3° Encontro Nacional de Quimica Terapeutica

3rct Portuguese Meeting on Medicinal Chemistry 1'1 Portuguese-Spanish-Brazilian Meeting on Medicinal Chemistry.

P27

Electrochemical behavior of hydroxyxanthones versus ROS and RNS scavenging activities

Clementina M. M. Santos,a,b M. Beatriz Q. Garcia,c Artur M. S. Silva,b Eduarda Fernandesc

"Department of Vegetal and Production Technology, School ofAgriculture, Polytechnic Institute of Braganfa, 5301-855 Bragan,·a, Portugal; ''Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal; cREQUIMTE, Department of Ch emical Sciences, Faculty of Pharmacy, University of

Porta, Rua de forge Viterbo Ferreira n." 228, 4050-313 Porta, Portugal

Xanthones are a class of naturally-occurring oxygenated heterocyclic compounds with a broad spectrum of biological activities of great interest for potential therapeutic applications. 1 IJ Thus, over the last decades a great number of publications have emerged focusing on the isolation and s~nthesis of these natural compounds or even in the search for novel bioactive derivatives.1- 1

Electrochemical methodologies can be applied to establish correlations between structure and oxidation potential of electroactive species and therefore to predict some of its biological activities. In fact, cyclic voltammetry has become an important and widely used electroanalytical technique in many relevant studies of redox ~rocesses for clarifying the electrochemical behavior of chemical and biochemical systems.1 1

Herein, we will report the electrochemical behavior of several phenolic and catecholic-substituted 2,3-diarylxanthones by cyclic voltammetry using a glassy carbon electrode, at pH 7.4, and the results obtained compared with their scavenging activities for reactive oxygen species (ROS) and reactive nitrogen species (RNS). 141 Useful considerations about oxidation mechanism will be highlighted and the electrochemical profile of xanthones will corroborate their biological propetties.

9.0

5.0

<t .:- 1.0

·3.0

·7.o.0.6 ·0.3 0.0 0.3 0.6 0.9

E I V (vs. Ag/AgCI)

Acknowledgments: Sincere thanks are expressed to Faculdade de Farmacia da Universidade do Porto, and to Universidade de Aveiro, Funda<;:ao para a Ciencia e a Tecno1ogia (Portugal) and FEDER for funding the Organic Chemistry Research Unit (project PEst-C/QUI/UI0062/20 11 ).

References [IJ (a) E1-Seedi, H.R. ; E1-Ghorab, D.M.H.; El-Barbary, M.A.; Zayed, M.F. ; Goransson, U.; Larsson, S. ;

Verpoorte, R. Curr. Med. Chem. 2009, /6, 2581. (b) Pinto, M. M. M.; Sousa, M. E.; Nascimento, M. S. J. Curr. M eel. Chem. 2005, / 2, 2517.

[2] (a) Pinto, M. M. M. ; Castanheiro, R. A. P. Curr. Org. Chem. 2009, 13, 1215. (b) Pinto, M. M. M. ; Sousa, M. E.; Curr. Med. Chem. 2005, /2 , 2447.

[3] Teixeira. J. G. ; Dias, C. B.; Teixeira, D. M. Electroanalysis 2009, 21 , 2345. (b) Boza1 , B. ; Us1u, B.; Ozkan, S. A. Int. J. Electrochemistry 2011 , 1. (c) Heinze, J. Angew. Clw n. lnt. Ed. Engl. 1984, 23, 831.

[4) Santos, C. M. M.; Freitas, M.; Ribeiro, D.; Gomes, A. ; Si1va, A. M. S. ; Cava1eiro, J. A. S. ; Fernancles, E. Bioorg. Med. Chem. 2010, 18, 6776.

1c and 3a Ipa / Ipc = 1

Electrochemical reversible process

Clementina M. M. Santos,a,b M. Beatriz Q. Garcia,c Artur M. S. Silvab and Eduarda Fernandesc aDepartment of Vegetal and Production Technology, School of Agriculture, Polytechnic Institute

of Bragança, 5301-855 Bragança, Portugal. Email: clems@ipb.pt bDepartment of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal cREQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto,

Rua de Jorge Viterbo Ferreira n.º 228, 4050-313 Porto, Portugal

[1]. (a) El-Seedi, H.R.; El-Ghorab, D.M.H.; El-Barbary, M.A.; Zayed, M.F.; Göransson, U.; Larsson, S.; Verpoorte, R. Curr. Med. Chem. 2009, 16, 2581.

(b) Pinto, M. M. M.; Sousa, M. E.; Nascimento, M. S. J. Curr. Med. Chem. 2005, 12, 2517.

[2]. (a) Pinto, M. M. M.; Castanheiro, R. A. P. Curr. Org. Chem. 2009, 13, 1215. (b) Pinto, M. M. M.; Sousa, M. E.; Curr. Med. Chem. 2005, 12, 2447.

[3]. (a) Teixeira. J. G.; Dias, C. B.; Teixeira, D. M. Electroanalysis 2009, 21, 2345. (b) Bozal, B.; Uslu, B.; 蒐zkan, S. A. Int. J. Electrochemistry 2011, 1.

(c) Heinze, J. Angew. Chem. Int. Ed. Engl. 1984, 23, 831.

[4]. Santos, C. M. M.; Freitas, M.; Ribeiro, D.; Gomes, A.; Silva, A. M. S.; Cavaleiro, J. A. S.; Fernandes, E. Bioorg. Med. Chem. 2010, 18, 6776.

[5]. Santos, C. M. M.; Silva, A. M. S.; Cavaleiro, J. A. S. Eur. J. Org. Chem. 2009, 2642.

Sincere thanks are expressed to Faculdade de Farmácia da

Universidade do Porto, and to Universidade de Aveiro, Fundação

para a Ciência e a Tecnologia (Portugal) and FEDER for funding

the Organic Chemistry Research Unit (project PEst-

C/QUI/UI0062/2011).

Table 1

O2•-, 1O2, HOCl, •NO and ONOO- (with and without 25 mM NaHCO3) scavenging effects of the tested 2,3-

diarylxanthones and positive controls (IC50, mean ± SEM).

Compound

IC50 (ちM)

O2•- 1O2 HOCl NO•

ONOO- without

NaHCO3

ONOO- with

NaHCO3

2,3-diarylxanthones

1a NA25たM 27%*100たM 155 ± 25 39%*200たM 29%*50たM 48%*50たM

1b NA200たM 80 ± 11 72.1 ± 8.6 175 ± 36 1.55 ± 0.14 1.80 ± 0.39

1c 28.1 ± 2.3 6.0 ± 1.0 15.7 ± 1.1 1.88 ± 0.18 0.40 ± 0.03 0.54 ± 0.08

2a NA200たM 68.8 ± 6.2 53.8 ± 7.8 41%*200たM 2.66 ± 0.29 2.00 ± 0.15

2b 166 ± 16 58.4 ± 4.9 22.4 ± 2.5 108 ± 18 1.72 ± 0.21 0.97 ± 0.25

2c 20.3 ± 2.5 3.3 ± 0.7 14.7 ± 1.3 0.42 ± 0.05 0.26 ± 0.05 0.78 ± 0.26

3a 76 ± 11 4.5 ± 0.6 10.8 ± 0.4 1.22 ± 0.21 0.37 ± 0.09 0.67 ± 0.09

3b 31.3 ± 3.2 6.8 ± 0.5 7.5 ± 0.7 0.62 ± 0.10 0.22 ± 0.03 0.89 ± 0.18

3c 10.4 ± 0.8 2.5 ± 0.2 1.2 ± 0.02 0.39 ± 0.05 0.17 ± 0.01 0.33 ± 0.06

Positive controls

Tiron 273 ± 32 __ __ __ __ __

Quercetin __ 1.8 ± 0.1 __ __ __ __

Dihydrolipoic acid __ __ 2.3 ± 0.3 __ __ __

Rutin __ __ __ 2.53 ± 0.37 __ __

Ebselen __ __ __ __ 0.50 ± 0.03 2.01 ± 0.22

NANo activity was found up to the highest tested concentration (in superscript) *Scavenging effect (mean %) at the highest tested concentration (in superscript)

The oxidation peak at higher potential values can be attributed to the phenolic groups.

The absence of the corresponding reduction peaks also pointed to the irreversibility of the

redox reaction of the oxidized compounds generated in the forward scan.

Cyclic voltammograms of compounds 1c, 2c, 3a-c showed the permanent presence of a

low oxidation peak attributed to the catechol group oxidation, in an electrochemical

reversible process.

In the Pearson test, excellent correlations are observed for O2•−, •NO and ONOO−, as

expected for scavenging reactions involving electron transfer mechanisms. Significant

correlations are also found for HOCl and 1O2, highly reactive oxygen species which are

known for scavenging mechanisms involving structural features.

Xanthone 3c with two catechol units presented the lowest anodic potential voltage (Epa =

0.15 V) and proved to be the most effective ROS and RNS scavenger.

Scheme 1. Chemical structures of the studied 2,3-diarylxanthones

O

O

R2

R11a-c

O

O

OH

R2

R12a-c

O

O

OH

OH

R2

R13a-c

a) R1 = R2 = H

b) R1 = OH, R2 = H

c) R1 = R2 = OH

a) R1 = R2 = H

b) R1 = OH, R2 = H

c) R1 = R2 = OH

a) R1 = R2 = H

b) R1 = OH, R2 = H

c) R1 = R2 = OH

BC

D

E

A

Xanthones are a class of naturally-occurring oxygenated heterocyclic compounds presenting a broad spectrum of

biological activities of great interest for potential therapeutic applications [1]. Thus, over the last decades a great number

of publications have emerged focusing on the isolation and synthesis of these natural compounds or even in the search

for novel bioactive derivatives [2].

Electrochemical methodologies can be applied to establish correlations between structure and oxidation potential of

electroactive species and therefore to predict some of its biological activities. In fact, cyclic voltammetry has become an

important and widely used electroanalytical technique in many relevant studies of redox processes for clarifying the

electrochemical behavior of chemical and biochemical systems [3].

Herein, we report the electrochemical behavior of several phenolic and catecholic-substituted 2,3-diarylxanthones, by

cyclic voltammetry, using a glassy carbon electrode, at pH 7.4. The obtained results are then compared with their

scavenging activities for reactive oxygen species (ROS) and reactive nitrogen species (RNS) [4]. Useful considerations

about oxidation mechanisms are highlighted and the electrochemical profile of xanthones discussed in light of their

antioxidant properties.

Voltammetric experiments were carried out using an Autolab electrochemical system (Eco Chemie model PGSAT

10). Data were acquired using GPES (General Purpose Electrochemical System) software, version 4.9.

The working electrode was a glassy carbon electrode (GCE) (3.0 mm) whereas an Ag/AgCl (KCl 3 M) electrode

and a carbon electrode were used as reference and auxiliary electrodes, respectively. Before use in

electrochemical experiment and in order to obtain a clean renewed electrode surface, the glassy carbon working

electrode was hand-polished with a 0.075 たm alumina aqueous slurry using a polishing cloth and washed with

purified water.

Xanthones 1-3 were synthesized according to previously reported procedures [5] and the chemical structures are

presented in scheme 1. Stock solutions of 1a-c, 2a-c and 3a-c were prepared in DMSO and diluted in sodium

phosphate buffer solution pH 7.4 at the final concentration 0.1 mM, unless otherwise mentioned.

The cell volume was 10 mL and cyclic voltammograms were obtained in a single cycle performed at a scan rate

of 100 mV s-1, at room temperature. For the scan rate studies, the scanning speed varied from 10 to 200 mV s-1.

Voltammetric scans were recorded in the voltage range between -0.5 and +1.0 V versus Ag/AgCl.

0

40

80

120

160

200

240

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

O2.-

sca

ven

gin

g a

ctiv

ity

Epa / V

Pearson r = 0.9419 0

20

40

60

80

100

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

1 O2

scav

eng

ing

act

ivit

y

Epa / V

Pearson r = 0.7784 0

20

40

60

80

100

0.00 0.20 0.40 0.60 0.80

HO

Cl s

cave

ng

ing

act

ivit

y

Epa / V

Pearson r = 0.8383

0

40

80

120

160

200

0.00 0.20 0.40 0.60 0.80

NO

. sca

ven

gin

g a

ctiv

ity

Epa / V

Pearson r = 0.9680 0

1

2

3

0.00 0.20 0.40 0.60 0.80

ON

OO

- wit

h N

aHC

O3

scav

eng

ing

act

ivit

y

Epa / V

Pearson r = 0.8693 0

1

2

3

0.00 0.20 0.40 0.60 0.80

ON

OO

- wit

ho

ut

NaH

CO

3 sc

aven

gin

g a

ctiv

ity

Epa / V

Pearson r = 0.9623

-7.0

-5.0

-3.0

-1.0

1.0

3.0

5.0

7.0

9.0

-0.6 -0.4 -0.2 -1E-15 0.2 0.4 0.6 0.8 1

I / ち

A

E / mV vs. Ag/AgCl

2c 0.1 mM

inversion at 1.0 V

inversion at 0.5 V

-7.0

-5.0

-3.0

-1.0

1.0

3.0

5.0

7.0

9.0

-0.6 -0.4 -0.2 -1E-15 0.2 0.4 0.6 0.8 1

I / ち

A

E / mV vs. Ag/AgCl

3b 0.1 mM

inversion at 1.0 V

inversion at 0.5 V

-4.0

-2.0

0.0

2.0

4.0

6.0

-0.4 -0.2 0 0.2 0.4 0.6 0.8

I / ち

A

E / V vs. Ag/AgCl

1c 0.1 mM

3a 0.1 mM

-2.0

0.0

2.0

4.0

6.0

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

I / ち

A

E / mV vs. Ag/AgCl

1b 0.1 mM

2a 0.1 mM

2b 0.1 mM

-3.0

-1.0

1.0

3.0

5.0

7.0

9.0

11.0

-0.4 -0.2 0 0.2 0.4 0.6 0.8

I / ち

A

E / V vs. Ag/AgCl

3c 0.1 mM

Epa = 0.16 V

Epc = 0.12 V

Epa = 0.18 V

Epc = 0.14 V

Epa = 0.56 V

Epa = 0.64 V

Epa = 0.58 V

Epa1 = 0.16 V

Epa2 = 0.69 V

Epc = -0.12 V

Epa1 = 0.18 V

Epa2 = 0.65 V

Epc = -0.11 V

Epa1 = 0.15 V

Epa2 = 0.37 V

Epc = -0.06 V

Inversion of potential scan before the

second oxidation peak (at 0.5 V),

2c and 3b Ipa / Ipc = 1

Electrochemical reversible process

O

O

OH

OH

OH

OH3c

Lowest Epa value!

Compound 3c presents

the lowest IC50 values for all ROS and RNS tested!

Scheme 2. Correlations between the Epa at pH 7.4 and the scavenging activity against ROS and RNS of

hydroxyxanthones 1-3a-c.

3° Encontro Nacional de Quimica Terapeutica 3rd Portuguese Meeting on Medicinal Chemistry

151 Portuguese-Spanish-Brazilian Meeting on Medicinal Chemistry

Has participated at the 3'd Portuguese Meeting on Medicinal Chemistry and 1st Portuguese-Spanish-Brazilian Meeting on Medicinal Chemistry and has presented a poster communication entitled "Electrochemical behavior of hydroxyxanthones versus ROS and RNS scavenging activities"

Portugal, Aveiro, 28t11 to 3o'11 November 2012

~~ Prof. Artur M. S. Silva

Chairman

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l