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Biochimie 92 (2010) 1123e1129

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Biochimie

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Research paper

A spectroelectrochemical and chemical study on oxidation of 7,8-dihydroxy-4-methylcoumarin (DHMC) and some related compounds in aprotic medium

Rita Petrucci a,*, Luciano Saso b, Vineet Kumar c,d, Ashok K. Prasad c, Sanjay V. Malhotra d,Virinder S. Parmar c, Giancarlo Marrosu a

aDipartimento di Ingegneria Chimica Materiali Ambiente, Sapienza Università di Roma, Via del Castro Laurenziano 7, I-00161, Rome, ItalybDipartimento di Fisiologia e Farmacologia “Vittorio Erspamer”, Sapienza Università di Roma, p.le Aldo Moro 5, I-00185, Rome, ItalycBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi- 110 007, Indiad Laboratory of Synthetic Chemistry, Development Therapeutics Program Support, National Cancer Institute-Frederick, SAIC-Frederick,1050 Boyles Street, Federick, MD 21702, USA

a r t i c l e i n f o

Article history:Received 7 January 2010Accepted 9 June 2010Available online 17 June 2010

Keywords:AntioxidantsPolyphenols oxidationRadical anionsSemiquinonesSpectroelectrochemistry

* Corresponding author. Tel.: þ39 649766855; fax:E-mail address: rita.petrucci@uniroma1.it (R. Petru

0300-9084/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.biochi.2010.06.008

a b s t r a c t

Electrochemical and chemical oxidation of 7,8-hydroxy-4-methylcoumarin (DHMC 1) and 7,8-diacetoxy-4-methylcoumarin (DAMC 4) were studied to investigate the mechanisms occurring in their antioxidantactivities in acetonitrile, under electron transfer and H-atom transfer conditions. Electrolysis andchemical reactions were followed on-line by monitoring the UV spectral changes with time.

The anodic oxidation of DHMC, studied by cyclic voltammetry and controlled potential electrolysis,occurs via a reversible one-step two-electrons process, yielding the corresponding stable phenoxoniumcation. Moreover, the chemical oxidation with an H-atom acceptor also follows a similar path, yieldingthe stable neutral quinonic product. Intermediates were never evidenced in both cases. Only in thepresence of a strong base, an anodic oxidation product mono-electronic was evidenced, likely the DHMCradical anion.

However, the anodic oxidation of the acetoxy derivative DAMC occurs at very high potential values,ruling out the possibility that the antioxidant activity observed in vivo might occur via an electrontransfer mechanism; no reactions were evidenced with an H-atom acceptor.

� 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction

Considerable progress has been made in the last decade aboutthe pathogenic role of free radicals, especially ROS (ReactiveOxygen Species), in different diseases [1e12]. There are strongevidences for the antioxidant and radical scavenging effects ofsome classes of natural polyphenols, in particular flavonoids andphenylpropanoids like hydroxycinnamic acids, which are impor-tant minor dietary constituents [13e21].

Widely distributed in the plant kingdom, coumarins are quitesimilar to flavonoids and structurally may be considered as lactone-derivatives of hydroxycinnamic acids. Several beneficial pharma-cological effects of 4-methylcoumarins have been reported [22,23]and their antioxidant and radical scavenging properties have beenrecently successfully demonstrated by our group and some others[24e29]. Furthermore, we have also demonstrated that the 7,8-

þ39 649766749.cci).

son SAS. All rights reserved.

dihydroxy-4-methylcoumarin (DHMC) and 7,8-diacetoxy-4-meth-ylcoumarin (DAMC) have a strong activity in vivo as inhibitors ofmembrane lipid peroxidation [26]. The fundamental role of a cate-chol moiety on the molecular structure has been reported fordifferent classes of phenolic antioxidants [30e32], whereas theactivity of diacetoxy derivatives appeared unexpected and lessobvious [33,34]. A path via phenoxyl radical for both DHMC andDAMC has been proposed [26], but actually the mechanismsinvolved in the oxidative processes are not yet well understood.Their possible pro-oxidant activity and their metabolic fate inhuman body are scarcely known till now, limiting the possibleemployment of these natural compounds in the preventive medi-cine as well as in the recognized pathology.

As a continuation of our work on the mechanisms involvedin the activity of these polyphenols, we are reporting an elec-trochemical and chemical study in acetonitrile on DHMC 1,DAMC 4 and some related coumarins, to investigate on the basicoxidative process under electron transfer and H-atom transferconditions. Structures of investigated coumarins are reported inFig. 1, Table 1.

Fig. 1. Structures of 4-methylcoumarins 1e6.

R. Petrucci et al. / Biochimie 92 (2010) 1123e11291124

2. Materials and methods

2.1. Materials

Reagents and anhydrous acetonitrile were purchased fromSigmaeAldrich. Tetraethylammonium perchlorate (TEAP) wasdried under vacuum after recrystallisation. Lead dioxide wasfreshly prepared as reported in literature [35].

Compounds 1e6 were synthesized and characterized by usfollowing procedure reported in literature. 1 (DHMC) was synthe-sized by using Pechmann condensation of pyrogallol with ethylacetoacetate in the presence of concentrated sulfuric acid [36,37]whereas 2 was synthesized as previously described by us [38]and by Chakravarti [39] and 3 according to our reported proce-dure [40].

Compounds 4e6 were prepared by acetylation of compounds1e3 using acetic anhydride/pyridine following our previous report[38,40,41].

2.2. Electrochemical experiments

Voltammetric measurements were performed with a three-electrode multipolarograph AMEL 472 coupled with a digital x/yrecorder AMEL 863, with a static glassy-carbon (GC) workingelectrode and a glassy-carbon Radiometer rotating disk electrode(BM-EDI 101) coupled with a Radiometer speed control unit (CTV101), Ag e AgClO4 (0.1 mol L�1)/MeCN e fine porosity fritted glassdisk e MeCN/TEAP (0.1 mol L�1) e sintered glass disk [42] asreference and a platinum wire as counter electrode. All experi-ments were carried out at room temperature on nitrogen purgedsolution of anhydrous acetonitrile containing 0.1 mol L�1 TEAP assupporting electrolyte, 1 � 10�3 mol L�1 substrate and increasingamounts of 2,6-lutidine and tetraethylammonium hydroxide(TEAOH) as deprotonating agents. Scan rates varied from 20mV s�1

to 2000 mV s�1. The accuracy of the potentials is �5 mV.Spectroelectrochemical experiments were carried out with

a diode array spectrophotometer HP 8452A and a potentiostatAMEL 552 coupled with an integrator AMEL 731 and an x/yrecorder LINSEIS L250E for controlled potential electrolyzes, usinga three-electrode modified UV cell, a platinum wire as workingelectrode, an Ag e AgClO4 (0.1 mol L�1)/MeCN e fine porosityfritted glass disk e MeCN/TEAP (0.1 mol L�1) e sintered glass disk

Table 1Structures of 4-methylcoumarins 1e6.

R3 R5 R6 R7 R8

1 DHMC H H H OH OH2 CH2CO2Et H H OH OH3 CH2CH2CO2Et H H OH OH4 DAMC H H H OAc OAc5 CH2CO2Et H H OAc OAc6 CH2CH2CO2Et H H OAc OAc

as reference and a platinumwire (placed on the inner wall of a glasstube containing MeCN/TEAP 0.1 mol L�1 and connected to the testsolution via a sintered glass disk) as auxiliary electrode. The solu-tion was stirred by purging with a continuous nitrogen flux.Experiments were carried out at room temperature on solution ofanhydrous acetonitrile containing TEAP 0.1 mol L�1 as supportingelectrolyte and 1 � 10�4 e 5 � 10�4 mol L�1 substrate.

2.3. Chemical oxidation

Chemical oxidation was carried out by adding directly in the UVcell small amounts of freshly prepared PbO2 [35] to solutions ofanhydrous acetonitrile containing 1 � 10�4 e 5 � 10�4 mol L�1

substrate.

3. Results and discussion

3.1. Cyclic voltammetry

The electrochemical behaviour of 7,8-dihydroxy-4-methyl-coumarin (DHMC, 1) was studied by cyclic voltammetry at a staticGC electrode in anhydrous acetonitrile, in the absence as well as inthe presence of a deprotonating agent. Cyclic voltammograms ofDHMC 1 exhibit two well defined irreversible anodic peak (seeFig. 2, solid line): the first one at Eap1 ¼ þ0.83 V, corresponding toa bit less than a two-electrons process, and the second one atEap2 ¼þ1.73 V, corresponding to a more than two-electrons process.

The simple models of catechol and ferrocenewere chosen asmono-electronic reference in the same medium.

In the reverse cathodic scan, a broadened cathodic wave wasobserved at Ecp ¼ þ0.21 V, for low switching potential El ¼ þ1.00 V(Fig. 2, dotted line ---) as well as for high switching potentialEl ¼ þ1.95 V (Fig. 2, solid line), suggesting that the first oxidationproduct of DHMC 1 must be involved in the reductive process;further, the ratio cathodic current/first anodic current (ic/ia1) of 0.25increased of about 42% when the solutionwas electrolyzed for 30 s,at the startingoxidative potential ofþ1.0V, before the cathodic scan.

When increasing amounts of 2,6-lutidine as deprotonatingagent were added to the tested solution, the first anodic peak ofDHMC at Eap1 ¼ þ0.83 V (Fig. 3, solid line) decreased, while another

Fig. 2. Cyclic voltammograms of DHMC 1 (switching potential El ¼ þ1.95 V, solid line;switching potential El ¼ þ1.00 V, dotted line - - - ) and DAMC 4 (dotted line - - -), inanhydrous acetonitrile 0.1 mol L�1 TEAP, at a static GC electrode, vs Ag/AgClO4

(ESCE ¼ EAg=AgClO4þ 310 mV, experimental value); scan rate 200 mV s�1.

Fig. 3. Cyclic voltammograms of DHMC 1 in anhydrous acetonitrile 0.1 mol L�1 TEAP,at a static GC electrode, vs Ag/AgClO4 (ESCE ¼ EAg=AgClO4

þ 310 mV, experimentalvalue), in the absence of deprotonating agent (solid line), in the presence of half-equivalent of 2,6-lutidine (dotted line - - -), in the presence of one equivalent of 2,6-lutidine (dotted line e$e$e$); scan rate 200 mV s�1.

Table 2Electrochemical data of DHMC 1 in anhydrous acetonitrile 0.1 mol L�1 TEAP, ata static GC electrode, in the absence and in the presence of deprotonating agents;scan rate 200 mV s�1; potential values vs Ag/AgClO4 (ESCE ¼ ESCE ¼ EAg=AgClO4

þ310 mV, experimental value).

DHMC DHMC/2,6-Lutidine DHMC/TEAOH

Eap1 /Ecp(V) þ0.830/þ0.210 þ0.180/�0.180 �1.080/�1.180

Eap2*/Ecp2*(V) e e �0.420/�0.490

*Potential values relative to the second peak in the two-steps two-electrons processof the DHMC dianion.

R. Petrucci et al. / Biochimie 92 (2010) 1123e1129 1125

anodic peak with increasing height was observed at a lower anodicpotential Eap1* ¼ þ0.18 V (Fig. 3, dotted lines), indicating an increasein the nucleophilicity of the compound [43]. The complete disap-pearance of the first peak atþ0.83 Vwas observed upon addition ofone equivalent of base. In Fig. 3 is shown the behaviour respectivelyfor half-equivalent of 2,6-lutidine added (Fig. 3, dotted line ---) andone equivalent of 2,6-lutidine added (Fig. 3, dotted line e$e$e$).

Also in the reverse scan, the cathodic peak of DHMC atEcp ¼ þ0.21 V (Fig. 3, solid line) completely shifted to less positivepotential (Ecp* ¼ �0.18 V) for one equivalent of 2,6-lutidine added(Fig. 3, dotted lines), with an increased final ratio ic*/ia* ¼ 0.70.

The same result was obtained when small increasing amounts,till one equivalent, of a stronger base as TEAOH were added to thesolution, suggesting that the new anodic peak likely corresponds tothe bi-electronic oxidation of the phenolate mono-anion of DHMC1 to the corresponding quinoid product, with a quasi-reversibleprocess (ratio ic*/ia* ¼ 0.70) at the anodic potential Eap1* ¼ þ0.18 V(Ecp* ¼ �0.18 V, ΔEac ¼ 360 mV).

When a further equivalent of TEAOH was added to the solution,cyclic voltammograms of DHMC 1 changed, evidencing a reversible

Fig. 4. Cyclic voltammograms of DHMC 1 in anhydrous acetonitrile 0.1 mol L�1 TEAP,at a static GC electrode, vs Ag/AgClO4 (ESCE ¼ EAg=AgClO4

þ 310 mV, experimentalvalue), in the presence of two equivalent of TEAOH: (4a) anodic scan of the dianion ofDHMC (Q��), starting potential �1.5 V; (4b) cathodic scan of the oxidation product ofDHMC (Q) electrochemically generated at the starting potential of 0.0 V, without (4b,solid line) and with (4b, dotted line) a 30 s electrolysis before starting the scan; scanrate 200 mV s�1.

two-steps two-electrons process at negative potentials(Eap1** ¼ �1.08 V, Eap2** ¼ �0.42 V), as expected for the oxidation of thedianion of an hydroquinone. This is evidenced in Fig. 4, where arereported cyclic voltammograms of DHMC in the presence of twoequivalents of TEAOH: voltammogram 4a (Fig. 4) was recorded inanodic scan (starting potential �1.5 V), evidencing the two-stepsoxidation of the dianion of DHMC, while voltammogram 4b (Fig. 4)was recorded in cathodic scan (starting potential 0.0 V), evidencingthe two-steps reduction of the quinoid oxidation product of DHMCelectrochemically generated at the starting potential of 0.0 V(Figure 4, 4b, solid line), even when solution was electrolyzed for30 s before the cathodic scan (Figure 4, 4b, dotted line). Electro-chemical data of DHMC 1 are resumed in Table 2.

The second anodic peak of DHMC 1 at Eap2 ¼þ1.73 V (Fig. 2, solidline) resulted to be unaffected by the presence of a base, indicatingthat protons are not involved. This suggests another independentoxidative process for DHMC occurring at higher potential values,likely at the aryl-olefinic site C3-C4. This may be supported byelectrochemical data in the same medium of 7,8-diacetoxy-4-methylcoumarin (DAMC) 4, with the same basic structure of DHMC1 but with no eOH moiety available: in fact, cyclic voltammogramof DAMC 4 evidenced an irreversible anodic peak at the highlypositive potential value Eap ¼ þ2.05 V (Fig. 2, dotted line ---).

DHMC 1 was also studied in the same medium at a GC rotatingdisk electrode (RDE) and data, reported in Table 3, were elaboratedby Levich equation [44] and discussed according to criteria sug-gested by the literature [45]: assuming the same diffusion coeffi-cient D for both DHMC and ferrocene (Dferrocene ¼ 2.30 � 10�9

m2 s�1, experimental value in anhydrous acetonitrile), a value of1.47 e� was found for the anodic oxidation of DHMC while,considering DDHMC ¼ Dferrocene � 20%, a value in the range 1.3e1.7electrons was found. These data suggest a chemical step interferingwith the electrochemical one [44,46].

C-3 substituted DHMC 2 and 3 were also studied at a static androtating disk electrode (RDE): cyclic voltammograms evidenceda behaviour very similar to DHMC 1, with a bi-electronic oxidativeprocess at less positive potential values (respectively þ0.65 V and0.70 V, as shown in Table 4), while a process corresponding to oneelectron was evidenced from RDE data (Table 3).

Table 3Electrochemical data at a GC RDE of DHMC 1 and C3 substituted derivatives 2 and 3in anhydrous acetonitrile 0.1 mol L�1 TEAP, vs Ag/AgClO4 (ESCE ¼ EAg=AgClO4

þ310 mV, experimental value).

u (rad s�1) iL/4,23�C�u1/2 (DHMC) 1 iL/4,23�C�u1/2 2 iL/4,23�C�u1/2 3

52.50 2.48 � 10�6 1.54 � 10�6 1.79 � 10�6

105.00 2.49 � 10�6 1.60 � 10�6 1.70 � 10�6

157.50 2.45 � 10�6 1.73 � 10�6 1.72 � 10�6

210.00 2.38 � 10�6 1.86 � 10�6 1.77 � 10�6

262.50 2.34 � 10�6 1.85 � 10�6 1.88 � 10�6

315.00 2.26 � 10�6 2.04 � 10�6 1.88 � 10�6

ne� 1.47 1.09 1.09

Table 4Electrochemical data of compounds 1e6, in anhydrous acetonitrile 0.1 mol L�1 TEAP,at a static GC electrode, scan rate 0.200 Vs�1, vs Ag/AgClO4 (ESCE ¼ EAg=AgClO4

þ 310mV, experimental value); substrate concentration 1 � 10�3 mol L�1.

Compound Eap (V)* Ecp (V)

oedihydroxy1 (DHMC) þ 0.830 þ 0.2102 þ 0.650 þ 0.2003 þ 0.700 þ 0.150

oediacetoxy4 (DAMC) þ 2.050 e

5 þ 1.780 e

6 þ 1.750 e

*Eap corresponds to the anodic potential of the first anodic peak evidenced by cyclicvoltammetry for each compound.

Scheme 1.

R. Petrucci et al. / Biochimie 92 (2010) 1123e11291126

Also C-3 substituted DAMC 5 and 6 evidenced, like for DAMC 4,an irreversible anodic peak at high potential values at cyclic vol-tammetry, while no significant RDE data were obtained for DAMC 4and C-3 substituted compounds 5 and 6 because of adsorptionphenomena.

3.2. Electrolysis of DHMC 1 in UV cell

In order to verify the number of electrons involved in theoxidative process, controlled potential electrolysis of DHMC 1 wascarried out in anhydrous acetonitrile in a modified UV cell ata platinum electrode and spectrawere recorded at short intervals oftime during electrolysis.

Electrolysis of DHMC 1 (lmax (MeCN)/nm 220, 260 and 314) wascarried out at þ0.9 V till the current value reached a constantplateau, corresponding to about 2 e�. The yellowish final solutionshowed lmax (MeCN)/nm 216, 284 and 456 (Fig. 5). The sameoxidized solution was then reduced at a slightly negative experi-mental potential and after the consumption of an equal amount ofcurrent, the uncolored solution of DHMC 1 was obtained and thefinal spectrum overlapped the starting one (Fig. 5). Neither stableintermediate nor transient compounds were detected in theoxidation or reduction process (in a previous work of us, thesemiquinone of p-benzoquinones electrochemically generated wasevidenced via UV spectrophotometry [47]).

3.3. Chemical oxidation in UV cell

DHMC 1, DAMC 4 and the C-3 substituted related compounds 2,3, 5, 6 were chemically oxidized with PbO2 in anhydrous acetoni-trile and reactions were monitored by UV spectrophotometry.

Fig. 5. UV spectral changes relative to the anodic oxidation and successive reversible cathelectrode: starting substrate (a); anodic oxidation product (b); reversible reduction produc

The oxidation products of DHMC 1 and C-3 substitutedcompound 2 and 3 exhibit a similar behaviour with strongabsorptions at 280 nm (DHMC 1), 280 nm (2) and 288 nm (3)respectively (Fig. 6). A little blue shift was observed for the chemicaloxidation product of DHMC (lmax 280 nm) compared to the elec-trochemical oxidation product of DHMC (lmax 284 nm).

Our attempts to oxidize the acetoxy substituted coumarinsDAMC 4, 5 and 6 with an H-atom acceptor as PbO2 failed, asexpected because of the lack of the donateable H-atom in thesesubstrates.

From our electrochemical and spectroelectrochemical data, itmay be assumed that the anodic oxidation in aprotic medium ofDHMC 1 proceeds via a reversible one-step two-electrons mecha-nism coupled with a proton transfer and leading to the corre-sponding phenoxonium cation (see scheme in Fig. 5). Theirreversibility of the anodic process evidenced in cyclic voltam-metry could be due to a slow proton exchange equilibrium inaprotic medium, as supported by the broadened shape of thecathodic wave, the dependence of the Ecp values as well as thecathodiceanodic peak current ratio from scan rates and from theabsence or the presence of a deprotonating agent. Also RDE datasuggest a chemical reaction interfering with the electrochemicalprocess. The calculated ne� changes from 1.47 (DHMC) to 1.0 (2, 3),indicating that the chemical reaction, likely the proton transfer, inthe case of 2 and 3 must be slow enough to be run out from theelectrode surface in our range of rotation speeds. However, a slowerdeprotonation is expected because of the stabilizing effect on thephenoxonium cation of the electron-donor side-chain.

Furthermore, in the presence of 2,6-lutidine as deprotonatingagent, the bi-electronic oxidation of the mono-anion occursthrough a quasi-reversible process.

In the presence of two equivalents of the stronger base TEAOH,when the dianion of DHMC (Q��) is generated, the anodic processchanges into a two-steps two-electrons mechanism, proceedingthrough the radical anion (the semiquinone of DHMC, Q��) first andthen to the final neutral quinoid structure (Q), as shown in theScheme 1.

odic reduction of DHMC 1 in anhydrous acetonitrile 0.1 mol L�1 TEAP at a Pt workingt (c).

Fig. 6. UVspectral changes relative to thechemical oxidationwithPbO2 in anhydrous acetonitrileofDHMC1 (a), 2 (b) and3 (c): starting substrates (spectra 1), oxidationproducts (spectra 2).

R. Petrucci et al. / Biochimie 92 (2010) 1123e1129 1127

In this last case, we may observe that a comproportionationchemical reaction may occur between the neutral oxidized form ofDHMC (Q) and the dianion (Q��) to yield the semiquinone (Q��). Thereaction, thermodynamically supported by the potential values, isexperimentallyevidencedbythe increasedheightof the secondanodicpeak corresponding to the oxidation of the semiquinone (Fig. 4a).When electrolysis was carried out for 30 s at 0.0 V before the cathodicscan, themono-electronic redox process of quinone/semiquinonewaswell evidenced,while comproportionationmay be run out because ofa mass effect and/or the near inversion potential (Fig. 4b).

The second anodic peak of DHMC at Eap2 ¼ þ1.73 V may be

assigned to a different oxidative process, independent from the firstone and not involving a proton transfer, that likely occurs at the C3-C4 aryl-olefinic structure; this is supported by cyclic voltammo-grams of DAMC 4 and C-3 substituted derivatives 5 and 6, exhib-iting a very similar irreversible anodic peak characterized by higher

potential values (þ1.77 V/þ2.05 V), as expected because of thenature of ring substituents. A light shift to less positive potentialvalues respect to the unsubstituted DHMC and DAMCwas observedfor all C-3 substituted coumarins, where the electron-donor side-chain may stabilize the radical cation or the dication produced inthe anodic oxidation. In fact, according to Ross et al. [48],aryl-olefins may undergo a one-step two-electrons oxidation to thecorresponding dication or a mono-electronic oxidation to the cor-responding radical cation, that may be further oxidized tothe dication or undergo chemical reaction according to the exper-imental conditions and/or the stability of the radical cation.

The chemical oxidation with a H-atom acceptor of all studiedcompounds followed a path in agreement with electrochemicalresults.

In fact, dihydroxy coumarins 1, 2 and 3 reacted with PbO2

yielding oxidation products with very similar UV spectra, whose

R. Petrucci et al. / Biochimie 92 (2010) 1123e11291128

stronger absorption falls in the same l range (around 280 nm); thechemical oxidation product of DHMC 1 showed the main strongadsorption only slightly blue shifted with respect to the electro-chemical oxidation product, to support further that the electro-chemical product should be the phenoxonium cation, characterizedby a more extended conjugation than the neutral quinone obtainedafter oxidation with PbO2. Also during these experiments no stableor transient intermediates were observed during the on-linemonitored reaction, this evidencing that even if the chemicaloxidation occurred via the neutral phenoxy radical, this might betoo reactive to be detected in the time range of UV experiments (UVrecorded at 1e5 s time intervals).

When PbO2 was added to solutions containing DAMC 4 andrelatedcompounds5and6, no changes inUVspectrawereobserved,as expected because of the unavailability of easily donateable H-atoms, which is in agreement with data discussed above.

4. Conclusion

In the present work, we have studied the electrochemicalbehaviour of 7,8-dihydroxy-4-methylcoumarin DHMC 1 to inves-tigate the oxidative process involved in its antioxidant activity.Because of the antioxidant properties evidenced in vivo also for therelated diacetoxy substituted coumarin DAMC 4, the electro-chemical behaviour of DAMCwas also studied in the samemedium.The chemical oxidation of both DHMC and DAMC was also studiedunder H-atom transfer conditions, to evidence the stability ofpossible radical intermediates.

Electrochemical and spectroelectrochemical data suggested andsupported a good antioxidant activity for the o-dihydroxycoumarinDHMC, whose anodic potential Eap lies in the same range offlavonoids like baicalein, quercetin or catechin (unpublished data)and HCAs [49], measured in the same omogeneous aproticmedium. The strong shift to less positive potential values, inducedby bases, measured for the phenolate anion may be also connectedto the antioxidant activity of DHMC, for example when environ-ment and free radical reactants are favourable to a sequentialproton-loss electron transfer mechanism [50].

Chemical and electrochemical experiments evidenced, forDHMC, a similar oxidative path both under electron transfer and H-atom transfer conditions, the oxidation product having a reversiblequinoid structure. It is interesting to mention that for the ring openstructure of caffeic acid a different behaviour was observed in thechemical oxidation, involving the phenoxy radical [49]. No inter-mediates were evidenced by cyclic voltammetry and UV experi-ments in the chemical and electrochemical oxidation of DHMC.

On the other hand, our data ruled out any possibility for DAMCto act as an antioxidant, either via electron transfer or H-atomtransfer mechanism. The biological activity observed for DAMCmust be consequent to the hydrolysis to DHMC, with a possiblesynergical effect due to the involvement of the acetoxy group ina side (or even main) reaction.

The important role of the catechol moiety on the molecular struc-ture of a potential antioxidant has been oncemore evidenced. It is ouropinion that the lactone structure of o-hydroxy-4-methylcoumarinsmay present some advantages over hydroxycinnamic acids [49], maybe because of the reversibility of the system quinone-hydroquinonetype both under electron transfer and H-atom transfer conditions.

Acknowledgments

We thank the Ministero dell’Università e dellaRicerca Scientificae Tecnologica (MURST) for financial support.

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