Chain Breaking Antioxidant Activity of Heavy (S, Se

Home , Block (periodic table), Butylated hydroxytoluene, Chalcogen, Oxide, Sulfur

Review ReviewChain Breaking Antioxidant Activity of Heavy (S, Se, Chain Breaking Antioxidant Activity of Heavy Te) Chalcogens Substituted Polyphenols (S, Se, Te) Chalcogens Substituted Polyphenols Caterina Viglianisi and Stefano Menichetti * Caterina Viglianisi and Stefano Menichetti * Department of Chemistry “Ugo Schiff”, University of Florence, Via Della Lastruccia 3-13, 50019 Sesto Fiorentino,Department Italy; of Chemistry [email protected] “Ugo Schiff”, University of Florence, Via Della Lastruccia 3-13, 50019 Sesto Fiorentino, *Italy; Correspondence: caterina.viglianisi@unifi.it [email protected]; Tel.: +39-055-4573535 * Correspondence: stefano.menichetti@unifi.it; Tel.: +39-055-4573535 Received: 30 September 2019; Accepted: 15 October 2019; Published: date Received: 30 September 2019; Accepted: 15 October 2019; Published: 16 October 2019 Abstract: Polyphenols are probably the most important family of natural and synthetic chain- Abstract:breaking Polyphenolsantioxidants. are Since probably long the ago, most chemists important have family studied of natural how and structural synthetic chain-breaking (bioinspired) modificationsantioxidants. can Since improve long ago, the chemistsantioxidant have activity studied of howthese structural compounds (bioinspired) in terms of modifications reaction rate withcan improve radical thereactive antioxidant oxygen activity species of these (ROS), compounds catalytic in termscharacter, of reaction multi-defence rate with radicalaction, hydrophilicity/lipophilicity,reactive oxygen species (ROS), biodistribution catalytic character, etc. In multi-defencethis framework, action, we will hydrophilicity discuss the /effectlipophilicity, played onbiodistribution the overall antioxidant etc. In this profile framework, by the weinsertion will discuss of heavy the chalcogens effect played (S, onSe theand overallTe) in the antioxidant phenolic profileskeleton. by the insertion of heavy chalcogens (S, Se and Te) in the phenolic skeleton.

Keywords: sulfur;sulfur; selenium; tellurium; polyphenols; chain breaking antioxidants

1. Introduction Phenols represent represent the the more more important important family family of of chain-breaking chain-breaking antioxidants antioxidants able able to toinhibit inhibit or orretard retard the the autoxidation autoxidation of oforganic organic compounds compounds in includingcluding biomolecules biomolecules in in living living organisms and α α handcraft materialsmaterial [s1 –[1–5].5]. Thus, Thus,α-tocopherol -tocopherol [6–11 [6–11]](α-TOH, ( -TOH, 1a), the 1a), more the potent more lipophilic potent antioxidant lipophilic knownantioxidant in nature known (and in nature the more (and abundant the more component abundant ofcomponent Vitamin E), of catechinVitamin (2),E), catechin a potent (2), antioxidant a potent memberantioxidant of themember ﬂavonoids of the familyflavonoids [12– 15family] (often [12–15 indicated] (often as indicated Vitamin as P) Vitamin and resveratrol P) and resveratrol [16–18] (3) can[16–18] be paradigmatically(3) can be paradigmatically indicated as indicated models foras themodels thousands for the of thousands natural polyphenols of natural polyphenols involved in theinvolved protection in the of protection living tissues of living from oxidativetissues from stress oxidative (Figure stress1). Indeed, (Figure the 1). dangerous Indeed, the increasing dangerous of oxidantsincreasing species of oxidants concentration species inconcentration tissues, such in as ti Reactivessues, such Oxygen as Reactive Species Oxygen (ROS), hasSpecies been (ROS), associated has withbeen manyassociated diﬀerent with pathologies many different and pathologies aging itself [and1–5]. aging On the itself other [1–5]. hand, On butylatedthe other hydroxytoluenehand, butylated (BHT,hydroxytoluene 4) or butylated (BHT, hydroxyanisole 4) or butylated (BHA, hydroxyanisole 5) represent (BHA, the core 5) skeletonrepresent of phenolicthe core antioxidantskeleton of additivesphenolic worldwideantioxidant used additives for increasing worldwide shelf lifeused and for protecting increasing from shelf autoxidation life and plastics, protecting lubricants, from oils,autoxidation solvents andplastics, many lubricants, other handwork oils, solvents materials and [ 19many–21] other (Figure handwork1). materials [19–21] (Figure 1). OH HO HO O OH O OH OH 1a: α-Tocopherol (α-TOH) 2: (+)-catechin

OH OH OH HO

OMe OH 3: resveratrol 4: BHT 5: BHA FigureFigure 1. Structure1. Structure of of model model naturalnatural and and synthetic synthetic phenolic phenolic antioxidants. antioxidants. BHT—butylated BHT—butylated hydroxytoluene; HBA—butylatedhydroxytoluene; hydroxyanisole. HBA—butylated hydroxyanisole. Although the situation at the biological level is, obviously, much more complex, the more important and peculiar feature of phenolic antioxidants is their ability in transferring a hydrogen atom (H ) from • Antioxidants 2019, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/antioxidants Antioxidants 2019, 8, 487; doi:10.3390/antiox8100487 www.mdpi.com/journal/antioxidants Antioxidants 2019, 8, 487 2 of 22 the ArOH to the radical chain propagating species, typically a peroxyl radical ROO , thus, blocking or, • at least, retarding the oxygen mediated autoxidation of organic molecules. Equations (1)–(4) report a simpliﬁed scheme of autoxidation that is applied when R is one of the unsaturated chains of a cell membrane, as well as when R is the aliphatic chain of a polyoleﬁn etc. [22,23].

R R (1) −→ • R + O ROO (2) • 2 −→ • ROO + R-H ROOH + R (3) • −→ • ROO + ArOH ROOH + ArO (4) • −→ • The formation of the first radical (R , Equation (1)) can be the result of the natural metabolism of • a living organism, or due to an oxidative event associated with heat, radiation, injury, inflammation etc. If the radical is formed in the presence of molecular oxygen, a very fast reaction occurs (the kinetic constant for this reaction is similar to that of diffusion 109 M 1s 1) with the unavoidable ≥ − − formation of a peroxyl radical ROO (Equation (2)). Peroxyl radicals are reactive enough to extract a • H from organic molecules (for example from allylic positions of unsaturated fatty acids) causing the • formation of a hydroperoxide ROOH and a new radical R (Equation (3)) in the propagation step of • the autoxidation process. Substituted phenols ArOH can react with peroxyl radicals faster than the great part of organic molecules R avoiding its oxidation. This reaction affords hydroperoxides ROOH and aryloxy radicals ArO (Equation (4)). The latter, if properly substituted, are stable enough to not extract a H from the • • surrounding. Thus, the chain oxidation is broken, or at least retarded, until the phenol can continue in its action of sacrificial reductant. Typically, antioxidant phenols are able to quench two ROO radicals • (i.e., the number n of ROO quenched is 2). This can occur by the reaction of ArO with a second • • equivalent of ROO , as in the case of tocopherols, or with the extraction of a second H , as in the case • • of catechin and related polyphenols. In both cases, after the second reaction, the phenol is transformed in a non-radical species, unable to further propagate the autoxidation or other undesired oxidative paths. Hydroperoxides, ROOH are unavoidably formed during the process (Equations (3) and (4)) and represent, in turn, potentially dangerous oxidants. These species require quenching mechanisms not involving phenolic species, and, as we will describe in the next chapters, heavy chalcogens derivatives containing, mainly Se and Te, can efficiently serve for this scope. The efficiency of phenols as chain-breaking antioxidants depends upon the Bond Dissociation Enthalpy (BDE) of ArO-H bond involved in the H transfer process and upon the kinetic constant • (k ) of the reaction of ArOH with ROO (Equation (4)) [22,23]. Indeed, for homologous families inh • of phenols, BDE and log kinh have a linear correlation: the lower the BDE, the higher the kinh, the faster the H transfer process, the better the chain antioxidant activity. Thanks to the pioneering • work of K.U Ingold, G. F. Pedulli and co-workers, solid procedures for measuring BDE, by Electron Paramagnetic Resonance (EPR) techniques, and kinetic constants kinh, quantifying the O2 consumption in the autoxidation of model hydrocarbons, of differently substituted phenols have been settled out. This allowed elucidating the role of phenolic structure and reaction media on the antioxidant activity and, to design more potent derivatives [22,23]. Briefly, parent phenol PhOH has a BDE of 87 kcal/mol; a value too high for ensuring a fast reaction with ROO (BDE of ROO-H is 88 kcal/mol). Additionally, once formed, parent phenoxyl radical PhO • • could extract a H from organic molecules (for example bis-allylic carbons in poly-unsaturated fatty • acids have a C-H BDE of 89 kcal/mol) acting as the actual promoter of the chain oxidation. Considering the modifications required for lowering BDE (i.e., increasing the kinh) it must be considered that while a phenolic OH residue is, overall, an electron-donating (ED) group, the corresponding phenoxyl radical O group, is, instead, strongly electron-withdrawing (EW). Hence, substituents with an EW character •− are able to stabilize the phenol ArOH, while ED groups are able to stabilize the ArO (and vice-versa). • AntioxidantsAntioxidants 20192019,, 88,, 487x FOR PEER REVIEW 33 of of 22

to stabilize the ArO• (and vice-versa). Thus, ED groups destabilize the ArOH level and stabilize the Thus,• ED groups destabilize the ArOH level and stabilize the ArO level with an overall decreasing of ArO level with an overall decreasing of the BDE, as expected in• a good chain-breaking antioxidant the(Figure BDE, 2). as Of expected similar in importance a good chain-breaking is the role played antioxidant by the (Figure stabilization2). Of similarof ArOH importance by the formation is the role of playedinter- or, by above the stabilization all, intramolecular of ArOH hydrogen by the formation bond (inter-HB of inter- and or, aboveintra-HB). all, intramolecular Thus, an ED group hydrogen able bondto act (inter-HB also as an and intra-HB intra-HB). acceptor Thus,an (for ED example, group able a tomethoxy act also group) as an intra-HB will decrease acceptor the (for BDE example, more aefficiently methoxy when group) in willparadecrease than when the in BDE ortho more position, eﬃciently since whenin the informerpara thansituation, when the in ortho‘electronicallyposition, sincedriven’ in destabilization the former situation, of ArOH the ‘electronicallyplus stabilization driven’ of ArO destabilization•, is reduced ofby ArOH the intra-HB plus stabilization stabilization of ArO , is reduced by the intra-HB stabilization of the ArOH (Figure2). of the• ArOH (Figure 2).

Figure 2. Electronic (red arrows) and intra-HB (green arrows) effects in modifying the ArO-H BDEFigure value 2. Electronic in substituted (red arrows) phenols. and ED—electron-donating; intra-HB (green arrows) EW—electron-withdrawing; effects in modifying the BDE—BondArO-H BDE Dissociationvalue in substituted Enthalpy. phenols. ED—electron-donating; EW—electron-withdrawing; BDE—Bond Dissociation Enthalpy. These general concepts help to rationalise why α-TOH (1a, Figure1) is the more potent natural lipophilicThese chain-breaking general concepts antioxidant. help to rationalise The aromatic why α phenolic-TOH (1a, ring Figure has four1) is EDthe alkylmore groupspotent natural and an alkoxyllipophilic oxygen chain-breakingpara to the antioxidant. phenolic OH. The Calculatedaromatic phenolic BDE for ringα-TOH has four is 77 ED kcal alkyl/mol groups with a andkinh anof 6 1 1 α 3.2alkoxyl10 oxygenM− s− para. Indeed to theβ phenolic-, γ, and OH.δ-TOH Calculated (1b–c, Figure BDE for1) with-TOH two is or77 onekcal/mol methyl with groups a kinh are,of 3.2 in × 6 × –1 –1 β γ δ turn10 M potents . Indeed polyphenolic -, , and antioxidants, -TOH (1b–c, yet Figure showed 1) with a lower two orkinh onethan methyl fully groups methylated are, in 1a turn (Figure potent3). Additionally,polyphenolic inantioxidants, tocopherols, yet alkoxy showed oxygen a lower is part kinh of than a six-membered fully methylated benzo-fused 1a (Figure chromane 3). Additionally, ring. This situationin tocopherols, forces alkoxy the oxygen oxygen lone is pair part to of lay a six-membered almost parallel benzo-fused to the aromatic chromaneπ system, ring. maximizing This situation the abilityforces ofthe resonance oxygen lone stabilization pair to lay of almostα-TO radical.parallel Toto betterthe aromatic understand π system, how conformationsmaximizing the can ability modify of • theresonance ArO stability, stabilization the 2,3,5,6-tetramethyl-4-methoxy of α-TO• radical. To better understand phenol 6, bearinghow conformations for alkyl and can a para-alkoxy modify the • group,ArO• stability, has a BDE the higher 2,3,5,6-tetramethyl-4-methoxy than those of 1a–d. In fact, thephenol steric 6, hindrance bearing broughtfor alkyl about and methyla para-alkoxy groups ongroup, C5 and has C6a BDE favours higher the than conformation those of 1a–d. with In the fact Oxygen-Methyl, the steric hindrance bond laying brought almost about perpendicular methyl groups to theon C5 aromatic and C6 ring. favours In this the situation, conformation the lone with pair the on Oxygen-Methyl methoxy oxygen bond in no laying longer almost in the perpendicular right position forto the an earomaticfficient ArO ring.stabilization In this situation, (Figure the3). lone pair on methoxy oxygen in no longer in the right • positionTo further for an efficient demonstrate ArO• howstabilization conformations (Figure can3). contribute to the rate of H transfer process, • IngoldTo reportedfurther demonstrate that the insertion how conformations of the para-alkoxy can oxygencontribute in ato five-membered the rate of H• ring, transfer i.e., process, moving fromIngold a reported chromane that to athe dihydrobenzo[b]furane insertion of the para-alkoxy system, oxygen causes in a a further five-membered increase ofring,kinh i.e.,. This moving was rationalisedfrom a chromane considering to a dihydrobenzo[b]furane that a five-membered ringsystem, is more causes rigid a and,further consequently, increase of increasingkinh. This was the alignmentrationalised between considering the oxygen that a lone five-membered pair and the ring aromatic is moreπ system. rigid and, This consequently, observation was increasing used for the preparationalignment between of very potentthe oxygen chain lone breaking pair and antioxidants. the aromatic For π example, system. compoundThis observation 7 showed was aused BDE for of 76the kcal preparation/mol of and of very a k potentof 5.0 ch10ain6 Mbreaking1s 1 discernibly antioxidants. better For than example, the α -TOHcompound performances 7 showed [ 24a BDE–26] inh × − − (Figureof 76 kcal/mol4). Noteworthy, of and a k similarinh of 5.0 conformational × 106 M–1s–1 discernibly considerations better than have the been α-TOH exploited performances for chalcogens [24–26] substituted(Figure 4). Noteworthy, phenols [27,28 similar] [8P, 8P conformational0]. considerations have been exploited for chalcogens substituted phenols [27,28] [8P, 8P’]. AntioxidantsAntioxidants 2019 2019, 8, ,x 8 FOR, x FOR PEER PEER REVIEW REVIEW 4 of4 22of 22

FollowingFollowing these these general general criteria criteria and and considering considering the the simplicity simplicity and and cost/efficiency cost/efficiency issues, issues, syntheticsynthetic BHT BHT (4) (4) and and BHA BHA (5) (5) were were selected selected as theas the core core skeletons skeletons of theof the worldwide-used worldwide-used antioxidant antioxidant additivesadditives in inplastics, plastics, lubricants, lubricants, oil oiletc etc [19–21]. [19–21]. In Infact, fact, the the aromatic aromatic rings rings are are substituted substituted with with ED ED groupsgroups and and in BHAin BHA a methoxy a methoxy group group is placedis placed para para position position to theto the phenolic phenolic OH. OH. This This allows allows a quite a quite lowlow BDE BDE (BDE (BDE for for BHT BHT and and BHA BHA are are 77 77and and 80 80kcal/mol kcal/mol respectively, respectively, see see Figure Figure 5) and5) and a fast a fast reaction reaction • withwith ROO ROO• as requiredas required for for the the protection protection of materialsof materials from from auto-oxidation. auto-oxidation. BHT BHT and and BHA BHA are are in turnin turn • ableable of quenchingof quenching two two ROO ROO• equivalents equivalents (n =(n 2) = and,2) and, additionally, additionally, the the stability stability of theof the corresponding corresponding • ArOArO• phenoxyl phenoxyl radicals radicals is furtheris further increased increased by byth eth sterice steric protection protection brought brought about about by bythe the two two ortho ortho tert-butyltert-butyl groups. groups. It Itmust must be beunderlined underlined that that the the BDE BDE lowering lowering of ofthe the ArO-H ArO-H bond, bond, obtainable obtainable with with the the introductionintroduction of theof the right right substituent(s) substituent(s) in thein the right right position(s), position(s), cannot cannot push push as lowas low as possible.as possible. In fact,In fact, withwith BDE BDE values values lower lower than than 72 72kcal/mol kcal/mol ArO-H ArO-H could could directly directly react react with with molecular molecular oxygen oxygen to giveto give a a radical cation ArO-H•+ and superoxide radical anion O2•–. Such a phenol would be unstable to air radical cation ArO-H•+ and superoxide radical anion O2•–. Such a phenol would be unstable to air andand useless useless as asan anantioxidant. antioxidant. Thus, Thus, the the design design of ofnew new antioxidants antioxidants requires requires the the ability ability to toprepare prepare • compoundsAntioxidantscompounds able2019 able, 8to, 487 reactto react very very fast fast with with ROO ROO• and and other other dangerous dangerous ROS, ROS, giving giving stable stable safe safe products products4 of 22 but unable to react with O2. but unable to react with O2.

• FigureFigure 3. Role 3. Role of conformationsof conformations on onresonance resonance stabilization stabilization of ArO of ArO•. . •

Figure 4. Effect of ring size on BDE of benzo-fused oxygen heterocycles. FigureFigure 4. Effect 4. Effect of ringof ring size size on onBDE BDE of benzo-fusedof benzo-fused oxygen oxygen heterocycles. heterocycles. Following these general criteria and considering the simplicity and cost/efficiency issues, synthetic The fine-tuning of phenols structure/antioxidant activity relationship has been investigated in BHTThe (4) fine-tuning and BHA (5)of werephenols selected struct asure/antioxidant the core skeletons activity of the relationship worldwide-used has been antioxidant investigated additives in detail by many groups around the world with several excellent results in term of performances, detailin plastics, by many lubricants, groups around oil etc [ 19the–21 world]. In fact,with the several aromatic excellent rings areresults substituted in term withof performances, ED groups and stability and access feasibility [29–34]. In this focussed review, the main achievements reached using stabilityin BHA and a methoxyaccess feasibility group is [29–34]. placed Inpara thisposition focussed to review, the phenolic the main OH. achievements This allows areached quite low using BDE heavy chalcogens (S, Se and Te) as substituents of the phenolic skeleton will be considered and heavy(BDE chalcogens for BHT and (S, BHASe and are Te) 77 as and substituents 80 kcal/mol of respectively, the phenolic see skeleton Figure5 will) and be a fastconsidered reaction and with discussed. The aim of the review is to report how the stereoelectronic features of chalcogen discussed.ROO as The required aim of for the the review protection is to of materialsreport how from the auto-oxidation. stereoelectronic BHT features and BHAof chalcogen are in turn • able of quenching two ROO equivalents (n = 2) and, additionally, the stability of the corresponding • ArO phenoxyl radicals is further increased by the steric protection brought about by the two ortho • tert-butyl groups. It must be underlined that the BDE lowering of the ArO-H bond, obtainable with the introduction of the right substituent(s) in the right position(s), cannot push as low as possible. In fact, with BDE values lower than 72 kcal/mol ArO-H could directly react with molecular oxygen to give a radical cation ArO-H + and superoxide radical anion O . Such a phenol would be unstable to air and useless as • 2•− an antioxidant. Thus, the design of new antioxidants requires the ability to prepare compounds able to react very fast with ROO and other dangerous ROS, giving stable safe products but unable to react • with O2. The fine-tuning of phenols structure/antioxidant activity relationship has been investigated in detail by many groups around the world with several excellent results in term of performances, stability and access feasibility [29–34]. In this focussed review, the main achievements reached using heavy chalcogens (S, Se and Te) as substituents of the phenolic skeleton will be considered and discussed. Antioxidants 2019, 8, x FOR PEER REVIEW 5 of 22 substituents can modify the chain-breaking antioxidant ability of phenols, and how the introduction of chalcogens can be exploited for the preparation of new potent antioxidants and for a better understanding of the red-ox biological processes where chalcogen containing phenols are involved with. Reaction media (solvent, pH, additives, etc.) can dramatically modify the antioxidant performances of phenols [35–39]. However, insights acquired reaction media effects remain completely true for chalcogens substituted phenols, thus the discussion about the role of S, Se or Te on the antioxidant activity can be carried out independently. Additionally, several different procedures to measure and quantify the antioxidant activity of phenols are available nowadays [40– 42]. In this review, we will consider exclusively the chain-breaking activity of phenols evaluated using the rigorous physicochemical approaches settled out by K. U. Ingold, G.F Pedulli [22] and L. Engman (vide infra). Research on chalcogens containing phenolic antioxidants has been developed in the last four decades or so, with new achievements on derivatives bearing, usually, one of the three elements. However, compounds with a similar structure, yet substituted with S or Se or Te, have been reported as well and studied for comparing the effect of each chalcogen. In the hope of facilitating the discussion and valorising similarities and differences, we have decided to describe initially the chemistry of sulfur-containing antioxidants, to then move to those having Se or Te. Antioxidants 2019, 8, 487 5 of 22 2. Discussion

2.1.The Sulfur aim Containing of the review Phenolic is to Antioxidants report how the stereoelectronic features of chalcogen substituents can modify the chain-breaking antioxidant ability of phenols, and how the introduction of chalcogens A solid quantification of the effect of sulfur substituents on BDE, and kinh, of phenolic can be exploited for the preparation of new potent antioxidants and for a better understanding of the antioxidants, is relatively recent [43]. Pedulli, Menichetti and co-workers reported that in compound red-ox biological processes where chalcogen containing phenols are involved with. Reaction media 8 the thio-analogue of BHA, a para-thiomethyl group decreases the BDE of about 3.6 kcal/mol, a value (solvent, pH, additives, etc.) can dramatically modify the antioxidant performances of phenols [35–39]. that is lower than the 4.4 kcal/mol measured for a para-methoxy group. On the other hand, the ortho- However, insights acquired reaction media eﬀects remain completely true for chalcogens substituted thiomethyl group in 9 decreases the BDE of about 0.8 Kcal/mole being just 0.2 kcal/mol the phenols, thus the discussion about the role of S, Se or Te on the antioxidant activity can be carried out contribution of an ortho-methoxy group (Figure 5). In other words, when in para position, the independently. Additionally, several diﬀerent procedures to measure and quantify the antioxidant stabilization of ArO• and destabilization of ArOH, i.e., the overall ED character effect, is less activity of phenols are available nowadays [40–42]. In this review, we will consider exclusively the important for a -SCH3 than for a -OCH3 group. Considering the ortho- substitution, the weaker intra- chain-breaking activity of phenols evaluated using the rigorous physicochemical approaches settled HB stabilization offered by an adjacent SCH3 group causes an overall superior decrease of BDE (0.8 out by K. U. Ingold, G.F Pedulli [22] and L. Engman (vide infra). vs 0.2 kcal/mol) moving from sulfur to oxygen [43].

OH OH

OMe 4 (BHT): BDE = 80.1 kcal/mol 5 (BHA): BDE = 77.4 kcal/mol 4 1 1 5 1 1 kinh =1.1x10 M s kinh =1.1x10 M s

OH OH SMe

SMe

8: BDE = 78.3 kcal/mol 9: BDE = 82.2 kcal/mol 4 1 1 4 1 1 kinh =3.0x10 M s kinh =0.4x10 M s

6 1 1 10: kinh =1.1x10 M s FigureFigure 5. Performances 5. Performances of ofSulfur Sulfur anal analoguesogues of ofBHA BHA 8, 9 8, and 9 and all allracrac-1-thio--1-thio-α-tocopherolα-tocopherol 10. 10.

However,Research taking on chalcogens into consideration containing that, phenolic for example, antioxidants an ortho has-methyl been group developed decreases in the the last ArO- four H decadesBDE of about or so, 2.0 with kcal/mol, new achievements the introduction on derivatives of thioalkyl bearing, residues usually, seems, one neither of the in three para- elements.nor in orthoHowever,-position, compounds convenient with for a increasing similar structure, the chain yet br substitutedeaking antioxidant with S or Seactivity or Te, of have phenols. been reported Indeed, as similarwell andconsideration studied for emerged comparing after the etheﬀect synthetically of each chalcogen. demanding In the preparation hope of facilitating of all-rac the-1-thio- discussionα- tocopheroland valorising 10, and similarities related species, and diﬀ carriederences, out we by have Ingold decided more to describethan 30 initiallyyears ago the [44,45]. chemistry The of sulfur-containing antioxidants, to then move to those having Se or Te.

2. Discussion

2.1. Sulfur Containing Phenolic Antioxidants

A solid quantification of the effect of sulfur substituents on BDE, and kinh, of phenolic antioxidants, is relatively recent [43]. Pedulli, Menichetti and co-workers reported that in compound 8 the thio-analogue of BHA, a para-thiomethyl group decreases the BDE of about 3.6 kcal/mol, a value that is lower than the 4.4 kcal/mol measured for a para-methoxy group. On the other hand, the ortho-thiomethyl group in 9 decreases the BDE of about 0.8 Kcal/mole being just 0.2 kcal/mol the contribution of an ortho-methoxy group (Figure5). In other words, when in para position, the stabilization of ArO and • destabilization of ArOH, i.e., the overall ED character effect, is less important for a -SCH3 than for a -OCH3 group. Considering the ortho- substitution, the weaker intra-HB stabilization offered by an adjacent SCH3 group causes an overall superior decrease of BDE (0.8 vs 0.2 kcal/mol) moving from sulfur to oxygen [43]. However, taking into consideration that, for example, an ortho-methyl group decreases the ArO-H BDE of about 2.0 kcal/mol, the introduction of thioalkyl residues seems, neither in para- nor in ortho-position, convenient for increasing the chain breaking antioxidant activity of Antioxidants 2019, 8, x FOR PEER REVIEW 6 of 22 substitution, in the benzo-fused chromane ring, of oxygen with sulfur (i.e., from 1a to 10) decreased the kinh to 1.1 × 106 M–1s–1, which is about one third of that of 1a, demonstrating the reduced ability of the thiochromane para-sulfur atom in stabilizing tocopheryl radical (α-TO•) as quantified two decades after [43] (Figure 5). We have reported a practical preparation of phenolic compounds with a 2,3- dihydrobenzo[1,4]oxathiine skeleton with valuable antioxidant activity [46–50]. The synthetic opportunity was exploited to prepare, for example, derivatives 11 and 12 with identical structures but a sulfur or an oxygen atom para- to the phenolic OH involved in H• transfer process (Figure 6). Indeed, kinh of 11 was higher than that of 12 boosting the better ability of a para-conjugated oxygen atom in stabilizing phenoxyl radicals ArO•. Having the possibility to prepare a great variety of benzoxathiine antioxidants and to manipulate their structure allowed us to achieve a number of additional insights about the role of the sulfur atom on the activity of these polyphenolic compounds. For example, oxidation at sulfur [43,48], with the formation of either sulfoxides, like 13 and 14, or sulfones, 15 and 16, sensibly depletes the H• transfer ability of these species above all when the sulfur atom is directly conjugated with the phenolic OH. This was expected due to the transformation of the ED group (the sulfide) into an EW group (the sulfoxide or sulfone) that will be particularly effective when the sulfur atom is directly conjugated with the -OH/-O• group (i.e., like in compounds 14 and 16). Additional considerations emerged considering derivative 17, and related species, that possesses exactly the aromatic skeleton of α-TOH (1a, Figure 1) with a sulfur atom in the place of a CH2 in position 4. This compound showed a kinh of 1.3 × 106 M–1s–1 and a BDE of 79 kcal/mol [51]. Thus, the introduction of the sulfur atom in positionAntioxidants 4 decreases2019, 8, 487the chain breaking antioxidant activity as when it was inserted in position 16 (see of 22 derivative 10 Figure 5). This was rationalised considering that introducing a sulfur atom in a benzo- fusedphenols. heterocyclic Indeed, ring similar means consideration increasing the emerged flexibility after of the the synthetically system (due demanding to the two preparation long sulfur- of carbonall- bonds).rac-1-thio- Asα -tocopheroldemonstrated 10, andby an related X-ray, species, at least carried in the out solid-state, by Ingold the more benzoxathiine than 30 years skeleton ago [44,45 of]. 17 showedThe substitution, a reduced in planarity the benzo-fused when compared chromane ring, with of chromane oxygen with system sulfur of (i.e., tocopherols. from 1a to 10)Thus, decreased in 17, the endocyclicthe k to 1.1oxygen106 loneM 1 spair1, which is less is parallel about one to thirdthe aromatic of that of π 1a, system demonstrating than in 1a, the causing reduced a abilityreduced of inh × − − abilitythe in thiochromane stabilizing thepara ArO-sulfur• radical. atom inIn stabilizingother words, tocopheryl introducing radical a sulfur (α-TO atom) as quantified in the chromane two decades ring • (i.e., afterhaving [43 a] (Figurebenzoxathi5). ine ring) means increasing the flexibility of the system i.e., in the opposite direction Weof increasing have reported the antioxidant a practical preparationactivity (compare of phenolic Figures compounds 4 and 6, i.e., with 1a a vs 2,3-dihydrobenzo[1, 7 vs 17). 4]oxathiineBenzoxathiines skeleton derivatives, with valuable like antioxidant 11, 12 and activity 17, were [46– prepared50]. The synthetic via a Diels-Alder opportunity reaction was exploited of a dienicto ortho prepare,-thioquinone for example, with derivatives electron-ric 11 andh styrene 12 with used identical as a dienophile. structures but Thus, a sulfur the proper or an oxygen decoration atom para- to the phenolic OH involved in H transfer process (Figure6). Indeed, k of 11 was higher of both reaction partners allowed the synthesis• of derivatives possessing a tocopherol-likeinh and a catechin-likethan that structure of 12 boosting [46–51]. the Indeed, better ability derivatives of a para lik-conjugatede 18 and 19 oxygenbring together atom in the stabilizing characteristics phenoxyl of radicals ArO . two of the more important• families of natural polyphenolic antioxidants (Figure 6).

FigureFigure 6. A 6.selectionA selection of 2,3-dihydrobenzo[1,4]oxathiine of 2,3-dihydrobenzo[1,4]oxathiine multi-defence multi-defence antioxidants. antioxidants.

Having the possibility to prepare a great variety of benzoxathiine antioxidants and to manipulate their structure allowed us to achieve a number of additional insights about the role of the sulfur atom on the activity of these polyphenolic compounds. For example, oxidation at sulfur [43,48], with the formation of either sulfoxides, like 13 and 14, or sulfones, 15 and 16, sensibly depletes the H transfer • ability of these species above all when the sulfur atom is directly conjugated with the phenolic OH. This was expected due to the transformation of the ED group (the sulfide) into an EW group (the sulfoxide or sulfone) that will be particularly effective when the sulfur atom is directly conjugated with the -OH/-O • group (i.e., like in compounds 14 and 16). Additional considerations emerged considering derivative 17, and related species, that possesses exactly the aromatic skeleton of α-TOH (1a, Figure1) with a sulfur atom in the place of a CH in position 4. This compound showed a k of 1.3 106 M 1s 1 and 2 inh × − − a BDE of 79 kcal/mol [51]. Thus, the introduction of the sulfur atom in position 4 decreases the chain breaking antioxidant activity as when it was inserted in position 1 (see derivative 10 Figure5). This was rationalised considering that introducing a sulfur atom in a benzo-fused heterocyclic ring means increasing the flexibility of the system (due to the two long sulfur-carbon bonds). As demonstrated by an X-ray, at least in the solid-state, the benzoxathiine skeleton of 17 showed a reduced planarity when compared with chromane system of tocopherols. Thus, in 17, the endocyclic oxygen lone pair is less parallel to the aromatic π system than in 1a, causing a reduced ability in stabilizing the ArO radical. • In other words, introducing a sulfur atom in the chromane ring (i.e., having a benzoxathiine ring) means increasing the flexibility of the system i.e., in the opposite direction of increasing the antioxidant activity (compare Figures4 and6, i.e., 1a vs 7 vs 17). Antioxidants 2019, 8, 487 7 of 22

Benzoxathiines derivatives, like 11, 12 and 17, were prepared via a Diels-Alder reaction of a dienic ortho-thioquinone with electron-rich styrene used as a dienophile. Thus, the proper decoration of both reaction partners allowed the synthesis of derivatives possessing a tocopherol-like and a catechin-like structure [46–51]. Indeed, derivatives like 18 and 19 bring together the characteristics of two of the Antioxidantsmore important 2019, 8, x FOR families PEER REVIEW of natural polyphenolic antioxidants (Figure6). 7 of 22 Actually, the multi-defence antioxidant activity of hybrid derivatives is particularly appealing Actually, the multi-defence antioxidant activity of hybrid derivatives is particularly appealing since the synergism between structurally different antioxidants is a mandatory requisite, at a biological since the synergism between structurally different antioxidants is a mandatory requisite, at a level, for transforming high oxidant radical species into safe unreactive derivatives through a cascade biological level, for transforming high oxidant radical species into safe unreactive derivatives through of red-ox quenching processes [52–59]. a cascade of red-ox quenching processes [52–59]. When studying the reactivity of benzoxathiine derivatives, we faced the problem to quantify the When studying the reactivity of benzoxathiine derivatives, we faced the problem to quantify the H transferability of OH groups laying in ortho- or in para-position to the endocyclic benzoxathiine H• transferability• of OH groups laying in ortho- or in para-position to the endocyclic benzoxathiine sulfur [60]. As defined, a thioalkyl residue reduces the BDE of an ortho-OH group of about 0.8 kcal/mol sulfur [60]. As defined, a thioalkyl residue reduces the BDE of an ortho-OH group of about 0.8 as the balance between ArOH/ArO stabilization by ED effect and ArOH stabilization by intra-HB [43]. kcal/mol as the balance between ArOH/ArO• • stabilization by ED effect and ArOH stabilization by However, as it was reported long ago, a strong intra-HB with ortho-thioalkyl groups is possible only intra-HB [43]. However, as it was reported long ago, a strong intra-HB with ortho-thioalkyl groups is when the S-alkyl bond lays perpendicular to the aromatic phenolic plane [61–65]. When the S-alkyl possible only when the S-alkyl bond lays perpendicular to the aromatic phenolic plane [61–65]. When bond is structurally forced (near) parallel to the phenolic ring the intra-HB strength sensibly decreases, the S-alkyl bond is structurally forced (near) parallel to the phenolic ring the intra-HB strength hence it decreases the stabilization of ArOH and the ArO-H BDE (Figure7). This phenomenon sensibly decreases, hence it decreases the stabilization of ArOH and the ArO-H BDE (Figure 7). This is typical of heavy chalcogens, while oxygen is able to give strong intra-HB almost independently phenomenon is typical of heavy chalcogens, while oxygen is able to give strong intra-HB almost upon the conformation considered. As we will discuss later on, this is directly related to “σ-hole independently upon the conformation considered. As we will discuss later on, this is directly related and chalcogen-bond” issues that must be considered since to give a complete frame of S, Se and Te to “σ-hole and chalcogen-bond” issues that must be considered since to give a complete frame of S, stereoelectronic contribution to H transfer processes [61–65]. Measuring kinh and BDE of a set of Se and Te stereoelectronic contribution• to H• transfer processes [61–65]. Measuring kinh and BDE of a different substituted benzoxathiines (20–23, Figure7) with the OH group ortho or para to the sulfur set of different substituted benzoxathiines (20–23, Figure 7) with the OH group ortho or para to the or the oxygen atom of benzo-fused heterocycle, we could quantify this contribution, demonstrating sulfur or the oxygen atom of benzo-fused heterocycle, we could quantify this contribution, that a sulfur atom inserted in a six-membered ring decrease the BDE of an ortho-OH group of about demonstrating that a sulfur atom inserted in a six-membered ring decrease the BDE of an ortho-OH 3.1 kcal/mol [60]. Indeed, a very week intra-HB is observed, as validated by Infra Red (IR) spectra group of about 3.1 kcal/mol [60]. Indeed, a very week intra-HB is observed, as validated by Infra Red and ab-initio calculations, in compounds 20 and 23, this sensibly decreases the ArOH stabilization and (IR) spectra and ab-initio calculations, in compounds 20 and 23, this sensibly decreases the ArOH also the corresponding BDE. As a matter of fact, boosting the evidence of a very week intra-HB, X-ray stabilization and also the corresponding BDE. As a matter of fact, boosting the evidence of a very analysis of derivative 23, showed that in the solid-state, the phenolic proton lays far from the sulfur week intra-HB, X-ray analysis of derivative 23, showed that in the solid-state, the phenolic proton atom pointing in the direction of the ortho-alkyl group. lays far from the sulfur atom pointing in the direction of the ortho-alkyl group.

FigureFigure 7. Conformational 7. Conformational and and substitution substitution considerations considerations on onintra-HB intra-HB strength strength of ofacyclic acyclic and and cyclic cyclic orthoortho-thioalkyl-thioalkyl substituted substituted phenols, phenols, and and benzoxathiines benzoxathiines 20–23 20–23 prepared prepared to quantify to quantify the the role role of ofthis this

phenomenonphenomenon on onkinhk andinh and ArO-H ArO-H BDE. BDE.

ThisThis observation observation was was used used to tosuggest suggest a rationale a rationale for for the the cysteine-tyrosine cysteine-tyrosine post-transactional post-transactional aryl- aryl-S S bond formationformation in in the the galactose galactose oxidase oxidase active active site. site. This This enzyme enzyme catalyses catalyses the oxidation the oxidation with molecular with molecularoxygen, oxygen, of the primary of the primary alcohol residuealcohol ofresidue galactose of galactose to the corresponding to the corresponding aldehyde aldehyde and hydrogen and hydrogenperoxide. peroxide. The catalytic The cyclecatalytic foresees cycle the foresees formation, the formation, on the tyrosine on the residue, tyrosine of anresidue,ortho-thiosubstituted of an ortho- thiosubstitutedphenoxyl radical phenoxyl that represents radical that the represents key oxidative the key intermediate oxidative intermediate of the whole process.of the whole We postulatedprocess. Wethat postulated the rotation that around the rotation the aryl-sulfur around the bond, aryl-sulfur modifying bond, the BDEmodifying of the tyrosinethe BDE OH of the (with tyrosine the thioalkyl OH (with the thioalkyl group perpendicular to the aromatic ring: strong intra-HB, stable ArOH high BDE; with the thioalkyl group on the plane of the aromatic ring: weak intra-HB, stable ArO• low BDE) facilitates the red/ox cycles. Probably, the arrival in the active site of the reduced galactose and its departure as oxidised galactose triggers the rotation around the aryl-sulfur bond modifying the red/ox potentials as required for the enzyme action [60]. At the same time, this observation indicates that in a sulfur atom inserted in a benzoxathiine ring, an efficient substituent to be placed ortho- to a phenolic OH involved in the H• transfer event. Antioxidants 2019, 8, 487 8 of 22

group perpendicular to the aromatic ring: strong intra-HB, stable ArOH high BDE; with the thioalkyl group on the plane of the aromatic ring: weak intra-HB, stable ArO low BDE) facilitates the red/ox • cycles. Probably, the arrival in the active site of the reduced galactose and its departure as oxidised galactose triggers the rotation around the aryl-sulfur bond modifying the red/ox potentials as required for the enzyme action [60]. At the same time, this observation indicates that in a sulfur atom inserted in a benzoxathiine Antioxidantsring, an 2019 eﬃ,cient 8, x FOR substituent PEER REVIEW to be placed ortho- to a phenolic OH involved in the H transfer8 of event. 22 • Indeed, exploiting this opportunity, we designed properly substituted derivatives and prepared very Indeed, exploiting this opportunity, we designed properly substituted derivatives and prepared very potent chain-breaking benzoxathiine antioxidants, for example compounds 24–26, with abilities near potent chain-breaking benzoxathiine antioxidants, for example compounds 24–26, with abilities near or superior to those of α-TOH 1a, also maintaining the catechin-like character [66] (Figure8). or superior to those of α-TOH 1a, also maintaining the catechin-like character [66] (Figure 8).

FigureFigure 8. Optimized 8. Optimized benzoxathiines benzoxathiines phenolic phenolic chain chain breaking breaking antioxidants antioxidants 24–26. 24–26.

AsAs already already reported reported in inthe the introduction introduction [22,23], [22,23 increasing], increasing the the number number of ofalkyl alkyl groups groups does does not not automaticallyautomatically mean mean increasing increasing kinhk inh(or(or decreasing decreasing BDE). BDE). In fact, In fact, chai chainn breaking breaking antioxidant antioxidant 25 25showed showed performancesperformances worse worse than than 24 24 despite despite the the additional methylmethyl groupgroup on on C7. C7. In In fact, fact, this this methyl methyl laying layingortho orthoto theto the methoxy methoxy group group disfavours, disfavours, by steric by steric hindrance, hindrance, the conformation the conformation with with the O-Me the O-Me bond bond parallel parallelto the to aromatic the aromatic ring required ring required for an eforfficient an efficient ArO stabilization ArO• stabilization (see Figure (see3). Figure Such steric 3). Such repulsion steric is • repulsionimportant is important enough to enough prefer a situationto prefer withouta situatio substituentsn without substituents in position 7 in that position is the skeleton7 that is chosen the skeletonfor derivative chosen for 24 andderivative catechin 24/tocopherol and catechin/tocopherol hybrid 26 both hybrid showing 26 kbothinh higher showing than kinh those higher of αthan-TOH those1a. of As α expected,-TOH 1a. compoundAs expected, 26 compound was capable 26 ofwas quenching capable of four quenching equivalents four ofequivalents ROO radicals of ROO• (n = 4) • radicalspossessing (n = 4) both possessing the tocopherol-like both the tocopherol-like and the catechol-like and the catechol-like portion operative portion [66 operative]. [66]. It hasIt has already already been been reported reported that moving that moving from six from to five-membered six to five-membered rings in phenolic rings in species phenolic bearingspecies benzofused bearing benzofused oxygen substituted oxygen heterocycles substituted heterocyclesmeans increasing means the increasing rigidity of the system, rigidity with of the a benefitsystem, on with the achain-breaking benefit on the antioxidant chain-breaking activity antioxidant [24,25,28]. activity Actually, [24 ,25a ,similar28]. Actually, trend was a similar not observedtrend was for 5-hydroxydihydrobenzo[ not observed for 5-hydroxydihydrobenzo[b]thiophenes of typeb]thiophenes 27–29 (Figure of type9) which 27–29 demonstrated (Figure9) which to be demonstratedpotent chain-brea to beking potent antioxidants chain-breaking but did antioxidants not overwhelm but the did performances not overwhelm of α the-TOH. performances We could of rationaliseα-TOH. Wethis could result rationalise having this developed result having a new developed procedure a new procedurefor the preparation for the preparation of 7- of hydroxydihydrobenzo[7-hydroxydihydrobenzo[b]thiophenesb]thiophenes based based on an on acid an acid promot promoteded transposition transposition of ofbenzoxathiines. benzoxathiines. Thus,Thus, these these latter latter heterocycles,heterocycles, are, are, at theat the same same time, time, an interesting an interesting skeleton skeleton for antioxidant for antioxidant polyphenols polyphenolsand starting and material starting for material new phenolic for new phenolic derivatives derivatives [67]. When [67]. carried When outcarried on structurallyout on structurally properly properlydesigned startingdesigned materials, starting the 2,3-dihydrobenzo[1,4]oxathiine materials, the 2,3-dihydrobenzo[1,4]oxathiinehydroxydihydrobenzo[b]thiophene → → hydroxydihydrobenzo[transposition allows theb]thiophene preparation transposition of potent chain allows breaking the pr antioxidantseparation of like potent 30–32, chain taking breaking advantage antioxidantsof the ortho -endocycliclike 30–32, sulfurtaking eadvantageffects discussed of the before, ortho namely- endocyclic (i) the sulfur ED eff ecteffects on ArOdiscussedstabilization before, by • namelythe para (i)-alkoxy the ED andeffectortho on-sulfide ArO• stabilization groups; (ii) the by weakthe para ArOH-alkoxy stabilization and ortho by-sulfide negligible groups; intra-HB (ii) the [67 ] weak(Figure ArOH9). stabilization by negligible intra-HB [67] (Figure 9). As a matter of fact, moving from derivatives 24–26 to 30–32, i.e., decreasing the ring size of the benzofused heterocyclic ring, we observe a tiny decrease of kinh (compare data of Figures8 and9). This was explained considering that in dihydrothiopene derivatives (like 27–32) the two long sulfur bonds allowed enough flexibility to vanish the expected increase in rigidity after ring constraint. Indeed, X-ray analysis of derivative 30 indicates that the dihedral angle among the aromatic and the heterocyclic ring is very similar to that measured in benzoxathiines like 23 or 24, supporting, at least in the solid-state, the above justification. However, it must be underlined that the transformation reported in Figure9 used for the synthesis of compounds 30–32, represents the one-step conversion of a non-phenolic derivative with no antioxidant activity into a potent phenolic chain breaking antioxidant [67].

Figure 9. Hydroxy-2,3-dyhydrobenzo[b]thiophene antioxidants.

As a matter of fact, moving from derivatives 24–26 to 30–32, i.e., decreasing the ring size of the benzofused heterocyclic ring, we observe a tiny decrease of kinh (compare data of Figure 8 and Figure 9). This was explained considering that in dihydrothiopene derivatives (like 27–32) the two long sulfur bonds allowed enough flexibility to vanish the expected increase in rigidity after ring Antioxidants 2019, 8, x FOR PEER REVIEW 8 of 22

Indeed, exploiting this opportunity, we designed properly substituted derivatives and prepared very potent chain-breaking benzoxathiine antioxidants, for example compounds 24–26, with abilities near or superior to those of α-TOH 1a, also maintaining the catechin-like character [66] (Figure 8).

Figure 8. Optimized benzoxathiines phenolic chain breaking antioxidants 24–26.

As already reported in the introduction [22,23], increasing the number of alkyl groups does not automatically mean increasing kinh (or decreasing BDE). In fact, chain breaking antioxidant 25 showed performances worse than 24 despite the additional methyl group on C7. In fact, this methyl laying ortho to the methoxy group disfavours, by steric hindrance, the conformation with the O-Me bond parallel to the aromatic ring required for an efficient ArO• stabilization (see Figure 3). Such steric repulsion is important enough to prefer a situation without substituents in position 7 that is the skeleton chosen for derivative 24 and catechin/tocopherol hybrid 26 both showing kinh higher than those of α-TOH 1a. As expected, compound 26 was capable of quenching four equivalents of ROO• radicals (n = 4) possessing both the tocopherol-like and the catechol-like portion operative [66]. It has already been reported that moving from six to five-membered rings in phenolic species bearing benzofused oxygen substituted heterocycles means increasing the rigidity of the system, with a benefit on the chain-breaking antioxidant activity [24,25,28]. Actually, a similar trend was not observed for 5-hydroxydihydrobenzo[b]thiophenes of type 27–29 (Figure 9) which demonstrated to be potent chain-breaking antioxidants but did not overwhelm the performances of α-TOH. We could rationalise this result having developed a new procedure for the preparation of 7- hydroxydihydrobenzo[b]thiophenes based on an acid promoted transposition of benzoxathiines. Thus, these latter heterocycles, are, at the same time, an interesting skeleton for antioxidant polyphenols and starting material for new phenolic derivatives [67]. When carried out on structurally properly designed starting materials, the 2,3-dihydrobenzo[1,4]oxathiine → hydroxydihydrobenzo[b]thiophene transposition allows the preparation of potent chain breaking antioxidants like 30–32, taking advantage of the ortho- endocyclic sulfur effects discussed before, namelyAntioxidants (i) the2019 ED, 8, 487effect on ArO• stabilization by the para-alkoxy and ortho-sulfide groups; (ii) the9 of 22 weak ArOH stabilization by negligible intra-HB [67] (Figure 9).

Antioxidants 2019, 8, x FOR PEER REVIEW 9 of 22 constraint. Indeed, X-ray analysis of derivative 30 indicates that the dihedral angle among the aromatic and the heterocyclic ring is very similar to that measured in benzoxathiines like 23 or 24, supporting, at least in the solid-state, the above justification. However, it must be underlined that the transformation reported in Figure 9 used for the synthesis of compounds 30–32, represents the one- step conversion of a non-phenolicFigureFigure 9. Hydroxy-2,3-dyhydrobenzo[ 9. Hydroxy-2,3-dyhydrobenzo[ derivative with no antioxidantb]thiopheneb]thiophene activity antioxidants. antioxidants. into a potent phenolic chain breaking antioxidant [67]. AsEven a matter more of fact, interestingly, moving from when derivatives a proper 24–26 structural to 30–32, arrangement i.e., decreasing is respected the ring size and of placed the Even more interestingly, when a proper structural arrangement is respected and placed under benzofusedunder harsher heterocyclic reaction ring, conditions, we observe a tiny second decrease rearrangement of kinh (compare could data take of Figure place 8 transformingand Figure harsher reaction conditions, a second rearrangement could take place transforming 7- 9).7-hydroxydihydrobenzo[ This was explained consideringb]thiophenes, that likein dihydr 30–32,othiopene into the corresponding derivatives (like aromatic 27–32) 7-hydroxybenzo[ the two long b] hydroxydihydrobenzo[b]thiophenes, like 30–32, into the corresponding aromatic 7- sulfurthiophenes bonds 33–35allowed [68] (Figureenough 10 flexibility). Many years to vanish ago, Ingold the demonstratedexpected increase that comparing in rigidity the after antioxidant ring hydroxybenzo[b]thiophenes 33–35 [68] (Figure 10). Many years ago, Ingold demonstrated that activity of 5-hydroxydihydrobenzo[b]furanes (like 7, Figure4) with the corresponding aromatic comparing the antioxidant activity of 5-hydroxydihydrobenzo[b]furanes (like 7, Figure 4) with the 5-hydroxybenzo[b]furanes (like 36, Figure 10) an important decrease of the kinh, almost an order of corresponding aromatic 5-hydroxybenzo[b]furanes (like 36, Figure 10) an important decrease of the magnitude, can be observed [45]. This was explained by considering that, with aromatization, the kinh, almost an order of magnitude, can be observed [45]. This was explained by considering that, with lone pair on oxygen, required for ArO stabilization, is actually engaged in the 10 electrons aromatic aromatization, the lone pair on oxygen, required• for ArO• stabilization, is actually engaged in the 10 system. Thus, despite the increased rigidity and perfect planarity reached, aromatization caused a electrons aromatic system. Thus, despite the increased rigidity and perfect planarity reached, signiﬁcant decrease of the H transfer process rate [45] (Figure 10). aromatization caused a significant• decrease of the H• transfer process rate [45] (Figure 10).

HO HO

O O

6 1 1 6 1 1 7: kinh =5.2x10 M s 36: kinh =0.5x10 M s

S strong acid S S

O Ar OH OH

MeO S MeO S MeO S

OH OH OH

6 1 1 6 1 1 6 1 1 33: kinh =5.0x10 M s 34: kinh =5.9x10 M s 35: kinh =3.9x10 M s

MeO S MeO S + -hole + -hole O O H very weak intra-HB high ArO stabilization

FigureFigure 10. EﬀEffectect of aromatizationof aromatization on chain breakingon chain antioxidant breaking activity antioxidant of dihydrobenzo[ activityb]furane of / dihydrobenzo[benzo[b]furaneb]furane/benzo[vs dihydrobenzo[b]furaneb]thiophenes vs dihydrobenzo[/benzo[b]thiophenesb]thiophenes/benzo[b]thiophenes couples. couples.

b k Surprisingly,Surprisingly, 7-hydroxybenzo[ 7-hydroxybenzo[b]thiophenes]thiophenes 33–35 33–35 showed showed kinh inhhigherhigher than than those those of ofthe the correspondingcorresponding 7-hydroxydihydrobenzo[ 7-hydroxydihydrobenzo[bb]thiophenes]thiophenes 30–32,30–32, indicating indicating that that when when moving moving from from oxygen oxygento sulfur, to sulfur, aromatization aromatization becomes becomes beneficial beneficial for thefor chainthe chain breaking breaking activity activity [68 [68].]. To To rationalise rationalise this this difference, we can consider that aromatization brings the complete planarity of the system, hence a very weak intra-HB, as confirmed by IR measurements, is present in derivatives 33–35. However, this does not seem enough to justify the observed kinh increasing. On the other hand, it has been reported that sulfur σ-hole, i.e., the electron-poor zone pointing along the direction of the carbon- sulfur bond, becomes more important with aromatization moving from dihydrothiophenene to thiophene systems [69–76]. Actually, the σ-hole is responsible of the very weak intra-HB at ArOH level, but it could be also responsible for an extra stabilization of the vastly electron-rich area around the phenoxyl radical ArO•. This speculation was corroborated by ab-initio calculations that indicated a similar, yet numerically more significant effect, moving from sulfur to selenium and tellurium, exactly the trend expected when a chalcogen-bond effect is operative. Indeed, we have suggested that, all the peculiar observations done on the ortho-thiosubstituted phenolic compounds prepared Antioxidants 2019, 8, 487 10 of 22 difference, we can consider that aromatization brings the complete planarity of the system, hence a very weak intra-HB, as confirmed by IR measurements, is present in derivatives 33–35. However, this does not seem enough to justify the observed kinh increasing. On the other hand, it has been reported that sulfur σ-hole, i.e., the electron-poor zone pointing along the direction of the carbon-sulfur bond, becomes more important with aromatization moving from dihydrothiophenene to thiophene systems [69–76]. Actually, the σ-hole is responsible of the very weak intra-HB at ArOH level, but it could be also responsible for an extra stabilization of the vastly electron-rich area around the phenoxyl radical ArO . This speculation was corroborated by ab-initio calculations that indicated a similar, • yet numerically more significant effect, moving from sulfur to selenium and tellurium, exactly the trend expected when a chalcogen-bond effect is operative. Indeed, we have suggested that, all the peculiar observations done on the ortho-thiosubstituted phenolic compounds prepared so far (role of conformations, intra-HB strength, ArOH/ArO stabilization) can be rationalised only taking in the • proper consideration the role that chalcogens σ-hole plays in all these mentioned features [69–76] (Figure 10).

2.2. Selenium and Tellurium Containing Phenolic Antioxidants The chemistry of selenium and tellurium phenolic antioxidants has received a crucial contribution with the work of Professor L. Engman and co-workers. Actually, as a result of a collaboration between this group and the group of Professor G.F. Pedulli, the synthesis was published, very demanding indeed, and the evaluation of the antioxidant activity of allrac-1-seleno-α-tocopherol 36 [77] (Figure 11). Compound 36 showed a k of 1.2 106 M 1s 1 with a BDE of 78 kcal/mol almost superimposable to inh × − − those of the corresponding sulfur analogue 10, see Figure5, being both less potent than natural α-TOH 1a. More recently, the same authors were able to quantify the contribution that acyclic alkyl chalcogen substituents, as for example in derivatives 37–42, have on BDE of phenolic antioxidants as function relative chalcogen/-OH relative ortho or para position [78,79] (Figure 11). Thus, when chalcogen alkyl substituents are in para position, the better performances, i.e., the higher decrease of BDE, are produced by sulfur substituents, then selenium and tellurium. This can be explained considering that ED the effect in ArO stabilization decreases with increasing atomic weight. On the other hand, an opposite • trend is observed for alkyl chalcogen substituents in ortho position. In this case, the contribution of the chalcogen σ-hole seems to be the key to rationalising the result. In fact, a weaker intra-HB and a better ArO stabilization are expected when moving from sulfur to tellurium due to the increasing • chalcogen-Bond effect with atomic weight increasing [78]. For Se and Te substituted phenolic antioxidants, we can consider the different stereoelectronic contributions listed for sulfur derivatives. However, such discussion would be limited without considering the peculiar characteristic of many Se and Te substituted phenolic antioxidants i.e., the opportunity to be regenerated by a sacrificial reductant, typically a thiol, thus allowing to be used in a catalytic amount. Heavy chalcogens (Se, Te) are able to react with hydroperoxides to give a chalcogen oxide which, in turn, can be reduced back by two equivalents of a thiol with the formation of a disulfide and water (Figure 12, black frame). Due to the different red/ox potentials of chalcogens, this reaction is typically operative with tellurium derivatives, it can be operative with selenium derivatives, with rates significantly depending upon the whole structural features of the compound, while, generally, it occurs at a negligible rate with sulfur derivatives. With the obvious generalization, this is the process carried out by glutathione peroxidase (GPx) enzyme, and in particular by the selenocysteine residue operating in the enzyme active site. Indeed, GPx operates quenching hydroperoxides (ROOH) while a thiol (reduced glutathione, GSH) is oxidised to a disulfide [80–85] (oxidized glutathione, GSSG). However, studying the reaction of peroxyl radicals ROO with heavy chalcogen substituted phenols, we must • consider that two oxidizable sites are present and two different oxidative processes can take place: (i) the direct H transfer from ArOH and, (ii) the oxidation at chalcogen (mainly Se and Te). Both • the H transferability and the oxidation at chalcogen will depend by the whole molecular skeleton • Antioxidants 2019, 8, x FOR PEER REVIEW 10 of 22 so far (role of conformations, intra-HB strength, ArOH/ArO• stabilization) can be rationalised only taking in the proper consideration the role that chalcogens σ-hole plays in all these mentioned features [69–76] (Figure 10).

Antioxidants 2019, 8, x FOR PEER REVIEW 11 of 22

skeleton including the mutual influence of these groups. Additionally, the H• transfer process from FigureFigure 11. 11. allracallrac-1-Seleno--1-Seleno-α-tocopherolα-tocopherol 36, 36,and and model model chalcogen chalcogen substituted substituted polyphenols polyphenols used used to to ArOH to ROO• generates a hydroperoxide ROOH, able, in turn, to oxidize the chalcogen atom. quantifyquantify the the substitution substitution contribu contributiontion on on chain chain breaking breaking activity. activity.

For Se and Te substituted phenolic antioxidants, we can consider the different stereoelectronic contributions listed for sulfur derivatives. However, such discussion would be limited without considering the peculiar characteristic of many Se and Te substituted phenolic antioxidants i.e., the opportunity to be regenerated by a sacrificial reductant, typically a thiol, thus allowing to be used in a catalytic amount. Heavy chalcogens (Se, Te) are able to react with hydroperoxides to give a chalcogen oxide which, in turn, can be reduced back by two equivalents of a thiol with the formation of a disulfide and water (Figure 12, black frame). Due to the different red/ox potentials of chalcogens, this reaction is typically operative with tellurium derivatives, it can be operative with selenium derivatives, with rates significantly depending upon the whole structural features of the compound, while, generally, it occurs at a negligible rate with sulfur derivatives. With the obvious generalization, this is the process carried out by glutathione peroxidase (GPx) enzyme, and in particular by the selenocysteine residue operating in the enzyme active site. Indeed, GPx operates quenching hydroperoxides (ROOH) while a thiol (reduced glutathione, GSH) is oxidised to a disulfide [80–85] (oxidized glutathione, GSSG). However, studying the reaction of peroxyl radicals ROO• with heavy chalcogen substituted phenols, we must consider that two oxidizable sites are present and two different oxidative processes can take place: (i) the direct H• transfer from ArOH and, (ii) the oxidation at chalcogen (mainly Se and Te). Both the H• transferability and the oxidation at chalcogen will depend by the whole molecular Figure 12. Heavy chalcogens (Se, Te) possible catalytic cycles (black frame, red frame and blue frame) in theFigure presence 12. Heavy of hydroperoxides chalcogens (Se, or Te) peroxyl possible radicals catalytic as oxidants cycles (black and a frame, sacrificial red thiolframe as and co-antioxidant. blue frame) in the presence of hydroperoxides or peroxyl radicals as oxidants and a sacrificial thiol as co- Simplifyingantioxidant. the possible situations, we can imagine that the ROO initially extracts a H from • • the phenolic OH, as in all the examples seen up to now, with a rate depending by the ArOH skeleton includingSimplifying the effect the of possible the chalcogen situations, substituent we can (Figureimagin e12 that, red the frame). ROO• Then, initially the ROOHextracts formed a H• from can oxidizethe phenolic the chalcogen OH, as in to all the the corresponding examples seen chalcogen up to now, oxide with (Ch a rate=O). depending Eventually, by the the thiol ArOH regenerates skeleton theincluding starting the phenol effect thatof the behaves chalcogen as a substituent catalyst being (Figure the thiol 12, red the realframe). consumed Then, the co-antioxidant. ROOH formed can oxidizeHowever, the chalcogen in many to cases, the corresponding the substitution chalcogen pattern ofoxide the ArOH(Ch=O). cannot Eventually, justify thethe observedthiol regenerates rates of ROOthe startingdepletion, phenol with thatk behaves107 asM a1 scatalyst1. Thus, being recently, the thiol a di thefferent real mechanismconsumed co-antioxidant. has been suggested. • inh ≥ − − In thisHowever, new scenario, in many the cases, initial the reaction substitution of ROO patternoccurs of at the chalcogen ArOH cannot with the justify formation the observed of Ch=O rates and • anof ROO• alkoxyl depletion, radical RO with(Figure kinh ≥ 1210,7 blueM–1s–1 frame).. Thus, This recently, latter, a extracts, different typically mechanism in the has solvent been suggested. cage, a H • • fromIn this the new phenolic scenario, OH the giving initial the reaction ArO and of alcohol ROO• (ROH).occurs Eventually,at chalcogen the with thiol the reduces formation the chalcogen of Ch=O • oxideand an and alkoxyl the phenoxyl radical RO• radical (Figure regenerating 12, blue frame). the phenolic This latter, antioxidants. extracts, typically In this way, in the the solvent H transfer cage, • processa H• from can the be muchphenolic faster OH than giving those the described ArO• and in alcohol the red (ROH). frame andEventually, much less the sensitive thiol reduces to ArOH the wholechalcogen substitution oxide and pattern, the phenoxyl in particular radical when regenerating the phenolic the phenolic OH and antioxidants the chalcogen. In substituent this way, the where H• thetransfer RO processis formed can are be adjacent. much faster than those described in the red frame and much less sensitive to ArOH •whole substitution pattern, in particular when the phenolic OH and the chalcogen substituent where the RO• is formed are adjacent. It must be underlined that, independently upon the mechanism operative, heavy chalcogen containing antioxidants seems particularly indicated at the biological level being able of quenching peroxyl radicals (ROO•) and hydroperoxides (ROOH) giving water, alcohols and disulfides as the only reaction products. However, depletion of endogenous thiols can, in turn, be a potentially dangerous situation. For many phenols containing heavy chalcogens, the antioxidant activity and, in particular, their ability to act as catalytic antioxidants has been measured quantifying, by HPLC, the amount of conjugated diene formed from a polyunsaturated fatty acid in the presence of a radical initiator and molecular oxygen [86]. Thus, the amount of conjugated diene formed in the presence of the substrate under study, until its consumption, gives the inhibited rate of peroxidation (Rinh) and the inhibition time (Tinh) that taken together represent a solid quantification of the phenol ability to block chain oxidations. For example, under the standard conditions used (chlorobenzene as solvent, linoleic acid Antioxidants 2019, 8, 487 12 of 22

It must be underlined that, independently upon the mechanism operative, heavy chalcogen containing antioxidants seems particularly indicated at the biological level being able of quenching peroxyl radicals (ROO ) and hydroperoxides (ROOH) giving water, alcohols and disulfides as the • only reaction products. However, depletion of endogenous thiols can, in turn, be a potentially dangerous situation. For many phenols containing heavy chalcogens, the antioxidant activity and, in particular, their ability to act as catalytic antioxidants has been measured quantifying, by HPLC, the amount of conjugated diene formed from a polyunsaturated fatty acid in the presence of a radical initiator and molecular oxygen [86]. Thus, the amount of conjugated diene formed in the presence of the substrate under study, until its consumption, gives the inhibited rate of peroxidation (Rinh) and the inhibition time (Tinh) that taken together represent a solid quantification of the phenol ability to block chain oxidations. For example, under the standard conditions used (chlorobenzene as solvent, linoleic acid > 30 mM, antioxidant 40 µM) α-TOH (1a) showed a R 25 µM h 1, and T 90 min [87,88]. If the measure inh ≈ − inh ≈ is carried out in a two phases system in the presence of an excess of a proper thiol, usually N-acetyl cysteine (NAC) 1 mM, it is possible to verify the catalytic activity verifying the increase of the inhibition time Tinh. For example, for α-TOH, Tinh and Rinh are insensitive to the presence of a co-antioxidant thiol. Indeed, it is well known that thiols are unable to regenerate tocopherols after oxidation. For tellurium containing phenols, carrying on the measure in the absence of a co-antioxidant thiol, means not only that we do not observe any increase of Tinh, but quite low Rinh is measured. In other words, it seems that, without the reducing thiol, it also vanishes the ability of the tellurium substituted phenol to react with ROO . This phenomenon has been explained considering that commercially available linoleic • acid contains variable amounts, increasing with storing, of the corresponding hydroperoxides formed by autoxidation. As depicted in Figure 12 black frame, hydroperoxides are able to oxidize the tellurium derivatives to the corresponding telluroxides. Thus, in the absence of a thiol, the measured Rinh is not that of the Tellurium substituted ArOH, yet that, much worst, of the corresponding a telluroxide R2Te=O[28,79,87–91]. Focussing on Tellurium substituents (Figure 13), since the first studies on symmetrical diarylhydroxy tellurides like 43 [86], it was clear how para- and, above all, ortho-hydroxy derivatives work quite well with Rinh and Tinh better than that of α-TOH. Inserting the Tellurium atom in a 1 five-membered benzo-fused ring, like in 44, ensured a good Rinh while Tinh was quite short (22 µM h− and 60 min) [28]. More recently, phenolic skeletons that allow a very good chain breaking antioxidants, independently upon the presence of a chalcogen atom have been considered [78,79]. For example, compound 45, with several other structural related derivatives, or compound 46, bearing the Te atom on the skeleton of pyridoxine (Vitamin B6) have been prepared and their antioxidant activity studied [87,88,90,91] (Figure 13). Results obtained with these phenolic derivatives follow the above-discussed trend, thus compound 45 showed Rinh better than α-TOH and a very good catalytic activity in the presence of NAC with T 400 min [91]. On the other hand, tellurated Vitamin B6 analogue 46 showed an even better inh ≥ Rinh yet a worse catalytic activity [87]. For these compounds also, the presence of a co-antioxidant thiol is mandatory to maintain both activities. Recently, Prof Engman merging the above observations, designed and prepared a set of very potent diaryl telluro derivatives, like 47 and 48, [89] (Figure 13) whose structure allowed the synergetic action of the Te residue on the OH group H transfer process • aptitude, and the OH group in the oxidation potential of Tellurium. Thus, exploiting the ED donating effect of the oxygen atom in a five-membered benzo-fused ring and the whole system conjugation, it was possible to maximize the reaction of ROO at Te followed by the H transfer to RO as described • • 1 • in the blue frame of Figure 12. As a matter of fact, measured Rinh (0.4 and 0.3 µM h− ) and Tinh (up to 600 min) for 47 and 48 were the best ever reported. It is worth nothing that the Rinh performance of these derivatives is only partially lost in the absence of co-reductant thiol. For example, without 1 any co-antioxidant thiol, derivative 48 showed Rinh = 23 µM h− and Tinh = 136 min, performances superimposable to that of α-TOH confirming the success of the overall structural design [89]. Antioxidants 2019, 8, x FOR PEER REVIEW 12 of 22

> 30 mM, antioxidant 40 μM) α-TOH (1a) showed a Rinh ≈ 25 μM h–1, and Tinh ≈ 90 min [87,88]. If the measure is carried out in a two phases system in the presence of an excess of a proper thiol, usually N-acetyl cysteine (NAC) 1 mM, it is possible to verify the catalytic activity verifying the increase of the inhibition time Tinh. For example, for α-TOH, Tinh and Rinh are insensitive to the presence of a co- antioxidant thiol. Indeed, it is well known that thiols are unable to regenerate tocopherols after oxidation. For tellurium containing phenols, carrying on the measure in the absence of a co- antioxidant thiol, means not only that we do not observe any increase of Tinh, but quite low Rinh is measured. In other words, it seems that, without the reducing thiol, it also vanishes the ability of the tellurium substituted phenol to react with ROO•. This phenomenon has been explained considering that commercially available linoleic acid contains variable amounts, increasing with storing, of the corresponding hydroperoxides formed by autoxidation. As depicted in Figure 12 black frame, hydroperoxides are able to oxidize the tellurium derivatives to the corresponding telluroxides. Thus, in the absence of a thiol, the measured Rinh is not that of the Tellurium substituted ArOH, yet that, much worst, of the corresponding a telluroxide R2Te=O [28,79,87–91]. Focussing on Tellurium substituents (Figure 13), since the first studies on symmetrical diarylhydroxy tellurides like 43 [86], it was clear how para- and, above all, ortho-hydroxy derivatives work quite well with Rinh and Tinh better than that of α-TOH. Inserting the Tellurium atom in a five- membered benzo-fused ring, like in 44, ensured a good Rinh while Tinh was quite short (22 μM h–1 and 60 min) [28]. More recently, phenolic skeletons that allow a very good chain breaking antioxidants, independently upon the presence of a chalcogen atom have been considered [78,79]. For example, compound 45, with several other structural related derivatives, or compound 46, bearingAntioxidants the2019 Te , 8atom, 487 on the skeleton of pyridoxine (Vitamin B6) have been prepared and their13 of 22 antioxidant activity studied [87,88,90,91] (Figure 13).

FigureFigure 13. 13. A selectionA selection of potent of potent Tellurium Tellurium containing containing phenolic phenolic antioxidants antioxidants prepared prepared so far. so far.

ResultsSelenium obtained substituted with phenolsthese phenolic showed derivati performancesves follow in between the above-discussed those of analogues trend, containing thus compoundsulfurAntioxidants or tellurium, 201945 showed, 8, x FOR as PEER expectedRinh betterREVIEW for than the intermediateα-TOH and a red very/ox profilegood catalytic of Selenium. activity Indeed, in the selenotocopherol presence13 of 22 of NAC36 (see with Figure Tinh ≥ 11 400) was min a [91]. chain On breaking the other antioxidant hand, tellurated very similar Vitamin to theB6 analogue sulfur analogue 46 showed 10 (see an Figureeven 5) betterbothselenotocopherol showingRinh yet a performances worse 36 (see catalytic Figure worse activity11) thanwas natural[87].a chain For αbreaking -TOHthese (seecompounds antioxidant Figure1). also, Avery similar thesimilar presence trend to the was of sulfur observeda co- α antioxidantforanalogue derivatives 10 thiol (see 36is Figure andmandatory 41 5) [78 both,79 to ] showmaintain (Figureing 11performances both) as wellactivities. as selenopyridinol worse Recently, than naturalProf 49 Engman [ 90-TOH] (Figure merging (see 14 Figure) that the 1). showedabove A a similar trend was observed for derivatives 36 and 41 [78,79] (Figure 11) as well as selenopyridinol 49 observations,Rinh higher thandesigned those and of the prepared corresponding a set of telluriumvery potent derivatives diaryl telluro and veryderivatives, similar like to the 47 sulfurand 48, ones. [90] (Figure 14) that showed a Rinh higher than those of the corresponding tellurium derivatives and [89]Moreover, (Figure 13) none whose of these structure compounds allowed showed the synerget an effiiccient action catalytic of the Te activity residue with on the inhibition OH group times H•T inh very similar to the sulfur ones. Moreover, none of these compounds showed an efficient catalytic transfer(and inhibition process aptitude, rates Rinh and) almost the OH independent group in the upon oxidation the presence potential of a co-antioxidantof Tellurium. Thus, thiol. exploiting A great eff ort activity with inhibition times Tinh (and inhibition rates Rinh) almost independent upon the presence of thehas ED been donating devoted effect to rationalizing of the oxygen the activityatom in of a dihydrobenzo[ five-memberedb]selenophenes benzo-fused ofring type and 50–55 the [whole28,92– 96] a co-antioxidant thiol. A great effort has been devoted to rationalizing the activity of system(Figure conjugation, 14). For all it of was these possible compounds to maximize it appears the thatreaction the valueof ROO• of the at Te inhibition followed rate byR the H•can be dihydrobenzo[b]selenophenes of type 50–55 [28,92–96] (Figure 14). For all of these compoundsinh it transferrationalised to RO• considering as described the in ED the and blueortho frame-OH of eff Figureect (including 12. As a the matter contribution of fact, measured of Se σ-hole Rinh in (0.4 ArO inh appearsμ that–1 the value of the inhibition rate R can be rationalised considering the ED and ortho-OH • andstabilization). 0.3 M h ) and Additionally, Tinh (up to for600 all min) compounds, for 47 and R48 wereis almost the best insensitive ever reported. to the It presence is worth ofnothing a thiol as effect (including the contribution of Se σ-hole in ArO•inh stabilization). Additionally, for all compounds, thatco-reductant the Rinh performance during the of measure. these derivatives Thus, for theseis only selenium partially derivatives lost in the absence the oxidation of co-reductant to R Se=O, thiol. due to Rinh is almost insensitive to the presence of a thiol as co-reductant during the measure. Thus,2 for these μ –1 Forthe example, adventitious without presence any co-antioxidant of hydroperoxides, thiol, is derivative not able to 48 modify showed the R antioxidantinh = 23 M h kinetic and profileTinh = 136 either selenium derivatives the oxidation to R2Se=O, due to the adventitious presence of hydroperoxides, is min, performances superimposable to that of α-TOH confirming the success of the overall structural becausenot able itto does modify not the take antioxidant place or because kinetic itprofile occurs ei muchther because slower it than does the not reaction take place with or peroxyl because radicals. it design [89]. Inoccurs a recent much paper slower [94 than], Engman the reaction reported with that peroxyl 5-hydroxy- radicals. and In a 7-hydroxybenzo[ recent paper [94],b Engman]selenophenes reported 52 and Selenium substituted phenols showed performances in between those of analogues containing 53that (Figure 5-hydroxy- 14), i.e., and those 7-hydroxybenzo[ with a para-b and]selenophenesortho- arrangement 52 and 53 (Fig betweenure 14), the i.e., selenium those with atom a para and- and the OH sulfur or tellurium, as expected for the intermediate red/ox profile of Selenium. Indeed, group,ortho- arrangement are indeed ablebetween to be the regenerated selenium atom by a and thiol the with OHT group,inh from are four indeed to fiveable timesto be regenerated longer (504 min andby a 420thiol min with respectively) Tinh from four than to those five measuredtimes longer without (504 min the sacrificialand 420 min thiol. respectively) For these twothan derivatives, those 1 μ – themeasured measured withoutRinh the(15 sacrificial and 20 µ thiol.M h− Forrespectively) these two derivatives, are too fast the to measured be justified Rinh considering (15 and 20 M an h initial H1 respectively)transfer process are too from fast to the be ArOH justified to considering the ROO asan ininitial Figure H• 12transfer red frame. process Also from in the this ArOH case, to it was • • postulatedthe ROO• as that in theFigure first 12 reaction red frame. is the Also oxidation in this ca atse, selenium it was postulated by the peroxyl that radicalthe first followedreaction is by the the H • transferoxidation ‘in at the selenium solvent by cage’ the asperoxyl depicted radical in the follow blueed frame by the of FigureH• transfer 12. This ‘in allowedthe solvent rationalising cage’ as the orderdepicted of reactivity,in the blue being frame derivative of Figure 5312. (with This chalcogenallowed rationalisingortho-OH e theffects order plus of a site-favouredreactivity, being ‘in the solventderivative cage’ 53 (with H transfer) chalcogen that ortho showed-OH effects the better plus performancesa site-favoured (Figure ‘in the solvent14). cage’ H• transfer) that showed the• better performances (Figure 14).

FigureFigure 14. 14. AA selection selection of ofSelenium Selenium containing containing phenols phenols prepared prepared so far. so far.

In the sulfur series, we have shown that the aromatization of dihydrobenzo[b]thiophenes to benzo[b]thiophenes is beneficial for the chain breaking antioxidant activity [68]. Among the few examples available of benzo[b]selenophenes used as antioxidants, derivatives 57–61 are probably the more interesting [95,96] (Figure 15) whose skeletons can be associated with that of natural resveratrol (compound 3 Figure 1). Compounds 58–61 [96], and other related derivatives were prepared and tested for their antiproliferative activity, while the peroxides quencher ability was tested using, mainly, qualitative approaches that have not been considered in this focussed review. Antioxidants 2019, 8, 487 14 of 22

In the sulfur series, we have shown that the aromatization of dihydrobenzo[b]thiophenes to benzo[b]thiophenes is beneﬁcial for the chain breaking antioxidant activity [68]. Among the few examples available of benzo[b]selenophenes used as antioxidants, derivatives 57–61 are probably the more interesting [95,96] (Figure 15) whose skeletons can be associated with that of natural resveratrol (compound 3 Figure1). Compounds 58–61 [ 96], and other related derivatives were prepared and tested for their antiproliferative activity, while the peroxides quencher ability was tested using, mainly, Antioxidantsqualitative 2019 approaches, 8, x FOR PEER that REVIEW have not been considered in this focussed review. 14 of 22

FigureFigure 15. 15.A selectionA selection of benzo[ of benzo[b]selenophenesb]selenophenes antioxidants antioxidants reported reported so sofar. far.

OnOn the the other other hand, hand, compound compound 57 showedshowed a a remarkable remarkable chain-breaking chain-breaking antioxidant antioxidant activity activity better betterthan than the naturalthe natural inspiring inspiring model model 3 [95]. 3 After [95]. theAfter insertion the insertion of the selenium of the selenium atom the atom OH involved the OH in involvedthe H intransfer the H• process, transfer i.e., process, that with i.e., the that lower with BDE, the lower was the BDE, OH was on C7, theortho OH toon the C7, selenium ortho to the atom, • seleniumwhile the atom, more while reactive the in more resveratrol reactive 3 is in the resveratrol OH on C4 03, activatedis the OH by on the C4para′, activated-conjugated by double the para bond.- conjugatedDespite being double measured bond. with Despite two di ffbeingerent methodologies,measured with dihydroselenophenes, two different methodologies, like 52 or 53 [94 ], dihydroselenophenes,react with ROO radicals like 52 faster or 53 than [94], selenophene react with 57ROO• [95]. radicals This seems faster to bethan in selenophene contrast with 57 the [95]. result • Thisachieved seems to with be in sulfur contrast analogues with the that, result after achiev aromatization,ed with sulfur increased analogues their that, antioxidant after aromatization, activity. In our increasedopinion their this incongruityantioxidant inactivity. only apparent In our opinion since two this di ffincongruityerent oxidation in only mechanisms apparent are since operative. two differentIn dihydroselenophenes oxidation mechanisms 52 and are 53 theoperative. mechanism In dihydroselenophenes described in Figure 12 52 blue and frame 53 the is working,mechanism with describedthe initial in oxidationFigure 12 atblue selenium frame is and working, ‘in the solventwith the cage’ initial extraction oxidation of at H seleniumas the final and step. ‘in the On solvent the other • cage’hand, extraction benzoselenophene of H• as the 57 final reasonably step. On reacts the other with peroxylhand, benzoselenophene radicals ROO by 57 a directreasonably H extraction reacts • • withfrom peroxyl ArOH radicals because ROO• the oxidation by a direct at H• selenium extraction and from formation ArOH ofbecause a R2Se th=eO oxidation derivative at isselenium not more andfeasible formation after of aromatization. a R2Se=O derivative In other is words,not more very feasible probably, after also aromatization. for hydroxy In benzo[ otherb words,]selenophenes very probably,aromatization also for increases hydroxy the benzo[ stabilizationb]selenophenes of the corresponding aromatization ArO incr,eases mainly the by stabilizationσ-hole issues, of but the the • correspondingoxidation at SeleniumArO•, inmainly dihydrobenzo[ by σ-holeb]selenophenes issues, remainsbut the a favouriteoxidation path. at Selenium in dihydrobenzo[b]selenophenes remains a favourite path. 2.3. Chalcogens in Tocopherol Skeletons 2.3. Chalcogensα-Tocopherol in Tocopherol (α-TOH, Skeletons 1a Figure 1) is the main component of Vitamin E and the more potent lipophilicα-Tocopherol phenolic (α antioxidant-TOH, 1a Figure known 1) in is nature the main [6–11 component]. Despite theof Vitamin role of tocopherols E and the atmore the biologicalpotent level still being controversial [97–99], their antioxidant activity is clear as well as the ROO quenching lipophilic phenolic antioxidant known in nature [6–11]. Despite the role of tocopherols• at the biologicalmechanism level operated still being by controversial these compounds. [97–99], Thus, their antioxidant researchers activity involved is inclear the as design, well as synthesis the ROO• and quenchingapplication mechanism of new bio-inspired operated syntheticby these antioxidantscompounds. use Thus,α-TOH researchers as a model involved to imitate in [100 the], adesign, target to synthesisreach or, and possibly, application to overcome of new [bio-29,34inspired]. For example, synthetic derivative antioxidants 62 was use prepared α-TOH as to a add model the to features imitate of a [100],catechol a target residue to reach to that or, ofpossibly, 1a [29], to while overcome compound [29,34]. 63, For exploiting example, the derivative great ED e ff62ect was of thepreparedpara-amino to addsubstituent the features and of the a catechol pyridinol residue ring ability to that to of avoid 1a [29], the while spontaneous compound reaction 63, exploiting molecular the oxygen, great ED likely showed better antioxidant performances among α-TOH analogue with k 6 107 M 1s 1, up to effect of the para-amino substituent and the pyridinol ring ability to avoid theinh spontaneous≈ × − reaction− molecular20 times oxygen, higher likely than theshowed natural bett modeler antioxidant [34] (Figure performances 16). We alreadyamong α discussed-TOH analogue the properties with kinh of ≈ 6thiotocopherol × 107 M–1s–1, up 10 to [45 20], times and selenotocopherol higher than the 36natural [77] and model the reasons [34] (Figure for the 16). decreased We already activity discussed observed thefor properties these analogues. of thiotocopherol Having demonstrated 10 [45], and the se possibilitylenotocopherol to obtain 36 [77] benzoxathiines and the reasons antioxidants, for the we decreasedadapt the activity synthetic observed procedure for forthese the analogues. preparation Having of (2-ambo demonstrated-40R,80R)-tocopherol the possibility derivatives to obtain 64a–d benzoxathiines antioxidants, we adapt the synthetic procedure for the preparation of (2-ambo- 4′R,8′R)-tocopherol derivatives 64a–d possessing the aromatic skeletons of α-, β-, γ- and δ-tocopherol, i.e., the four saturated components of Vitamin E [101]. As previously explained, due to the flexibility of the benzoxathiine ring, compounds 64a–d were less potent than corresponding analogues 1a–d. Additionally, for these compounds also, BDE decreases, and kinh increases, with the number of methyl groups being 64a (analogue to α-TOH) the more potent and 64d (analogue to δ-TOH) the less potent chain breaking antioxidant in this series [101] (Figure 16). Antioxidants 2019, 8, 487 15 of 22

possessing the aromatic skeletons of α-, β-, γ- and δ-tocopherol, i.e., the four saturated components of Vitamin E [101]. As previously explained, due to the ﬂexibility of the benzoxathiine ring, compounds 64a–d were less potent than corresponding analogues 1a–d. Additionally, for these compounds also, BDE decreases, and kinh increases, with the number of methyl groups being 64a (analogue to α-TOH) the more potent and 64d (analogue to δ-TOH) the less potent chain breaking antioxidant in this Antioxidantsseries [101 2019], (Figure8, x FOR 16PEER). REVIEW 15 of 22

OH HO HO

O N N 62 63

HO HO

S Se 10 36

1 R ChR HO S HO

R2 O O

1 2 64b 1 2 64a:R =R =Me :R =Me,R =H, 65a: Ch = S,R=nBu 65b: Ch = S,R=nOct 1 2 64d 1 2 64c:R =H,R =Me :R =R =H 66a: Ch = Se,R=nBu 66b: Ch = Se,R=nOct 67a: Ch = Te,R=nBu 67b: Ch =Te,R=nOct

HO HO

RCh O RCh O

68a: Ch = S,R=nBu 68b: Ch = S,R=nOct 71a: Ch = S,R=nBu 71b: Ch = S,R=nOct 69a: Ch = Se,R=nBu 69b: Ch = Se,R=nOct 72a: Ch = Se,R=nBu 72b: Ch = Se,R=nOct 70a: Ch = Te,R=nBu 70b: Ch = Te,R=nOct 73a: Ch = Te,R=nBu 73b: Ch = Te,R=nOct

HO HO

S O S O 74 75 FigureFigure 16. 16.A selectionA selection of tocopherols of tocopherols analogues analogues prepared prepared so far. so far.

DerivativesDerivatives 10, 10,36 36and and 64 64were were not not assembled assembled using using a pre-organized a pre-organized chromane chromane skeleton skeleton and, and, indeed,indeed, required required significant significant synthetic synthetic efforts. efforts. On On the the other other hand, hand, derivatives derivatives 65–73 65–73 were were prepared prepared to to introduceintroduce sulfur, sulfur, selenium selenium or tellurium or tellurium alkyl alkyl residues residues in the in skeleton the skeleton of commercially of commercially available available β-, γ-β -, andγ -δ and-tocopherolδ-tocopherol exploiting exploiting for functionalizations for functionalizations the unsubstituted the unsubstituted position(s) position(s) on the aromatic on the aromatic ring [102,103].ring [102 Tellurium,103]. Tellurium compounds compounds like 67, like 70 67, and 70 73 and showed 73 showed inhibition inhibition rates rates RinhR forinh thefor thereaction reaction with with ROO , similar to those ofα α-TOH, but, differently fromα α-TOH, can be regenerated by a co-reductant ROO•, similar• to those of -TOH, but, differently from -TOH, can be regenerated by a co-reductant thiolthiol with with extra-long extra-long inhibition inhibition times times TinhT.inh After. After learning learning that that a benzo[ a benzo[b]thiopheneb]thiophene is probably is probably the the best best sulfur-containing residue to be inserted ortho to a phenolic OH involved in the H transfer process, we sulfur-containing residue to be inserted ortho to a phenolic OH involved in the H•• transfer process, wesynthesized synthesized derivatives derivatives 74 74 and and 75 as75 the as resultthe result of five of consecutive five consecutive electrophilic electrophilic processes processes occurring occurringone-pot one-pot from carefully from carefully designed designed benzoxathiines benzoxathiines [104]. Indeed, [104]. benzo[Indeed,b ]thiophenesbenzo[b]thiophenes 74 and 75 74 showed and 6 1 1 6 1 1 k of 6 10 M s6 –1and–1 9 10 M6 s–1 –1 respectively, to the best of our knowledge, the better 75 showedinh k×inh of 6 × −10 − M s and× 9 × 10 M− −s respectively, to the best of our knowledge, the better performancesperformances ever ever reported reported for for a sulfur a sulfur containing containing phenolic phenolic antioxidant. antioxidant. Additionally, Additionally, as asalready already reportedreported for for derivative derivative 63 63[34], [34 we], we demonstrated demonstrated that that the the structural structural modification modification brought brought about about the the constructionconstruction of the of the thiophene thiophene ring ring can canbe well be well tolerated tolerated at biological at biological level level and compounds and compounds 74 and 74 75 and showed75 showed a binding a binding with withα-tocopherolα-tocopherol transfer transfer protein protein (α-TTP) (α-TTP) very very similar similar to that to that of the of the natural natural substratesubstrate α-TOHα-TOH [104].- [104 ]. VeryVery recently, recently, the abilityability in in the the manipulation manipulati ofon heavy of heavy chalcogen chalcogen reagents reagents allowed theallowed preparation the preparationof di-tocopheryl of di-tocopheryl tellurides 76–78 tellurides [105] and 76–78 ditocopheryl [105] and sulfides ditocopheryl 79, 81, 83 sulfides and ditocopheryl 79, 81, 83 disulfides and ditocopheryl80, 82, 84 [106 disulfides] (Figure 1780,). 82, Ditocopheryl 84 [106] (Figure tellurides 17). 76–78 Ditocopheryl showed, as expected,tellurides a76–78 very goodshowed, inhibition as expected,rate Rinh aand very catalytic good inhibition activity inhibition rate Rinh and times catalyticTinh as activity already inhibition reported in times similar TinhTe-substituted as already reported phenols. in similar Te-substituted phenols. Ditocopheryl sulfides and disulfides were generally less reactive towards ROO• than the corresponding tocopherols. This was expected in light of previously discussed considerations about ortho-OH effect of acyclic sulfur substituents [43] (see Figures 5 and 7). In more detail, for each couple (δ,δ-, γ,γ-, β,β-) disulfides are less active than sulfides and δ,δ- ditocopheryl sulfide 79 was the compound with the higher kinh. The better activity of sulfides than disulfides can be rationalised, as confirmed by FT-IR studies and supported by ab-initio calculations, considering that in disulfides both phenolic OH are engaged in an intra-HB, while in sulfides only one phenolic OH is engaged, while the other can efficiently participate to the H• transfer process. Rationalization of the superiority of 79 when compared with 81 and 83, despite the lack of one ED Antioxidants 2019, 8, 487 16 of 22

Ditocopheryl sulfides and disulfides were generally less reactive towards ROO than the corresponding • tocopherols. This was expected in light of previously discussed considerations about ortho-OH effect of acyclic sulfur substituents [43] (see Figures5 and7). In more detail, for each couple ( δ,δ-, γ,γ-, β,β-) disulfides are less active than sulfides and δ,δ-ditocopheryl sulfide 79 was the compound with the higher kinh. The better activity of sulfides than disulfides can be rationalised, as confirmed by FT-IR studies and supported by ab-initio calculations, considering that in disulfides both phenolic OH are engaged in an intra-HB, while in sulfides only one phenolic OH is engaged, while the other can efficiently participate to the H transfer process. Rationalization of the superiority of 79 when • Antioxidantscompared 2019 with, 8, 81 x FOR and PEER 83, despite REVIEW the lack of one ED methyl group is less trivial. However, molecular16 of 22 dynamic calculations showed that in the absence of a methyl group ortho to the phenolic OH, as it methyl group is less trivial. However, molecular dynamic calculations showed that in the absence of occurs in δ,δ-79, the intra-HB strength is further decreased (Figure 18). In other words, the additional a methyl group ortho to the phenolic OH, as it occurs in δ,δ-79, the intra-HB strength is further ortho-methyl group in sulfides γ,γ-81 or β,β-83 favours conformations with the phenolic OH protons decreased (Figure 18). In other words, the additional ortho-methyl group in sulfides γ,γ-81 or β,β-83 pointing toward the sulfur atom, increasing the intra-HB strength hence reducing the chain breaking favours conformations with the phenolic OH protons pointing toward the sulfur atom, increasing the antioxidant activity [106]. intra-HB strength hence reducing the chain breaking antioxidant activity [106].

Figure 17. ChalcogensChalcogens containing containing ditoco ditocopherolspherols prepared so far.

Figure 18. Role of intra-HB in δ,δ-ditocopheryl sulfide 79 and δ,δ-ditocopheryl disulfide 80. Antioxidants 2019, 8, x FOR PEER REVIEW 16 of 22 methyl group is less trivial. However, molecular dynamic calculations showed that in the absence of a methyl group ortho to the phenolic OH, as it occurs in δ,δ-79, the intra-HB strength is further decreased (Figure 18). In other words, the additional ortho-methyl group in sulfides γ,γ-81 or β,β-83 favours conformations with the phenolic OH protons pointing toward the sulfur atom, increasing the intra-HB strength hence reducing the chain breaking antioxidant activity [106].

Antioxidants 2019, 8, 487 17 of 22 Figure 17. Chalcogens containing ditocopherols prepared so far.

Figure 18. RoleRole of of intra-HB intra-HB in δδ,,δδ-ditocopheryl-ditocopheryl sulfide sulfide 79 and δ,,δ-ditocopheryl disulfide disulfide 80. 3. Conclusions The fine-tuning of the chalcogens stereoelectronic features, the ability in design and synthesis of chalcogens containing compounds and the skills to meticulously evaluate the chain-breaking antioxidant activity allowed the preparation of a plethora of new polyphenolic antioxidants. The actual role of heavy chalcogens (S, Se and Te) in modulating the chain-breaking antioxidant activity has been reviewed and discussed. Very few insights are available about the toxicity/biocompatibility of the great part of the derivatives shown here. Since many of these compounds derive from natural precursors, the study of eventual change of toxicity after the introduction of the heavy chalcogens (above all Se and Te) would be of great interest. Additionally, the medicine industry always searches for new antioxidants with better chemical or biological properties, thus we hope this review could stimulate the readers interest and curiosity to face these research challenges.

Author Contributions: C.V. and S.M. have been engaged in writing, reviewing and editing of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

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