Resveratrol-based benzoselenophenes with an enhanced antioxidant and chain breaking capacity

Biomolecular Chemistry

PAPER

Cite this: Org. Biomol. Chem., 2015, 13, 5757 Received 30th January 2015, Accepted 13th April 2015 DOI: 10.1039/c5ob00193e www.rsc.org/obc

Resveratrol-based benzoselenophenes

with an enhanced antioxidant and chain

breaking capacity

†

Damiano Tanini,

Lucia Panzella,*

Riccardo Amorati,

Antonella Capperucci,*

Elio Pizzo,

Alessandra Napolitano,

Stefano Menichetti

and Marco d

’Ischia

The structural modification of the resveratrol scaffold is currently an active issue in the quest for more potent and versatile antioxidant derivatives for biomedical applications. Disclosed herein is an expedient and efficient entry to a novel class of resveratrol derivatives featuring an unprecedented 2-phenylbenzo-selenophene skeleton. The new compounds were obtained in good yields by direct selenenylation of resveratrol with Se(0) and SO2Cl2in dry THF. Varying the [Se : SO2Cl2: resveratrol] ratio resulted in the

for-mation of the parent benzoselenophene (1) and/or mono (2) and/or dichloro (3) benzoselenophene derivatives. All the benzoselenophene derivatives proved to be more efficient than resveratrol in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) assays, with 1 showing an activity nearly comparable to that of Trolox. 1–3 also proved to be more efficient inhibitors than the parent resveratrol in kinetic experiments of styrene autoxidation. DFT calculations of the O–H bond dis-sociation enthalpy (BDE) revealed that the introduction of the Se-atom causes a significant decrease of the BDE of 3-OH and 5-OH, with just a small increase of the 4’-OH BDE. Compounds 1–3 showed no cytotoxicity at 5μM concentrations on human keratinocyte (HaCaT) and intestinal (CaCo-2) cell lines.

Introduction

Natural or natural product-inspired phenolic antioxidants have attracted widespread interest as food supplements and as addi-tives for a broad range of applications. Because of the growing demand for cheap, efficient and non-toxic antioxidants on the part of food, pharmaceutical and polymer industries, consider-able efforts are currently devoted to develop practical manipu-lation strategies for potentiating and tailoring the antioxidant properties of natural phenolic scaffolds based on the struc-ture–property relationships.1

Among the polyphenol-based natural products, stilbene phytoalexin resveratrol is one of the most widely distributed. Isolated from a broad range of plants, resveratrol owes its

notoriety to its occurrence in red wine and its implication in the“French paradox”.2

Currently, it is on the market as a food supplement because of its manifold positive activities on human health3 and has been extensively investigated as a versatile platform for the design of natural product-like compounds.4Some of the bio-logical effects of resveratrol are related to its antioxidant activity, as it prevents the oxidation of LDL in vitro and reduces the markers of oxidative stress in vivo.5The antioxidant activity depends on the 4′-OH group, which exhibits a superior H-atom transfer and peroxyl radical scavenging capacity compared to the resorcinol-type OH groups, due to the olefinic double bond contribution to resonance stabilization of the phenoxyl radical.6 However, despite many favorable properties, resvera-trol antioxidant activity cannot be compared with that of other natural phenols. Accordingly, the development of new deriva-tives with a stronger chain breaking and free radical scaven-ging capacity is an important research goal for both scientific and practical interests.

†Electronic supplementary information (ESI) available: NMR spectra of 1–3; optimized stoichiometry ratios for the isolation of 1–3; computational data. See DOI: 10.1039/c5ob00193e

a_{Department of Chemistry“Ugo Schiff”, University of Florence, Via della Lastruccia}

13, I-50019 Sesto Fiorentino, Italy. E-mail: antonella.capperucci@unifi.it

b

Department of Chemical Sciences, University of Naples“Federico II”, Via Cintia 4, I-80126 Naples, Italy. E-mail: panzella@unina.it

c_{Department of Chemistry“G. Ciamician”, University of Bologna, Via S. Giacomo 11,}

I-40126 Bologna, Italy

d_{Department of Biology, University of Naples“Federico II”, Via Cintia 4, I-80126}

Naples, Italy

Published on 13 April 2015. Downloaded by Universita Studi di Firenze on 21/05/2015 14:43:51.

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It is well known that the electron-donating groups in the para and ortho positions to the phenolic hydroxyl lower the O–H bond dissociation enthalpy (BDE) and increase the rate of H-atom transfer to peroxyl radicals.7,8In addition, the incorporation of nitrogen atoms into the aromatic ring of phenolic compounds to obtain pyridinols and pyrimidinols has enabled the development of potent radical-trapping antioxidants, characterized by an increased stability under air with respect to the parent phenols.9

Several studies support the introduction of 3rdperiod and higher chalcogen atoms as another effective means of enhan-cing the antioxidant capacity of phenolic compounds.10,11 Sel-enium-substitution was found to enhance the chain breaking activity via a decrease in the BDE values of the phenolic groups.12,13Similarly, the introduction of an octyltelluro group into the β- or δ-tocopherol system resulted in more efficient quenching of peroxyl radicals via an unusual oxygen transfer process to the chalcogen in the presence of N-acetylcysteine.14

In this paper, we have reported selenium-substitution as a means of boosting the resveratrol antioxidant capacity via con-version to 2-phenylbenzoselenophene derivatives showing a Trolox-like antioxidant and chain-breaking capacity.

Results and discussion

Preparation of the benzoselenophene derivatives

The proposed approach for the preparation of the benzoseleno-phene derivatives hinges on an efficient selenenylation pro-tocol involving in situ generation of SeCl2, a convenient source

of electrophilic Se2+,15 by the reaction of Se(0) with SO2Cl2

(eqn (1)),16 neat for 10 min, for SO2 evolution, and then in

dry THF, followed by the reaction with resveratrol in dry DMF. Seþ SO2Cl2! SeCl2þ SO2 ð1Þ

SO2Cl2⇄ SO2þ Cl2 ð2Þ

Using a [Se : SO2Cl2: resveratrol] ratio of [1 : 0.8 : 0.4], the

reaction leads to the benzoselenophene derivative 1 in almost

50% yield (Scheme 1). Compared to other methods examined, the reaction is advantageous in terms of costs and expedient operational conditions, without the need for phenol group pro-tection, a key requirement for facile and scalable procedures. To the best of our knowledge, there is no precedent for one-pot reactions of stilbenes with an electrophilic selenyl species to obtain benzoselenophenes. Although several methods are described for the synthesis of benzoselenophene derivatives,17 the most efficient ones are based on electrophilic intramolecu-lar cyclization of alkynyl selenocompounds.18

The choice of suitable solvents required several attempts. In the first step of the reaction, THF was used since it signifi-canlty reduces the well-known disproportionation of mono-chalcogen dihalides into oligo-mono-chalcogen dihalides.15,16 For the second step of the reaction, among the few solvents able to dissolve resveratrol, protic solvents such as water and metha-nol, or acetone, were avoided because of their reactivity with SO2Cl2and the formation of electrophilic seleno-species. The

best results were eventually obtained with DMF that afforded a good solubility of resveratrol and was compatible with the reac-tion condireac-tions. On the other hand, when DMSO was used, selenophenes 1–3 (vide infra) were obtained in much lower yields.

Interestingly, analysis of the reaction mixture revealed the presence of small amounts of chlorinated side-products. Accordingly, suitable modification of the stoichiometry ratio between Se(0) and SO2Cl2allowed one to gain selective access

to two additional derivatives identified as the mono- and dichloro derivatives substituted on the resorcinol ring. Thus, the reaction of Se(0) (1 equiv.) with 1 equiv. of SO2Cl2 and

resveratrol (0.4 equiv.) led to the isolation of the 4-chlorobenzo-[b]selenophene derivative 2 in 71% yield (Table S1, ESI†). On the other hand, using a 1 : 2 ratio between Se(0) and SO2Cl2,

the dichlorinated derivative 3 became the major product and was isolated in 82% yield.

These results show that the distribution of the products 1–3 is strictly dependent on the amount of SO2Cl2used (see Fig. S1

in the ESI† for further details). Notably, the scale of reagents

Scheme 1 One-pot formation of selenophenes 1_{–3 from the reaction of resveratrol with Se(0)/SO}Cl.

did not affect the product distribution, which remained sub-stantially the same in reactions run with up to 1.5 g of resveratrol.

Formation of the benzoselenophene derivatives involves probably an electrophilic aromatic substitution as the first step, followed by an electrophilic addition to the double bond, and then HCl elimination leading to 1 (Scheme 1). The efficient ring closure of the selenophene ring implies that ‘in situ’ generated SeCl2reacts almost exclusively with the C-2

nucleophilic carbon of the resorcin moiety of resveratrol and, hence, that the reactivity at C2 is much higher than at C4. Due to the presence of Cl2in the reaction mixture (eqn (2))19a

sub-sequent chlorination may be envisaged to give 2 and 3. The actual intermediacy of 1 in the process has been demonstrated in separate experiments in which the formation of 2 and 3 was observed by reacting 1 with Se(0)/SO2Cl2. Reacting resveratrol

with commercially available Se2Cl2did not lead to any

detect-able selenophene formation. On the other hand, the use of SeCl4 led to some 3 (10–15% yield) supporting the superior

synthetic value of the adopted procedure.

Whenever chlorination occurred, only one regioisomer of the monochloro derivative was observed under all the reaction conditions examined. The assignment of structure 2 to the monochloro compound, as in Scheme 1, was based on a1H,

13_{C heteronuclear multibond correlation (HMBC) spectrum,}

showing distinct cross-peaks between the signal of the chlor-ine-bearing carbon atδ 107.7 and the signals of the 3-H and 6-H protons atδ 7.66 and 6.41, respectively (see the ESI†). The above formulation is in agreement with chemical shift argu-ments, based on the modest deshielding effect of chlorine on the ipso and meta carbons.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/ antioxidant power (FRAP) assay

The antioxidant capacity of 1–3 was assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assay20 _{against Trolox in}

methanol. The time course of DPPH decolorization (Fig. 1)

shows that all the selenophene derivatives are more efficient than resveratrol when tested under the same conditions.

Notably, the percentage of DPPH reduced after 10 min in the case of 1 was comparable to that obtained with Trolox (Table 1). The superior activity of selenophenes is also appar-ent from the data analysis (Table 1), with a ca. four-fold increase of the rate constant for the H-atom transfer in the fast step (k1value) in the case of 1 with respect to resveratrol. The

stoichiometry (ntot in Table 1) of 3.68 and 3.54 found for

Trolox and 1 respectively indicates that under the reaction con-ditions, the oxidation products of these compounds are still capable of reacting with DPPH, as observed in the case of other polyphenols.21

The reducing capacity of the selenophene derivatives was measured by the ferric reducing/antioxidant power (FRAP) assay.22 Table 1 reports the results expressed as Trolox equi-valents. 1 again showed an activity comparable to that of Trolox, while the chlorinated derivatives 2 and 3, though more active than resveratrol, proved to be less efficient.

Inhibited autoxidation studies

The chain-breaking antioxidant activity was tested by studying the inhibited autoxidation of styrene in chlorobenzene or acetonitrile (50% v/v) at 30 °C, initiated by AIBN (0.05 M), in the presence of variable amounts of 1–3 and resveratrol (indi-cated as ArOH in eqn (7) and (8)).11,23 2,2,5,7,8-Pentamethyl-6-chromanol (α-TOH), an α-tocopherol analogue lacking the phytyl tail, was used as the reference antioxidant.23

Initiator! R• ð3Þ R•þ O2! ROO• ð4Þ

ROO•þ RH ! ROOH þ R• ð5Þ ROO•þ ROO•! Non-radical products ð6Þ

ROO•þ ArOH !kinh

ROOHþ ArO• ð7Þ ROO•þ ArO•! Non-radical products ð8Þ

Fig. 1 Decrease in the absorbance at 515 nm of 200_{μM DPPH in the} presence of 50_{μM selenophene compounds, resveratrol or Trolox in} methanol. The mean ± SD values for three separate experiments are reported.

Table 1 Percentage of reduction, rate constant and number of H-atoms transferred for the reaction of 1–3 (50 μM each) with DPPH (200μM) and Trolox equivalents of 1–3 as determined by the FRAP assay Compd DPPHa FRAP DPPH reducedb_(%) k1 c (M−1s−1) ntotd Trolox equiv.e 1 93.1 ± 0.1 340 ± 16 3.54 ± 0.01 0.94 2 74.1 ± 0.4 146 ± 21 2.82 ± 0.02 0.84 3 81.3 ± 0.1 142 ± 13 3.10 ± 0.01 0.63 Resveratrol 55.6 ± 0.9 87 ± 3 2.12 ± 0.27 0.56 Trolox 96.5 ± 0.4 491 ± 5 3.68 ± 0.02 —

a_{Values are means ± SD (n = 3).} b_{Calculated after 10 min of the}

reaction. cRate constant for the fast step. dNumber of H-atoms transferred after 10 min. e_{Values are means of two separate}

experiments (SD < 5%).

The autoxidation was followed by monitoring the oxygen consumption in an oxygen uptake apparatus based on a differ-ential pressure transducer (Fig. 2). The slope of the oxygen consumption trace during the inhibited period afforded the rate constant for the reaction with peroxyl radicals (kinh, eqn

(7)), while its length enabled the determination of the stoichio-metric coefficient n (Table 2).

In the apolar solvent chlorobenzene, the kinhvalues of the

selenophene derivatives are larger than that of resveratrol, the order being 1 > 2> 3> resveratrol (Table 2). This reactivity order is believed to be derived from two overlapping effects: the elec-tron-donating activity of the Se-atom, which causes a lowering of the bond dissociation enthalpy (BDE) of the OH groups, and the H-bond accepting ability of chlorine atoms which increases the BDE of the nearby OH groups.24Compared to chlorobenzene, a reactivity decrease was observed in the polar solvent acetonitrile, showing that the reaction between benzo-selenophenes and ROO• is a H-atom transfer reaction which occurs more efficiently with the OH groups that are not H-bonded to the solvent.24In acetonitrile, the reactivity order is 2 ≥ 3> 1> resveratrol, reasonably because in chlorinated

derivatives, the OH groups are protected from the solvent by the intramolecular H-bond with the Cl-atom.24

Autoxidation experiments in chlorobenzene also provided the stoichiometry of the radical trapping (Table 2), which in all the Se-containing compounds were significantly smaller than 2, that is the value expected for antioxidants acting by eqn (7) and (8) (i.e. by H-atom transfer followed by radical–radical recombination).24It may be suggested that under autoxidation conditions, i.e. in the presence of peroxyl radicals and hydro-peroxides, the selenocompounds are partially converted into Se-oxides, which, however, are expected to be poor anti-oxidants (Table S2, ESI†). Kinetic data with peroxyl radicals in chlorobenzene are in agreement with the results of the DPPH assay indicating that the selenophenes are more reactive than resveratrol. However, the reactivity order among the seleno-phenes varies with the solvent, due to the effect of the inter-play between the solvent characteristics and the different reaction mechanisms for the reaction with ROO• and DPPH radicals.26

DFT calculations

To obtain deeper insight into the antioxidant activity of 1–3, the bond dissociation enthalpy (BDE) of the phenolic O–H bonds was investigated by DFT calculations at the B3LYP/ LANL2DZdp level.13 The BDE(OH) values were obtained by using an isodesmic approach, that consists of calculating ΔBDE between the investigated compounds and phenol, and by adding this value to the known experimental BDE(OH) of phenol in benzene (86.7 kcal mol−1).27 This procedure has been previously tested with phenols having alkyl-selenium substituents in the ortho or para positions, and it reproduced the experimental BDE(OH) values within ±0.3 kcal mol−1, and therefore, it was expected to provide accurate BDE values for the 3-OH and 5-OH groups. On the other hand, when compar-ing the calculated 4′-OH BDE of resveratrol to the experimental value (82.6 kcal mol−1 in benzene),28 it was found that this BDE value was underestimated by 1.8 kcal mol−1. Considering that an almost identical underestimation is obtained with other DFT-based methods also,28the values for 4′-OH reported in Table 3 and S2 (ESI†) were scaled by adding 1.8 kcal mol−1.

The results reported in Table 3 show that the introduction of the Se-atom causes a significant decrease of the 3-OH and 5-OH BDEs, and an increase of the 4′-OH BDE. The effect on the

Fig. 2 Oxygen consumption measured during the autoxidation of styrene (4.3 M) in chlorobenzene initiated by AIBN (0.05 M) at 30 °C in the absence of antioxidants (dashed line) and in the presence of: (a) 7 × 10−6 M resveratrol; (b) 1.5 × 10−5 M 3; (c) 1.9 × 10−5 M 2; (d) 1.9 × 10−5M 1.

Table 2 Rate constants (kinh) and number of radicals trapped (n) for the

reaction of 1–3 with peroxyl radicals at 30 °C

Compd. kinh/105(M−1s−1) na Chlorobenzene Acetonitrile 1 8.8 ± 1.8 0.24 ± 0.02 0.66 ± 0.06 2 4.9 ± 0.4 0.48 ± 0.05 0.83 ± 0.13 3 3.5 ± 0.5 0.44 ± 0.04 0.85 ± 0.09 Resveratrol 1.8 ± 0.2 0.10 ± 0.02 1.6 ± 0.2 α-TOHb ₃₂c _6.8d ₂c

a_{n Values were measurable only in chlorobenzene.} b

2,2,5,7,8-Pentamethyl-6-chromanol (reference antioxidant). c_{From ref. 23.} d_{From ref. 25.}

Table 3 Calculated BDE(OH) in the gas-phase at the B3LYP/ LANL2DZdp level

Compd

BDE (O–H) (kcal mol−1)a

4′-OH 3-OH 5-OH

1 83.8 82.4 83.0

2 84.5 82.0 84.4

3 84.8 83.5 83.4

Resveratrol 82.6 85.0 85.3

a_{For the numbering of the OH groups, see Fig. 3.}

3 and 5 positions can be interpreted as being derived from the electron-releasing, radical-stabilizing effect of the chalcogen atom.13Calculations showed that a significant spin density is delocalized on selenium (Fig. 3, structures (c) and (d)). However, for the 3 position, a larger BDE would be expected because the 3-OH is H-bonded to the Se-atom, as previously reported for 2-alkylselenophenols.13

The phenolic OH groups in ortho to hydrogen bond-accept-ing groups possess larger BDEs than the free ones because the cleavage of the O–H bond also involves the loss of the intra-molecular H-bond. Although the Se-atom is not considered a strong H-bond acceptor, nevertheless it has been reported that an ortho octyl-seleno substituent raises the BDE of a phenolic OH by about 3 kcal mol−1.13Notably, the calculations show that there is no H-bond between Se and the 3-OH, as the most stable structures have the 3-OH group pointing away from the Se-atom (Fig. 3). In the case of 1, for instance, the 3-OH“away” isomer is more stable by 1.5 kcal mol−1than the“toward” one, as already observed in benzo-fused heterocycles containing a sulfur atom ortho to a phenolic OH group.29For what concerns the 4′-OH, the small increase of the BDE(OH) observed on moving from resveratrol to benzoselenophenes may be explained as being due to the perturbation of the stilbene system caused by the Se-atom, which causes a decrease of spin delocalization (Fig. S2, ESI†). The introduction of a Cl-atom in 2 had a small BDE-lowering effect on the 3-OH, in line with the reported effect of chlorine on the BDE of phenols,7_{while it}

increased the BDE of the 5-OH by 1.4 kcal mol−1because of the formation of a weak intra-molecular H-bond, as Cl-atoms are not good H-bond acceptors. Similarly, the BDE of the 3-OH and 5-OH in 3 are larger than those in 1 because both OH are involved in weak intramolecular H-bonds with the Cl-atoms.

Cytotoxicity evaluation

With the aim of evaluating the possible use of the selenoderi-vatives of resveratrol as topical or systemic antioxidants, the cytotoxicity of 1–3 was preliminarily evaluated in comparison with resveratrol by measuring the proliferation of human keratinocyte cells (HaCaT) and human intestinal (CaCo-2) cells in a time-dependent assay. The results are illustrated in Fig. 4, which reports no significant decrease in cell sur-vival measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) reduction after 24, 48 and 72h of incubation following the addition of benzoselenophene compounds at 5μM final concentration. No alterations in cell morphology (light microscopy, data not shown) could be observed.

Fig. 3 Optimized geometry of the most stable conformations of 1 (a) and of the phenoxyl radicals obtained from the abstraction of the H-atom from the 4’-OH (b), 3-OH (c) and 5-OH (d); spin density in the radicals is also shown.

Fig. 4 E_{ffect of resveratrol and benzoselenophenes 1–3 on the viability} of HaCaT and CaCo-2 cells. The mean values ± SD from three indepen-dent experiments run in triplicate are shown.

Conclusions

Resveratrol manipulation by selenium chemistry leads to benzoselenophene derivatives as promising lead structures for innovative antioxidants with Trolox-like capacity. Ring closure allowed installment of the selenium center onto the resorcinol moiety enhancing the H-atom transfer ability of the 3-OH and 5-OH groups with respect to the main resveratrol active site, the 4′-OH group, via a consistent decrease of their BDE(O–H). The marked solvent-dependence of the chain-breaking activity of unsubstituted 1 compared to chlorinated derivatives under-scored the role of chlorine atoms as modulators of the deacti-vating effect of H-bonding with polar solvents on the H-atom donor capacity. The possible advantages of the new benzoseleno-phene antioxidants relative to the parent compound is the combination within a single scaffold of the H-atom donor groups (OH) with a hydroperoxide-scavenging center (Se) and the availability of activity-tuning sites, e.g. the chlorine substi-tuents, on the active resorcinol moiety.

Experimental section

All the commercial materials were used as received without further purification. THF and DMF were dried using a solvent purification system (Pure-Solv™). NMR spectra were recorded in CD3OD at 200, 300 or 400 MHz, 50 or 100 MHz, 38 or

76 MHz for1H,13C and77Se NMR, respectively. The chemical shifts were referenced according to residual solvent signals at 3.31 ppm (1H) and 44.9 ppm (13C). For77Se spectra, PhSeSePh was used as the external reference (461 ppm). J values are given in Hz. Mass spectra were determined by electrospray ionization (ESI) in negative ion mode. Flash column chrom-atography was performed using silica gel (230–400 mesh). The purity of the isolated products was estimated by1H NMR ana-lysis. All products were obtained with≥95% purity.

General procedure for preparation of the benzoselenophene derivatives

All the reactions were carried out using oven-dried glassware under an inert atmosphere (N2). Fresh distilled SO2Cl2

(0.8 mmol, 1 mmol or 2 mmol to obtain 1, 2 or 3 as major pro-ducts, in that order) was added dropwise to selenium powder (79 mg, 1 mmol) and stirred at rt for 10 min, and then 2.5 mL of distilled THF was added. After 1 h, resveratrol (94 mg, 0.4 mmol) dissolved in 0.8 mL of dry DMF was added. The mixture was stirred for 24 h at rt. The brownish red product was filtered over Celite before extraction with ethyl acetate (3 × 15 mL), and the organic layer was washed with water and brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum to

afford the crude product, which was purified by flash column chromatography (chloroform/methanol 9 : 1, 1% acetic acid). 2-(4-Hydroxyphenyl)benzo[b]selenophene-5,7-diol (1)

Following the general procedure, from 200 mg of resveratrol, compound 1 was obtained together with 2 (70 : 30 ratio

deter-mined by NMR), and purified by flash column chromato-graphy to give a hygroscopic brownish red powder (128 mg, 48% yield). Found C 54.7; H 3.6%; ESI-MS: m/z 304 ([M− H]−). C14H10O3Se requires C, 55.1%, H, 3.3%; M, 305.19. NMR: δH(400 MHz, CD3OD, Me4Si) 6.25 (1H, d, J = 2.2 Hz, 6-H), 6.72 (1H, d, J = 2.2 Hz, 4-H), 6.81 (2H, d, J = 8.0 Hz, 3′-H, 5′-H), 7.44 (1H, s, 3-H), 7.48 (2H, d, J = 8.0 Hz, 2′-H, 6′-H); δC(50 MHz, CD3OD, Me4Si) 99.6 (C-6), 103.1 (C-4), 116.6 (C-3′, C-5′), 122.4 (C-3, C-7a), 128.7 (C-2′, C-6′), 129.4 (C-1′), 146.7 (C-3a), 149.1 (C-2), 155.2 (C-5 or C-7), 157.7 (C-7 or C-5), 158.9 (C-4′); δSe

(76 MHz, CD3OD, PhSeSePh) 462.5. Compound 1 was exposed

to Se/SO2Cl2under the standard reaction conditions, and the

products formed were analyzed by TLC (eluant chloroform/ methanol 9 : 1, 1% acetic acid).

4-Chloro-2-(4-hydroxyphenyl)benzo[b]selenophene-5,7-diol (2) Following the general procedure, starting from 200 mg of resveratrol, compound 2 was obtained as an 85 : 15 mixture (ratio determined by NMR) with 3. After flash chromato-graphy purification, 2 (211 mg, 71% yield) was isolated as a hygroscopic dark brownish green compound. Found C 49.8; H 2.4%; ESI-MS: m/z 339 ([M− H]−). C14H9ClO3Se requires C,

49.5%, H, 2.7%; M, 339.63. NMR: δH (300 MHz, CD3OD, Me4Si) 6.41 (1H, s, 6-H), 6.85 (2H, d, J = 9.0 Hz, 3′-H, 5′-H), 7.47 (2H, d, J = 9.0 Hz, 2′-H, 6′-H), 7.66 (1H, s, 3-H); δC (100 MHz, CD3OD, Me4Si) 101.2 (C-6), 107.7 (C-4), 117.7 (C-3′, C-5′), 120.8 (C-7a), 121.2 (C-3), 129.8 (C-2′, C-6′), 129.9 (C-1′), 144.6 (C-3a), 151.7 (C-2), 153.6 (C-5 or C-7), 154.7 (C-7 or C-5), 160.2 (C-4′); δSe(76 MHz, CD3OD, PhSeSePh) 498.6. 4,6-Dichloro-2-(4-hydroxyphenyl)benzo[b]selenophene-5,7-diol (3) Following the general procedure, starting from 200 mg of resveratrol, after flash column chromatography purification, 3 (269 mg, 82% yield) was obtained as a hygroscopic dark brownish compound. Found C 44.7; H 2.4%; ESI-MS: m/z 373 ([M − H]−). C14H8Cl2O3Se requires C, 44.95%, H, 2.1%; M, 374.08. NMR: δH (400 MHz, CD3OD, Me4Si) 6.83 (2H, d, J = 8.8 Hz, 3′-H, 5′-H), 7.50 (2H, d, J = 8.8 Hz, 2′-H, 6′-H), 7.65 (1H, s, 3-H);δC(100 MHz, CD3OD, Me4Si) 108.2 (C-6), 109.3 (C-4), 117.7 (C-3′, C-5′), 120.9 (C-3), 129.5 (C-1′), 129.9 (C-2′, C-6′), 133.0 (C-7a), 142.2 (C-3a), 149.7 (C-2), 150.4 (C-5 or C-7), 152.1 (C-7 or C-5), 160.4 (C-4′); δSe (76 MHz, CD3OD, PhSeSePh) 510.9. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay

The assay was performed as described.20Briefly, to 1.98 mL of 200μM DPPH in methanol, 20 μL of 5 mM methanolic solu-tion of compounds 1, 2, 3 or resveratrol were added and rapidly mixed. The reaction was followed by spectrophoto-metric analysis measuring the absorbance at 515 nm every 30 s for 10 min. Trolox was used as the standard.

Ferric reducing/antioxidant power (FRAP) assay

The assay was performed as described.22To 3.6 mL solution of FRAP reagent, 10–30 μL of 5 mM methanolic solution of com-pounds 1, 2, 3 or resveratrol (10–70 μM final concentration)

were added. After 10 minutes, the absorbance at 593 nm was measured. Trolox was used as the standard. The FRAP reagent was prepared freshly by mixing 0.3 M acetate buffer (pH 3.6), 10 mM 2,4,6-tris(2-pyridyl)-s-triazine in 40 mM HCl, and 20 mM ferric chloride in water, in the ratio 10 : 1 : 1, in that order.

Kinetics with peroxyl radicals

The chain-breaking antioxidant activity of the title compounds was evaluated by studying the inhibition of the thermally initiated autoxidation of styrene in chlorobenzene or aceto-nitrile. Autoxidation experiments were followed by measuring the O2 consumption by using a gas-uptake recording

appar-atus. In a typical experiment, an air-saturated mixture of styrene in acetonitrile or chlorobenzene (50% v/v) containing AIBN (5 × 10−2M) was equilibrated with the reference solution containing also an excess of 2,2,5,7,8-pentamethyl-6-chroma-nol (α-TOH) in the same solvent at 30 °C. After equilibration, a concentrated solution of the antioxidant was injected into the sample flask (final concentration from 5 × 10−6to 5 × 10−5M), and the oxygen consumption in the sample was measured. From the slope of the oxygen consumption during the inhib-ited period, kinh values were obtained as previously reported,

while the n coefficient was determined from the length of the inhibited period usingα-TOH (n = 2) as a reference.11

Calculations

DFT calculations were carried out using the Gaussian03 system of programs.30Gas phase geometries were optimized at the B3LYP/LANL2DZ level,31with the added diffuse and polar-ization basis function, i.e. the B3LYP/LANL2DZdp level.32Basis sets were from the EMSL basis set library.33 This level has been previously adopted to calculate the geometries and bond dissociation enthalpies of S, Se and Te containing phenols.34 The nature of the located stationary points was determined by computation of harmonic vibrational frequencies (zero ima-ginary frequency). The enthalpies at 298 K were computed at the stationary points from frequency calculations, after scaling the results by a factor of 0.9809.35The geometries of the inves-tigated compounds are reported in the ESI.†

Cell viability assay

The cytotoxicity of resveratrol and benzoselenophene deriva-tives 1–3 was evaluated by performing the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Non-tumorigenic human keratinocyte cells (HaCaT) and human intestinal Caco-2 cells were obtained from the A.T.C.C. (American Type Culture Collection, Manassas, VA, U.S.A.) and grown in Dulbecco’s modified Eagle’s medium (DMEM) sup-plemented with 10% fetal bovine serum, 4 mM glutamine, 400 units per mL penicillin and 0.1 mg mL−1 streptomycin. Cell lines were maintained at 37 °C in a humidified incubator con-taining 5% CO2. The cells were plated on 96-well plates at a

density of 2.5 × 103cells per well in 100μL of medium contain-ing either 5μM resveratrol or 5 μM 1–3. After 24, 48 and 72 h of incubation, 10 μL of a 5 mg mL−1stock MTT solution in

PBS, corresponding to a final concentration of 0.5 mg L−1 in DMEM (final volume 100 μL) were added to the cells. After 4 h incubation, the MTT solution was removed and the MTT formazan salts were dissolved in 100 μL of 0.1 N HCl in anhydrous isopropanol. Cell survival was expressed as the absorbance of blue formazan measured at 570 nm with an automatic microplate reader.

Acknowledgements

This work was supported by a grant from Italian MIUR (PRIN 2010-2011 2010PFLRJR, PROxi project).

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