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Electrochimica Acta 56 (2010) 841–852

Contents lists available at ScienceDirect

Electrochimica Acta

journa l homepage: www.e lsev ier .com/ locate /e lec tac ta

tudy on the oxidation of C4-phenolic-1,4-dihydropyridines and its reactivityowards superoxide radical anion in dimethylsulfoxide

icardo Salazara, P.A. Navarrete-Encinab, J.A. Squellac, C. Barrientosb,. Pardo-Jiménezc, Luis J. Núnez-Vergarac,∗

Laboratorio de Electrocatálisis. Facultad de Química y Biología. Universidad de Santiago de Chile, Santiago, ChileLaboratorio de Sintesis Orgánica y Modelación Molecular, Santiago, ChileLaboratorio de Bioelectroquímica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 233, Santiago, Chile

r t i c l e i n f o

rticle history:eceived 24 May 2010eceived in revised form6 September 2010ccepted 18 September 2010vailable online 25 September 2010

eywords:olyhydroxyphenyl dihydropyridines

a b s t r a c t

Electrochemical characterization on glassy carbon electrode (GCE) and reactivity with superoxide radicalanion in aprotic medium of three new synthesized C4-phenolic-1, 4-dihydropyridines is reported.

Voltammetry, coulometry, controlled-potential electrolysis (CPE), UV–vis spectroscopy, 1H NMR tech-niques were employed for the characterization of title compounds.

The oxidation mechanism involves initially an oxidation process on the phenol moiety with the forma-tion of the corresponding quinone followed by a second one affecting the dihydropyridine ring to givethe pyridine derivative. Both processes appeared irreversible in character.

Cyclic voltammetry was used to generate O2•− by reduction on GCE of molecular oxygen in DMSO. The

•−

lectrochemical oxidationuinonesntioxidant indexuperoxide radical anion

reactivity of DHPs towards O2 was directly measured by the anodic current decay of the radical in thepresence of increasing concentration of tested 1,4-dihydropyridines and compared with the reaction ofthe reference antioxidant, Trolox. The linear correlations obtained between the anodic current of O2

•−

and compound concentrations in the range between 0.01 mM and 1.00 mM allowed the determination ofboth the DHP antioxidant index (AI) and the concentrations needed to consume 50% of O2

•−. SynthesizedC4-phenolic 1,4-dihydropyridines exhibited significant scavenging capacity towards superoxide radical

and t

anion higher than Trolox

. Introduction

1,4-Dihydropyridines (DHPs), an important class of drugs,re potent blockers of calcium (Ca2+) currents through voltage-ependent L-class Ca2+ channels. They induce relaxation of vascularmooth muscle, preferentially in arterial beds, and display a nega-ive inotropic effect on isolated cardiac muscle. In therapy, this classf drugs is principally used in the treatment of cardiac arrhythmias,eripheral vascular disorders, and hypertension [1–3].

Additionally, several in vitro findings reported that 1,4-DHPalcium antagonists display antioxidant properties [4–9]. Thisffect has not been clearly proved due to its Ca2+ channelntagonist effect, but it would rather be related with the reac-ivity of these structures towards different radical species [10].

nvestigations developed in our laboratory [11–13] have beenonducted to clarify how 1,4-DHPs interact with or scavenge rad-cals. In this same line of evidences, it has been reported thathe 2,6-dimethyl-3,5-diethoxycarbonyl-1,4-DHP possesses consid-

∗ Corresponding author. Tel.: +56 2 9782812; fax: +56 2 7371241.E-mail address: lnunezv@ciq.uchile.cl (L.J. Núnez-Vergara).

013-4686/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.electacta.2010.09.063

ested commercial 1,4-dihydropyridines.© 2010 Elsevier Ltd. All rights reserved.

erable antioxidant activity in the stabilization of edible oils [14]and exhibits synergistic properties with antioxidants, such as�-tocopherol [15] and 2,6-bis(tert-butyl)-4-methylphenol (BHT)[16].

Recently, synthesis, electrochemical characterization and reac-tivity towards free radicals of new C4-monophenolic andC4-methoxy-phenolic substituted 1,4-DHPs have been reportedby our laboratory [17,18]. Two redox centers coexisting in thesecompounds show mutual significant interactions affecting boththe electrochemical responses and reactivity towards free radicals.Based on these encouraging results, we decided to synthesize newderivatives containing dihydroxyphenyl- and trihydroxyphenylgroups at 4-position on the 1,4-dihydropyridine ring.

Phenols have been described to have greater antioxidant activitythan vitamins C and E. The mechanism of their action as antioxi-dants seems to involve the ability of phenols to scavenge radicalsby an electron transfer process by which the phenol is converted

into a phenoxyl radical [19].

Electrochemical oxidation of 1,4-dihydropyridines in aproticmedium have been extensively reported by us [20–24] and otherresearchers [25–33]. Also, the electrochemical characterization ofcompounds containing a phenolic moiety has been studied [32–36].

842 R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852

NH

OH

HO

O

O

O

O

NH

O

O

O

O

HO OH

NH

O

O

O

O

NH

OH

HO

O

O

O

O

OH

HO OHHO OH

OH

HO

OH

H3C

CH3

OOH

O

CH3

HO

CH3

4-Ph-DHP 3,5-OH-DHP

3,4-OH-DHP 3,4,5-OH-DHP

pyrogallol resorcin pyrocathecol

mical

Fa

nefstb

2

2

F

Trolox

Fig. 1. Che

urthermore, the electrochemical measurements have been used tossess antioxidant abilities of these types of compounds [37,38].

This paper reports both the electrochemical oxidation of someew C4-phenolic-1,4-dihydropyridines and its reactivity towardslectrogenerated superoxide radical anion (O2

•−) in dimethylsu-oxide (DMSO). Synthesized derivatives contain two aromatic ringystems with a number of conjugated double bonds and redox reac-ive NH and OH groups (Fig. 1). These features affect both the redoxehavior and its reactivity towards free radicals.

. Experimental

.1. Compounds

Chemical structures of synthesized compounds are shown inig. 1.

structures.

2,6-dimethyl-3,5-diethoxycarbonyl-4-phenyl-1,4-dihydropyridine (4-Ph-DHP) was synthesized according to amethodology previously reported [12,17].

3,4-OH-DHP, 3,5-OH-DHP and 3,4,5-OH-DHP were synthe-sized by the following general procedure: 10 mmol ethyl3-aminocrotonate and 5 mmol of the respective aldehyde(3,4-dihydroxy-benzaldehyde for 3,4-OH-DHP derivative; 3,5-dihydroxy-benzaldehyde for 3,5-OH-DHP derivative and 3,4,5-trihydroxy-benzaldehyde for 3,4,5-OH-DHP derivative) weredissolved in glacial acetic acid (30 mL) with vigorous stirring at60 ◦C. After 6 h heating, water is added until precipitation of the

dihydropyridine compound. The respective solids were filtered offand washed with ethanol/water (1:1). The compounds were recrys-tallized twice from absolute ethanol.

2,6-dimethyl-3,5-diethoxycarbonyl-4-(3,4-dihydroxyphenyl)-1,4-dihydropyridine (3,4-OH-DHP): Yield:

imica

31ı2R((((C

d428HR6N3(63

t331M4(N4(63

2aN5oido2mo

arf

2

(rAetdPtK

(

(

R. Salazar et al. / Electroch

2%. mp 195.0–197.0 ◦C. FT-IR �max (KBr) cm−1 3411.3, 1666.4,470.9, 1370.8, 1247.6, 1218.8, 1123.0, 1020.7 and 781.0. 1H NMR(300 MHz; acetone-d6), 1.2 (6 H, t, J = 7.4, R–CO–O–CH2–CH3),

.3 (6 H, s, R–CH3), 3.0 (1 H, s, R2–CH), 4.0 (4 H, q, J = 7.4,–CO–O–CH2–CH3), 4.9 (2 H, s, –OH), 6.6–7.6 (4H,m,Ar-H), 7.81-H, s, N-H). 13C-NMR ı (75 MHz; acetone-d6) 13.8 (2 × s), 17.92 × s), 38.6 (s), 58.9 (2 × s), 103.3 (2 × s), 114.4 (s), 115.1 (s), 119.1s), 140.5 (s), 144.3 (2 × s), 160.2 (s), 165.3 (s) and 205.5 (2 × s).from EtOH). Found: C, 62.95; H 6.40; N, 3.89. C19H23NO6 requires, 63.15; H, 6.41; N, 3.88%).

2,6-dimethyl-3,5-diethoxycarbonyl-4-(3,5-ihydroxyphenyl)-1,4-dihydropyridine (3,5-OH-DHP): Yield:0%. mp 245.0–247.0 ◦C (from EtOH). FT-IR �max (KBr) cm−1 3403.7,979.6, 1672.2, 1599.0, 1473.1, 1373.3, 1221.2, 1156.5, 1004.2,44.6, 800.3 and 619.5. 1H NMR ı (300 MHz; acetone-d6) 1.1 (6, t, J = 7.4, R–CO–O–CH2–CH3), 2.2 (6 H, s, R–CH3), 2.9 (1 H, s,2–CH), 3.9 (4 H, q, J = 7.4, R–CO–O–CH2–CH3), 4.8 (2 H, s, –OH),.0 (1 H, s, Ar-H), 6.2 (1 H, s, Ar-H), 7.7 (1 H, s, Ar-H), 7.8 (1-H, s,-H). 13C-NMR ı (75 MHz; acetone-d6) 13.8 (2 × s), 17.9 (2 × s),9.1 (s), 59.0 (2 × s), 100.2 (2 × s), 102.8 (s), 106.2 (2 × s), 144.6s), 150.4 (2 × s), 158.0 (s), 167.2 (s) and 205.4 (2 × s). Found: C,2.90; H 6.43; N, 3.89. C19H23NO6 requires C, 63.15; H, 6.41; N,.88%.

2,6-dimethyl-3,5-diethoxycarbonyl-4-(3,4,5-rihydroxyphenyl)-1,4-dihydropyridine (3,4,5-OH-DHP): (Yield:5%. mp 245.0–247.0 ◦C (from EtOH). FT-IR �max (KBr)/cm−1

400.5, 2960.2, 1682.2, 1660.2.0, 1654.5, 1449.9, 1374.5, 1228.1,159.4, 1054.5, 1033.5 and 783.7. 1H NMR ı (300 MHz; acetone-d6,e4Si) 1.1 (6 H, t, J = 7.3, R–CO–O–CH2–CH3), 2.2 (6 H, s, R–CH3),

.3 (4 H, q, J = 7.3, R–CO–O–CH2–CH3), 4.7 (1 H, s, R2–CH), 6.22 H, s, Ar-H), 7.1 (1 H, s, –OH), 7.6 (2 H, s, –OH), 7.9 (1-H, s,-H). 13C-NMR ı (75 MHz; acetone-d6) 17.0 (2 × s), 21.1 (2 × s),1.7 (s), 62.0 (2 × s), 106.2 (2 × s), 109.9 (2 × s), 134.2 (s), 142.62 × s), 146.5 (s), 148.4 (2 × s) and 170.56 (2 × s). Found: C,0.28; H 6.15; N, 3.72. C19H23NO7 requires C, 60.47; H, 6.14; N,.71%)

Commercial 1,4-dihydropyridines. Nifedipine: 1,4-dihydro-,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxyliccid dimethyl ester (Mintlab Laboratories, Santiago, Chile).isoldipine: (1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,-pyridine carboxylic acid methyl 2-methylpropyl ester) wasbtained from Sanitas Laboratories, Santiago, Chile. Nimodip-ne: (1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridineicarboxylic acid 2-methoxyethyl-1-methylethyl ester) wasbtained from Saval Laboratories, Santiago, Chile. Amlodipine:-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-ethyl-3,5-pyridine dicarboxylic 3-ethyl 5-methyl ester was

btained from Chile Laboratories, Santiago, Chile.Trolox, (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic

cid) was purchased from Sigma–Aldrich and it was used aseceived. A stock solution of Trolox was prepared in dimethylsul-oxide (DMSO) at a concentration of 10 mM.

.2. Voltammetry

Differential pulse voltammetry (DPV) and cyclic voltammetryCV) were performed with a BAS-CV 100 assembly. All voltammet-ic experiments were carried out with 1 mM solutions of 1,4-DHPs.

stationary glassy carbon electrode (GCE) was used as workinglectrode (0.07 cm2) for DPV and CV experiments. The surface of

he electrode was polished to a mirror finish with alumina pow-er (0.3 �m and 0.05 �m) before use and after each measurement.latinum wire was used as auxiliary electrode and all poten-ials were measured against an (Ag/AgCl) electrode in saturatedCl.

Acta 56 (2010) 841–852 843

2.2.1. Electrolytic mediumDMSO containing 0.1 M tetrabutylammonium hexafluorophos-

phate (TBAFP6).

2.2.2. Controlled-potential electrolysis (CPE)A three electrode circuit with an (Ag/AgCl) as a refer-

ence electrode, platinum as counter-electrode and a reticulatedglassy carbon working electrode was used. Exhaustive electroly-sis was carried out at the following potentials: Ep1 = 900 mV andEp2 = 1400 mV for 3,4-OH-DHP and 3,4,5-OH-DHP. The appliedpotentials for 3,5-OH-DHP were Ep1 = 1050 mV and Ep2 = 1400 mV.A BAS-CV 100 assembly was used to electrolyze the 1,4-DHPssolutions. Net charge was calculated including correction for theestimated background current.

2.3. Characterization of oxidation products from 3,4-OH-DHP,3,5-OH-DHP and 3,4,5-OH-DHP

All UV–vis spectra were recorded in the 200–1000 nm range byusing an Agilent spectrophotometer with diode array using 1 cmquartz cell. A quartz crystal glass spectroelectrochemical cell (140ACH Instrument, path length: 1 mm) of 0.1 mL with three electrodes,a Pt gauze as a working electrode (CH Instrument 011547), a nonaqueous (Ag/AgCl) as reference electrode (CH Instrument 010249)and a Pt as counter electrode (CH Instrument 011548) – were used.

a) Spectroelectrochemical experiments of 0.1 mM and 0.05 mMsolutions of compounds were used to electrolyze the dihy-dropyridine moiety and phenolic group and to characterizethe oxidation products produced during the electrolysis ofboth electroactive groups. Applied potentials were the same asdescribed in Section 2.2.

b) To assess the chemical nature of the oxidation productscorresponding to the phenolic moiety, 1 mM phenolic 1,4-DHP solutions in the presence of 5 mM hydroxylamine wereelectrolyzed. Reaction medium was a mixture of aqueous0.1 M Britton–Robinson buffer pH 5.5/acetonitrile (70/30)to assure the solubility of the compounds. Blank solutionswere prepared containing the phenolic 1,4-DHP derivatives at1 mM concentration + 1 mM hydroxylamine in 0.1 M aqueousBritton–Robinson buffer pH 5.5/acetonitrile (70/30). Time-course of reactions was followed between 200 nm and 1000 nmduring 45 min for each compound. All experiments were con-ducted at room temperature.

(c) Chemical oxidation of phenolic 1,4-DHP. 1 mM solution of eachphenolic 1,4-DHP was mixed with 0.1 M KIO4 solution in ace-tonitrile/water (1:1) and further reaction with hydroxylamine.Time-course of reactions was followed by UV–visible spec-troscopy in a range of 200–1000 nm. Spectral characteristicswere compared with those obtained through the CPE experi-ments.

2.4. Electrochemical experiments to assess the reactivity ofC4-substituted 1,4-DHPs with superoxide radical anion

Cyclic voltammetric (CV) experiments were performed in DMSO(Merck) with 0.1 M TBAPF6 purchased from Fluka. DMSO was driedwith 0.3 nm molecular sieves. Oxygen (99.8% pure) and nitrogen(99.9% pure) were purchased from AGA (Santiago, Chile). All themeasurements were carried out in a BAS CV-100 W voltammetricanalyzer with a three electrode measuring cell. A glassy carbon disc

electrode with an area of 0.071 cm2, a platinum wire counter elec-trode and an (Ag/AgCl/NaClsat) reference electrode were used forthe measurements. The glassy carbon disk working electrode waspolished using successively 0.3 �m and 0.05 �m alumina powderon a polishing cloth before each measurement.

844 R. Salazar et al. / Electrochimica

Table 1Oxidation peak potential values on glassy carbon electrode in aprotic medium(DMSO + 0.1 M TBAPF6).

Compound E vs. (Ag/AgCl) (mV)

Peak IOH Peak IIOH �Ep-OHa PeakNH �Ep-NHb

3,4-OH-DHP 780 −12 1172 +1083,5-OH-DHP 952 −140 1212 +1483,4,5-OH-DHP 600 −92 1200 +1364-Ph-DHP – – 1064 –Pyrocatechol 792 – – –Resorcin 1092 – – –Pyrogallol 692 928 – – –

a Differences of oxidation peak potential values of OH group (peak I) with respectt

r

omtafwebtrwta

3

33

3

Dps

7

This effect can be corroborated from Fig. 2A, where 3,4-OH-DHP

Fp

o the phenol reference derivative (pyrocatechol, resorcin and pyrogallol).b Differences of oxidation peak potential values of 1,4-dihydropyridine ring with

espect to 4-Ph-DHP derivative.

In oxygen media, O2 gas was bubbled directly into the cell inrder to obtain a fixed concentration of oxygen, and during theeasurement, O2 gas was flushed over the cell solution. In order

o maintain fixed oxygen concentration in the measurement celln apparatus consisting of two flow-meters (Cole Palmer 316SS)or oxygen and nitrogen, respectively, equipped with needle valvesere used. Knowing the oxygen solubility in DMSO [39] and by

stablishing the oxygen and nitrogen flow rates, it was possi-le to determine the concentration of oxygen in the gas passinghrough the measurement cell [40]. All cyclic voltammograms wereecorded at a constant temperature of 25 ◦C, the initial potentialas fixed at 0.0 V vs. (Ag/AgCl/NaClsat) and the scanning poten-

ial was reversed at −0.9 V vs. (Ag/AgCl/NaClsat). Concentration ofntioxidants varied between 0 and 50 mM.

. Results and discussion

.1. Electrochemical oxidation of 3,4-OH-DHP, 3,5-OH-DHP and,4,5-OH-DHP in DMSO + 0.1 M TBAFP6

.1.1. VoltammetryPV results revealed that compounds 3,5-OH-DHP and 3,4,5-OH-

HP exhibited two well-defined anodic peaks. In Table 1 oxidation

eak potential values and �Ep of different derivatives in DMSO areummarized.

The corresponding potential values for 3,4-OH-DHP were80 mV and 1172 mV and for 3,5-OH-DHP were 952 mV and

140012001000800600400

II

I

I

3,4,5-OH-DHP

3,5-OH-DHP

3,4-OH-DHP

5μA

II

II

I

E vs (Ag / AgCl)/ mV

A

ig. 2. Dp voltammograms in DMSO + 0.1 M TBAFP6 of: (A) 1 mM solutions of 3,4-OH-DHyrogallol solutions.

Acta 56 (2010) 841–852

1212 mV. The oxidation peak potential values corresponding to3,4,5-OH-DHP were 600 mV and 1200 mV (Fig. 2A, Table 1).

The electroactive groups responsible for these oxidation peakscould be assigned by comparison with reference compounds basedon the following considerations: (a) 4-Ph-DHP derivative, havingno OH groups at all, experimented only the oxidation of the dihy-dropyridine ring, which occurs at 1064 mV in the same electrolyticmedia (Fig. 2B, Table 1) (b) pyrocatechol (1,2-dihydroxybenzene)and resorcin (1,3-dihydroxybenzene) exhibited a single oxidationsignal at potentials of 792 mV and 1092 mV, respectively. (c) Pyro-gallol (1,2,3-trihydroxybenzene) shows two oxidation signals at692 mV and 928 mV. Considering that these compounds lack ofa dihydropyridine ring, the signals corresponded to the oxidationof the OH groups (Fig. 2B, Table 1). Consequently, the oxidationpeaks in the Fig. 2A can be assigned as follows: peak I appear-ing in the range 600–1000 mV belong to the oxidation of the OHgroups. Peak II correspond to the oxidation of the DHP ring. It canbe seen from Fig. 2A and B that the oxidation potential of 4-Ph-DHP (Ep = 1064 mV) is shifted towards higher oxidation potentialvalues related to the hydroxyphenyl-substituted 1,4-DHP, whichcan be explained considering the hydroxyphenyl moiety as anelectron-withdrawing group because its conversion to quinones(3,4-OH-DHP and 3,4,5-OH-DHP).

The coexistence of two redox centers (i.e. phenolic and DHPmoieties) in the same molecule, produced significant changes inthe oxidation potential respect to the molecules wherein the cen-ters are isolated (Fig. 2, Table 1). A shift toward lower oxidationpotential values for the phenolic groups in the C4-substituted 1,4-DHPs was observed. In contrast, the oxidation of the DHP ring inthese compounds occurred at higher oxidation potential values.The results above-summarized are consistent with the electron-donating effect of the dihydropyridine ring on the OH groupsoxidation. In the case of 3,4-OH-DHP, the DHP ring increases theelectronic density of the OH group in p-position (4-position ofphenyl moiety), facilitating the oxidation of this group. This behav-ior is consistent with previous results obtained by Michalkiewiczand Skorupa [41] indicating that the first stage of the anodic oxi-dation of 3,4-dihydroxyphenyl acetic acid (DOPAC), proceed on theOH group at 4-position of the aromatic ring.

exhibits a lower oxidation potential of the OH group than 3,5-OH-DHP derivative where the hydroxyl group are in meta-position withrespect to the DHP ring position (�Ep = 172 mV), in concordancewith studies that demonstrated that cathechol moiety is more eas-

B

140012001000800600400

*

4-Ph-DHP

pyrogallol

pyrocatechol

resorcin

10μA

E vs (Ag / AgCl) / mV

P, 3,5-OH-DHP and 3,4,5-OH-DHP. (B) 1 mM 4-Ph-DHP, pyrocatechol, resorcin and

R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852 845

A

1400120010008006004002000

3,4,5-OH-DHP

3,5-OH-DHP

3,4-OH-DHP

10 μA

E vs (Ag / AgCl) / mV

B3,4-OH-DHP 3,4,5-OH-DHP

I I

I

II

8006004002000-200

10 μA

8006004002000-200

10 μA

F HP in0

iOiwhdbsrtt

tatosh

sd(t−rtoTP

E vs (Ag / AgCl)/mV

ig. 3. Cyclic voltammograms of 1 mM solutions of synthesized C4-phenolic 1,4-D.1 V s−1.

ly oxidizable than resorcin [34]. Moreover, the oxidation of theH group at 4-position on the 3,4,5-OH-DHP derivative is eas-

er than that of 3,4-OH-DHP (�Ep = 180 mV), which is consistentith the interaction between the hydroxyl in 4-position with bothydroxyl groups at 3- and 5-position together with the electron-onating effects of the DHP ring at 1-position. The interaction maye hydrogen-bonding between the OH groups and also electronicubstituent effects on the hydroxyl group at 4-position. The finalesult of these interactions may explain the oxidation easiness ofhis derivative. In any case, the electrode oxidation at lower oxida-ion potentials produces the substituted quinones as final products.

In conclusion, for 3,4-OH-DHP and 3,4,5-OH-DHP derivativeshe first step involves the oxidation of the OH group at 4-position ofromatic ring generating a phenoxyl radical followed by the oxida-ion of the second hydroxyl group at 3-position with the formationf an ortho-quinone derivative [42,43], which could affect the sub-equent oxidation of the DHP ring. This fact produces a shift towardigher oxidation potential values for the second anodic peak.

Cyclic voltammetric experiments were carried out at differentweep rates ranging from 0.1 V s−1 up to 3 V s−1. Under these con-itions, two anodic signals were observed for three derivativesFig. 3A). From cyclic voltammograms in the range of −1000 mVo 1000 mV, reduction peaks at 185 mV for 3,4-OH-DHP and at85 mV for 3,4,5-OH-DHP, corresponding to the reduction of the

espective quinone (Fig. 3B) were found. Thus, these two deriva-ives were oxidized through a quasi-reversible process. In the casef 3,5-OH-DHP, no cathodic peak was found in the same range.herefore, this derivative is oxidized by an irreversible process.eak II corresponding to the oxidation of DHP ring (Fig. 3A) shows

E vs (Ag / AgCl)/mV

DMSO + 0.1 M TBAPF6 (A) Extended sweep (B) short sweep for peak I. Sweep rate:

no signal in the reverse scan for all compounds indicating that thisoxidation process is of irreversible character.

Plots of log (I/�A) vs. log(v/V s−1) had slopes between 0.5 and 1.0,indicating that the current is controlled by a mixed process (diffu-sion and adsorption). On the other hand, oxidation potential valueswere dependent on the sweep rates supporting the irreversiblecharacter of the oxidation of the compounds in this medium.

To understand the mechanism of oxidation in more detail andto determine the number of electrons transferred in the oxidationprocess of synthesized 1,4-DHPs, controlled-potential electrolysis(CPE) in DMSO + 0.1 M TBAFP6 were carried out. For all the syn-thesized C4-phenolic-DHP, the first oxidation process an averagen value of 2.25 ± 0.25 and an overall process with an average nvalue of 4.55 ± 0.30 were obtained. CPE of pyrocatechol, resorcinol,pyrogallol and 4-Ph-DHP derivatives exhibited an average n valueof 2.15 ± 0.15 electrons. In conclusion, the first oxidation processcomprises the transformation from the phenol to the respectivequinone, while the second value is compatible with the oxidationof both the phenolic moiety and the dihydropyridine ring.

In conclusion, for 3,4-OH-DHP and 3,4,5-OH-DHP derivativesthe oxidation involves a first stage of anodic oxidation proceedingon the OH group in position four of the aromatic ring and pro-duces a phenoxyl radical. This radical is chemically unstable andparticipates in the successive homogenous reaction. As a conse-

quence of this, only a small part of this product could takes partin the second stage of the electrode reaction. This fast charge-transfer step produces substituted ortho-quinone which undergoesthe cathodic reduction at the potential 185 mV for 3,4-OH-DHPand −85 mV for 3,4,5-OH-DHP (Fig. 3B). This interpretation is

846 R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852

F applU 1200

c[

33

3

bdsawtcos3tstGni

Tbp

ig. 4. (A) UV–visible spectra of the CPE of a 0.1 mM solution of 3,4-OH-DHP at anV–visible spectra of the CPE of electrolyzed solution (A) at an applied potential of

onsistent with previous reports on the oxidation of cathecols41,44–46].

.2. Characterization of oxidation products from 3,4-OH-DHP,,5-OH-DHP and 3,4,5-OH-DHP

.2.1. Spectroelectrochemical experiments3,4-OH-DHP shows two original UV–vis maxima absorption

ands near to 290 nm and 350 nm (Fig. 4A). CPE at 900 mV pro-uce significant changes in the UV–vis differential spectra as arehown in Fig. 4B. Three new bands at 272 nm, 312 nm and 406 nmppeared. A decrease in the UV–vis zone at 365 nm was also foundhich corresponds to the cathecol part of the molecule. This elec-

rolyzed solution was used for a second further electrolysis at aontrolled-potential of 1200 mV during 500 s (Fig. 4C). We canbserve the appearance of a new UV–vis band at 268 nm and theignificant decrease in the intensity of the original UV–vis band at43 nm (Fig. 4D). From these spectral changes it can be concludedhat: (a) at an applied potential of 900 mV the maximum at 406 nmupports the formation of the corresponding ortho-quinone deriva-ive from 3,4-OH-DHP, which is consistent with a previous work ofiacomelli et al. [47]. (b) At an applied potential of 1400 mV, theew maximum at 268 nm accounts for pyridine formation accord-

ng to previous works of us [13,17,25] and others [48].CPE of both pyrocatechol and 4-Ph-DHP were also carried out.

he original spectrum of pyrocatechol shows a single sharp UV–visand at 280 nm. The application of an oxidation potential at 900 mVroduced the appearance of maxima at 262 nm, 296 nm and 374 nm

ied potential of 900 mV during 500 s. (B) UV–visible differential spectra of (A). (C)mV during 500 s. (D) UV–visible differential spectra of the solution in (C).

(Fig. 5A). Clearly, these new maxima are related with the oxida-tion product, i.e. the quinone derivative, which exhibits a similarresponse to that shown in Fig. 4A and B for the electrolysis of 3,4-OH-DHP at an applied potential of 900 mV.

Furthermore, after the CPE of 4-Ph-DHP the most significantchanges on the UV–vis spectra were the decrease in the intensity ofthe original UV–vis band at 354 nm (which is due to the oxidationof the dihydropyridine nucleus) and the appearance of a new bandat 278 nm which corresponds to the oxidation product, i.e. the pyri-dine derivative (Fig. 5C and D). The above UV assignments provingthe oxidation of phenolic and 1,4-DHP moieties were confirmed bycomparison with the spectroelectrochemical results obtained withreference compounds having only one moiety in its structure.

To support quinone formation after the electrolysis at anapplied potential of 900 mV for 3,4-OH-DHP and 3,4,5-OH-DHPand at applied potential of 1050 mV for 3,5-OH-DHP, we used thewell-known chemical characterization reaction of ketones withhydroxylamine leading to oxime formation [49,50]. The idea behindthis characterization was to compare the chemically synthesizedDHP-oximes with the in situ electrochemically produced ones.Therefore, we can compare the UV–vis spectra obtained after theelectrochemical oxidation of the derivatives in the presence of5 mM hydroxylamine with the chemical oxidation of 1 mM phe-

nolic 1,4-DHP with 0.1 M KIO4 solution in acetonitrile/water (1:1)in the presence of hydroxylamine to produce the oxime derivative.

In Fig. 6A, the time-course of CPE corresponding to 3,4,5-OH-DHP after 15 min in the presence of hydroxylamine is shown. Ascan be seen from this figure, two bands at 288 nm and 350 nm

R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852 847

F applieU 200 m

aactctUaaetato

Fbos

ig. 5. (A) UV–visible spectra of the CPE of a 0.5 mM pyrocatechol solution at anV–visible spectra of CPE of 0.1 mM 4-Ph-DHP solution at an applied potential of 1

ppeared supporting the formation of the corresponding oximes a consequence of the reaction between the quinone electro-hemically generated and the hydroxylamine. Fig. 6B shows: (1)he UV–vis spectrum for the electrochemical oxidation and (2) thehemical oxidation with KIO4 for 3,4,5-OH-DHP compound, respec-ively. As can be seen, both oxidation products exhibited a similarV–vis spectrum. A similar behavior was shown by 3,4-OH-DHPnd 3,5-OH-DHP derivatives. Two bands with maxima at 286 nmnd 356 nm were observed in the case of 3,4-OH-DHP derivative

ither by electrochemical and chemical oxidation. For 3,5-OH-DHPhe absorbance maxima for the oxidation product were at 286 nmnd 355 nm. As expected, 4-Ph-DHP did not show any change inhe UV–vis spectrum at an applied potential of 900 mV either withr without addition of hydroxylamine. Taking into account the

550500450400350300250

0

1

2

6

5

4

3

2

1

A

Abs

orba

nce

/ a.u

.

λ / nm

ig. 6. (A) Time-course of CPE 0.1 mM 3,4,5-OH-DHP solution at an applied potential of 90uffer pH 5.5/acetonitrile (1/1). 1: 0 min, 2–6: 15–45 min. (B) 1: UV–visible spectrum of CPEf 1 mM of hydroxylamine at 15 min. 2: UV–vis spectrum for the reaction product betwolution (1:1) in the presence of 0.1 M of hydroxylamine after 15 min.

d potential of 900 mV during 500 s. (B) UV–visible differential spectra of (A). (C)V during 500 s. (D) UV–visible differential spectra of (C).

above results, in Scheme 1 the oxidation mechanism of C4-phenolicsynthesized compounds is depicted. Also, these results are in agree-ment with previous observations [26,28,32–34].

3.3. Reactivity of 1,4-dihydropyridines towards superoxideradical anion

3.3.1. NMR experiments. Assessment of the acid–base reactionbetween DHPs derivatives and superoxide radical anion

In order to test the acid–base reactivity of protons belonging tothe –NH– part of the dihydropyridine ring and hydroxyl groups onthe phenyl ring of 1,4-DHP compounds towards superoxide anion,their NMR spectra in DMSO-d6 in the absence and in the presenceof O2

•− were recorded. The 1H NMR spectra of substituted DHPs in

550500450400350300250

0

1

2

3

1

B

Abs

orba

nce

/ a.u

.

λ / nm

2

0 mV in the presence of 1 mM of hydroxylamine in 0.1 M aqueous Britton–Robinsonof 0.1 mM 3,4,5-OH-DHP solution at an applied potential of 900 mV in the presence

een 1 mM 3,4,5-OH-DHP solution and 0.1 M KIO4 solution in a acetonitrile/water

848 R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852

NH

OH

OH

OO

OO

NH

O

O

OO

OO

NH

NOH

NOH

OO

OO

-2H-2e, +

mV900

NH2OH

KIO1. 4 AcCN/H, 2 min.20ºC,25(1:1),O

NH2. 2OH

N

O

O

OO

OO

-2H-2e, +

mV1400

S ppinge

DiIWolpraa

cheme 1. Oxidation mechanism exemplified for 3,4-OH-DHP in DMSO and the traither after the electrochemical or the chemical oxidations.

MSO-d6 show signals between 8.2 ppm and 8.6 ppm correspond-ng to the hydroxyl and secondary amino groups of the molecules.n Fig. 7A this behavior is represented for 3,4-OH-DHP derivative.

hen we mix the C4-phenolic 1,4-DHP derivatives with an excessf O2

•− in DMSO-d6 an acid–base equilibrium is quickly estab-ished. After the addition of O2

•−, the signals in the range (8.2–8.6)

pm disappeared, evidencing anion formation (Fig. 7B). From theseesults it can be concluded that the superoxide anion acts as a basebstracting the proton at 1-position of the dihydropyridine ring andlso the hydroxyl group protons on the aromatic ring at C4-position.

56789

A C4-H

ppm

Ar-OHDHP-NH

Fig. 7. 1H NMR for 3,4-OH-DHP derivative in the a

by hydroxylamine of the oxidized products corresponding to the phenolic moiety

3.3.2. About the O2•− scavenging by 1,4-dihydropyridines

As it was demonstrated in a previous publication [13] the anionicform of DHPs does not led to products and therefore O2

•− scaveng-ing is carried out by the DHP itself.

Hydroxy phenyl DHPs have two redox centers (i.e. the hydrox-yphenyl and DHP rings) that scavenge O2

•− by two different•−

mechanisms: (a) the interaction of O2 and the hydroxyphenyl

ring leading to quinone type of compounds (I) and (b) the oxida-tion of the DHP ring to form pyridine derivatives (II) as is shown inScheme 2.

56789

BC4-H

ppm

bsence (A) and in the presence of O2•− (B).

R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852 849

NH

OH

OH

CO2EtEtO2C

N

O

O

CO2EtEtO2Cacid-base

equilibrium

HOO3+

O2 2

O3 2

hydroxyphenylbyscavenginggroup.

NH

O

O

CO2EtEtO2C(I)

HOO2+

O2 2

dihydropyridinebyscavengingring.

N

O

O

CO2EtEtO2C

(II)

Scheme 2. Mechanism involved in the scavenging activity of C4-phenolic 1,4-dihydropyridines.

-300-400-500-600-700-800

-40

-30

-20

-10

0

10

20

30 A

Ipa

0

O2

-.

O2

-. O2

O2

I / μ

A

E vs (Ag / AgCl)/mV-300-400-500-600-700-800

-40

-30

-20

-10

0

10

20

30

I / μ

A

B

10 mA

E vs (Ag / AgCl)/mV

Fig. 8. (A) Typical cyclic voltammogram of the electroreduction of O2 showing the electrode process and the parameters involved. (B) Cyclic voltamograms of the electrore-duction of oxygen in absence (solid line) and in the presence (dashed line) of 1.0 mM of 3,4,5-OH-DHP.

850 R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852

302520151050

0.0

0.2

0.4

0.6

0.8

1.0

0.50.40.30.20.10.00.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

(ipa

-ipa

)/ip

a

[1,4-DHP] / mM

[1,4-DHP] / mmol dm^-3

(ipa0 -ip

aS)/

ipa0

3,4,5-OH-DHP 3,4-OH-DHP 3,5-OH-DHP Trolox

Fig. 9. Dimensionless parameter related to the decrease of superoxide anodic cur-rtt

3r

dodDTdsavcaIsfl

aiioattscrvdai

Ot

rDorc

50403020100

0.0

0.2

0.4

0.6

0.8

1.0

AI 30

AI 50

[1,4-DHP] / mmol dm^-3

(ipa0 -ip

aS)/

ipa0

Phenyl-DHP Nifedipine Nisoldipine Nimodipine Amlodipine

explanation given is related to the ability of losing the proton of thehydroxyl group at 4-position of the phenyl group easier than theproton of hydroxyl at 3-position [55].

Table 2Parameters for the regression equations for the synthesized and commercial 1,4-dihydropyridines. .

Compound Antioxidant index (AI)a AI30 (mM) AI50 (mM) r2

3,4,5-OH-DHP 0.689 ± 0.076 – 0.11 0.9953,4-OH-DHP 0.583 ± 0.024 0.49 0.81 0.9933,5-OH-DHP 0.114 ± 0.019 2.00 3.78 0.9974-Ph-DHP 0.112 ± 0.017 0.30 50.00 0.996Nifedipine 0.213 ± 0.022 1.92 5.13 0.996Nisoldipine 0.188 ± 0.020 1.86 5.58 0.994

ent, [I0pa − IDHP

pa ]/I0pa vs. the concentration of synthesized 1,4-dihydropyridines and

he reference antioxidant, Trolox. Inset: Typical linear dependence at a concentra-ion level between 0.01 and 1 mM for 3,4,5-OH-DHP derivative.

.4. Reactivity of 1,4-DHPs towards electrogenerated superoxideadical anion assessed by cyclic voltammetry

Cyclic voltammetry can be employed for the generation andetection of O2

•− by the electrochemical reduction of molecularxygen. O2

•− is derived from the direct electro-reduction, in theiffusion layer of glassy carbon electrodes, of molecular oxygen inMSO medium (Fig. 8A), without inclusion of any other compound.he presence of the radical O2

•− is easily detected by its anodic oxi-ation current measured at the same electrode during the reversecan (Fig. 8A). It is known that O2

•− radical is stable in aprotic mediand dismutation does not occur during the time scale of the cyclicoltammetry in DMSO solution [51]. By using this procedure wean instantly follow the reaction of O2

•− with bioactive compoundst the electrode surface in the oxygen reduction potential range.n fact, several authors have reported studies on the reactivity ofuperoxide radical anion, O2

•− with 1,4-dihydropyridines [52,53],avonoids [37] and sulfur amino acids [54].

In the present paper, the reactivity of some new synthesizednd commercial 1,4-DHPs towards superoxide radical anion, O2

•−

s assessed by a cyclic voltammetric methodology. From the exper-mental point of view, single cyclic voltammograms in the absencef 1,4-DHPs were recorded to determine the current value I0

pa, as thenodic peak current of O2

•− (Fig. 8A). This value is directly relatedo the O2

•− concentration at the electrode surface and depends onhe solubility of oxygen in DMSO and the experimental parameterselected to record the cyclic voltammograms. The time-scale wasontrolled by the scan rate at 0.1 V s−1 and the working potentialange was settled from 0 mV to −900 mV vs. (Ag/AgClsat). Cyclicoltammograms of O2 reduction were recorded in the presence ofifferent concentrations of synthesized and commercial 1,4-DHPsnd the reference antioxidant, Trolox, to evaluate their antioxidantndex towards superoxide radical anion.

The increase of 1,4-DHPs concentration leads to a decrease of2

•− anodic peak current, IDHPpa , with no significant modification of

he cathodic current (Fig. 8B).So our observations suggest that the 1,4-DHP compounds

eacted with superoxide radical as H atom donors leading to OH-

HP• radical and the conjugated base of hydrogen peroxide. A seriesf IDHP

pa values were determined from the cyclic voltammogramsecorded at increasing concentrations of each tested compound. Asan be seen from Fig. 9, all the tested compounds exhibited a sim-

Fig. 10. Dimensionless parameter related to the decrease of superoxide anodic cur-rent, vs. the concentration of commercial 1,4-dihydropyridines.

ilar effect on the O2 reduction, i.e. a linear dependence betweenthe dimensionless parameter [I0

pa − IDHPpa ]/I0

pa and the 1,4-DHP con-centration range between 0.01 mM and 1.00 mM (inset in Fig. 9).Exceeding this concentration level, linearity is lost. Calculation ofantioxidant index, AI50 (concentration of 1,4-DHP needed to con-sume superoxide radical by a current decrease of 50% of the initialanodic current) were carried out in the concentration linear range(Fig. 10). The kinetics of the reaction between the 1,4-DHPs andO2

•− may be related to the slopes of straight lines obtained fromthe graphics of [I0

pa − IDHPpa ]/I0

pa vs. 1,4-DHP concentration (Inset,Fig. 9, i.e. the biggest slope means the most active compound).The corresponding slope values are shown in Table 2. As can beseen, commercial 1,4-DHPs were significantly less reactive thanthe C4-phenolic synthesized 1,4-DHPs and Trolox. 3,4,5-OH-DHPand 3,4-OH-DHP were respectively, 2.45 and 2.10-fold more reac-tive towards O2

•− than Trolox. A similar trend for the AI50 values isobserved, i.e., 3,4,5-OH-DHP has an AI50 value that is 31-fold lowerthan Trolox. In Table 2, it can be also observed that the 3,5-OH-DHPis the less reactive hydroxyl derivative.

The lower reactivity towards O2•− exhibited by 3,5-OH-DHP

compared with the 3,4-OH-DHP derivative, is in agreement withtheoretical and experimental studies which document that 3,5-dihydroxy phenyl group is significant less reactive than the3,4-dihydroxy phenyl group due to differences in the phenolic O–Hbond dissociation enthalpies of the OH groups at position 3 and 5in the phenyl with respect to the same OH at position 3 and 4. The

Nimodipine 0.222 ± 0.020 2.04 4.63 0.997Amlodipine 0.209 ± 0.019 2.04 5.35 0.998Trolox 0.281 ± 0.036 0.78 3.41 0.999

a Average slopes obtained from five independent runs for each compound.

R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852 851

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

1300120011001000900800700600500

s (A

AN

TIO

XID

AN

T C

AP

AC

ITY

Trolox

3,5-OH-DHP

4-P

h-D

HP

Nis

oldi

pine

Nife

dipi

neA

mlo

dipi

ne

Nim

odip

ine

F e oxidd

(pt

muaCaocc3pOtihad

4

rrnudc

hpci

E v

ig. 11. Relationship between antioxidant index and peak potential values for therivative (�).

The examination of inhibitory concentration of 1,4-DHPsTable 2) reveals that 3,4-OH-DHP and 3,4,5-OH-DHP were the mostotent compounds exhibiting the lowest AI50 values, meaning thathey have significant antioxidant index towards superoxide radical.

In Fig. 11, the relationship between the antioxidant index (AI,easured as O2

•− consumption) and oxidation peak potential val-es of phenolic DHPs, 1,4-DHP (with no substituents at phenyl ring)nd commercial DHPs are depicted. As can be seen, the synthesized4-phenolic DHPs and Trolox exhibit a nice dependence between AInd Ep ox, i.e., the most active compound towards O2

•− has a lowerxidation potential value. In contrast, commercial DHPs maintain aonstant relationship between AI and Ep ox. Low Ep ox values exhibitomparatively high AI properties of the synthesized 3,4,5-OH and,4-OH-DHPs and are consistent with the oxidation easiness of thehenolic part of the molecules to their respective quinones. 3,4,5-H-DHP may form the o-quinone easier than 3,4-OH-DHP while

he 3,5-OH-DHP does not form a quinonic compound demonstrat-ng poor antioxidant index. Commercial DHPs and 4-Ph-DHP haveigh Ep ox and low AI values and their antioxidant effects may bettributed to the oxidation of DHP ring to the respective pyridineerivative.

. Conclusions

The new synthesized C4-polyphenolic 1,4-DHP showing twoedox centers capable to be electrochemically oxidizable. Bothedox centers mutually interacted, i.e. the oxidation of the phe-olic part of the molecules was easier than the correspondingnsubstituted-phenolic compounds. The oxidation of the dihy-ropyridine ring was shifted towards more anodic potential valuesompared with the unsubstituted 4-Ph-DHP.

Regarding the characterization of the oxidation products, weave found that the oxidation of phenolic moieties of com-ounds gave rise to the corresponding quinone derivatives. Thehemical nature of these oxidation products was confirmed byts further reaction with hydroxylamine to generate the corre-

[

[

g/AgCl)/mV

ation of: OH group to quinone-type derivative (�) and 1,4-DHP ring to pyridine

sponding dioxime derivative. The 1,4-dihydropyridine ring wasoxidized to the pyridine derivative, which exhibited the charac-teristic UV–visible band with maxima at (270–278) nm.

All synthesized C4-polyphenolic 1,4-dihydropyridines weremore reactive than the tested commercial ones and even than thereference antioxidant, Trolox, towards superoxide radical anion.

Scavenging of O2•− by C4-phenolic 1,4-DHPs are related to the

existence of hydrogens at the N-position of the dihydropyridinering and hydroxyl groups on the phenyl ring according to NMRexperiments. It is well-known that the high reactivity of phenoliccompounds is based on the abstraction of H• by O2

•− [56] whichgives rise to the phenoxyl radical and the further formation of therespective quinone plus HOO− [42]. On the other hand, the oxida-tion of 1,4-dihydropyridine ring to the pyridine derivative supportthe participation of this moiety in the scavenging of this type ofradicals.

Acknowledgement

This work was supported by Grant from FONDECYT 1080132.

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