synthesis of