Antioxidant activity of synthetic resveratrol analogs: A structure-activity insight

Article abstract of DOI:10.2174/157018012801319508

This study evaluated the antioxidant activity of five resveratrol analogs using the DPPH method. The molecules activity was related with their chemical structure. Besides descriptive statistics, the analysis of variance (ANOVA) followed by Tukey's post hoc test were performed (p<0.05). The antioxidant activity of analogs A and B was statistically similar with each other and from the reference standard resveratrol, possibly due to the presence of 4-hydroxy grouping. The aromatic hydroxyl reduces reactive free radicals and produces phenoxyl radical, stabilized by resonance. Although the analog C has shown IC₅₀ value statistically different from the resveratrol (p<0.001), its antioxidant activity was considered satisfactory. The other analogs (D and E), which have a 4-acid grouping in place of 4-hydroxy grouping, showed lower antioxidant activity than resveratrol (p<0.001). Further studies to address possible advantages of analogs in relation to resveratrol should be conducted in order to make them feasible for therapeutic use.

Full text of DOI:10.2174/157018012801319508

676
Letters in Drug Design & Discovery, 2012, 9, 676-679
Antioxidant Activity of Synthetic Resveratrol Analogs: A Structure-
Activity Insight
Natalia Oliveira Calil1, Gustavo Senra Gonçalves de Carvalho2, Annelisa Farah da Silva1, Adilson
David da Silva*,2 and Nádia Rezende Barbosa Raposo*,1
1Núcleo de Pesquisa e Inovação em Ciências da Saúde (NUPICS), Faculdade de Farmácia, Universidade Federal de
Juiz de Fora, Juiz de Fora, M.G. 36036-330, Brazil; 2Departamento de Química, Instituto de Ciências Exatas,
Universidade Federal de Juiz de Fora, Juiz de Fora, M.G. 36036-330, Brazil.
Received March 07, 2012: Revised May 09, 2012: Accepted May 09, 2012
Abstract: This study evaluated the antioxidant activity of five resveratrol analogs using the DPPH method. The molecules
activity was related with their chemical structure. Besides descriptive statistics, the analysis of variance (ANOVA)
followed by Tukey’s post hoc test were performed (p<0.05). The antioxidant activity of analogs A and B was statistically
similar with each other and from the reference standard resveratrol, possibly due to the presence of 4-hydroxy grouping.
The aromatic hydroxyl reduces reactive free radicals and produces phenoxyl radical, stabilized by resonance. Although
the analog C has shown IC50 value statistically different from the resveratrol (p<0.001), its antioxidant activity was
considered satisfactory. The other analogs (D and E), which have a 4-acid grouping in place of 4-hydroxy grouping,
showed lower antioxidant activity than resveratrol (p<0.001). Further studies to address possible advantages of analogs in
relation to resveratrol should be conducted in order to make them feasible for therapeutic use.
Keywords: Analogs, Antioxidant Activity, Chemical Synthesis, Hydroxylation, Resveratrol, Structure-Activity Relationship.
INTRODUCTION
the high level of this compound in red wine (0.1 to 14.3
mg/L) is connected to low incidence of heart disease in some
regions of France [7-8].
The continuous production of free radicals during the
metabolic processes resulted in the development of
antioxidant defense mechanisms, in order to limit the
intracellular levels of these reactive species and to control
the damage caused by them [1]. The free radicals toxic
effects and the cell damages raised by them are responsible
for aging process and oxidative stress of the organism [2].
This oxidative stress is considered a major cause for diseases
such as cancer, diabetes, Alzheimer and Parkinson diseases,
atherosclerosis and rheumatoid arthritis [1-4].
Based on the hypothesis that 4-hydroxy grouping, in
trans conformation, is required for cell proliferation
inhibition and aiming to develop
a compound with
antiproliferative potential higher than resveratrol, Savio and
colleagues (2009) [6] synthesized the analog 4,4'-dihydroxy-
trans-stilbene (Figure 1). Compared with resveratrol, this
analog inhibited the efficiency of fibroblasts clonogenic cell
nine times more, but through a different mechanism. Cheng
and colleagues (2006) [8] observed that synthetic analogs of
resveratrol, with characteristic di-ortho-hydroxy, had
antioxidant and cytotoxicity activities against HL-60 cancer
cells, greater than resveratrol. Calil and colleagues (2012) [9]
evaluated the antioxidant activity of resveratrol analogs and
found that the hydroxyl grouping insertion is directly related
to the increase of antioxidant activity of compounds.
Resveratrol (3,5,4’-trihydroxystilbene) (Figure 1),
a
natural polyphenol, is a chemopreventive agent which has
attracted interest, especially in the last ten years, due to its
pharmacological properties. These include antioxidant, anti-
inflammatory, antiaging, cardioprotective, neuroprotective
and anticancer effects of this compound [5-8]. The search for
effective antiproliferative agents has been growing along
with the evidence that higher intake of flavonoids would be
associated with lower risk of cancer and that apoptosis
induction is an important component of chemoprevention of
this disease [5].
Resveratrol can be found in at least 70 species of plants,
besides being also present in the human diet components
such as grapes, berries, nuts and peanuts. It is believed that
Fig. (1). Chemical structure of resveratrol and 4,4'-dihydroxy-trans-
stilbene.
*Address correspondence to these authors at the NUPICS, Faculdade de
Farmácia, Universidade Federal de Juiz de Fora, Juiz de Fora, M.G. 36036-
330, Brazil; Tel/Fax: +55 32 21023809; E-mail: nadiafox@gmail.com.
Departamento de Química, Instituto de Ciências Exatas, Universidade
Federal de Juiz de Fora, Juiz de Fora, M.G. 36036-330, Brazil;
Thus, the present study aimed to evaluate the antioxidant
activity of five synthetic analogs of resveratrol, with or
without hydroxyl at position 4 of the aromatic ring, and to
relate the molecules activity with their chemical structure.
Tel: +55 32 21023310; E-mail: david.silva@ufjf.edu.br
1875-628X/12 $58.00+.00
© 2012 Bentham Science Publishers

Biological Activity of Resveratrol Analogs
MATERIAL AND METHODS
Chemistry
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 7 677
Statistical Package for Social Sciences (SPSS) version 14.0
for Windows software. It were compared the average values
of IC50 of resveratrol analogs among themselves and with the
standard (resveratrol), assuming a significance level of
p<0.05.
Five analogs of resveratrol were synthesized in the
Laboratory of Chemistry of Federal University of Juiz de
Fora (Juiz de Fora, Minas Gerais - Brazil) and their
antioxidant activities were evaluated. As
standard, resveratrol (trans-resveratrol 99.0%, Attivos
a reference
CONCLUSION
Results of resveratrol analogs IC50 are shown in Table 2.
The antioxidant activity of analogs A and B was statistically
similar to resveratrol standard (p=0.109 and p=1.000,
respectively) and among themselves (p= 0.079).
Magistrais, Brazil) was used (Table 1).
The resveratrol analogs A-E were synthesized by the
classical method of imine formation involving condensation
between aromatic amine (4-aminophenol and 4-amino-
salicylic acid) with a variety of aromatic aldehydes in
The use of resveratrol by mammals shows some
disadvantages such as low bioavailability and rapid clearance
from the circulation. The administration of high doses
(above 1g/kg body weight) to improve its effectiveness, can
cause an increase of toxic effects risk. According to Baur and
Sinclair (2006) [15], some alternatives for improving the
resveratrol bioavailability would be the block of its
metabolism, the discovery of more potent compounds (which
mimic its effects) or the development of analogs with better
bioavailability.
1
ethanol [10]. All compounds were characterized by H and
13C NMR I.R. and melting point (Table 1). It was performed
in accord with data in the literature [11-12].
The NMR experiments were performed at 300 MHz for
1H and 75 MHz for 13C in DMSO-d6 (ppm) and I.R.
experiments was performed at KBr support (cm-1).
ANTIOXIDANT ACTIVITY
Based on previous work in which the olefinic double of
resveratrol was replaced by an imino group and the products
obtained had significant antioxidant activity [9], it was also
changed in such present work the olefinic group by an imino
group. Imino groups may be able of forming more stable
intermediates that resveratrol [16].
The antioxidant activity was evaluated using the 2,2-
diphenyl-1-picrylhydrazyl (DDPH; Sigma-Aldrich, USA)
method, described by Sreejayan and Rao (1996) [13], with
minor modifications. This method is based on the DPPH
reduction in the presence of an antioxidant (AH) a proton
donor (H+) for a non-radical (DPPH-H) [14].
The analogs A and B showed antioxidant activity
statistically similar to resveratrol. The activity found is
possibly related to the hydroxy grouping insertion as a
differential in comparison to other samples tested. According
to Cerqueira and colleagues (2007) [17], the aromatic
hydroxyl radical has reducing power of reactive free radicals
and produces phenoxyl radical, stabilized by resonance. The
results found by Calil and colleagues (2012) [9] also confirm
that the degree of activity of antioxidant substances is
directly related to the number of hydroxyl groups present in
the molecule. Among five synthetic analogs of resveratrol,
the ones (A and B) which have a hydroxyl group in para
position of aromatic ring containing the nitrogen atom were
those that presented the best potential for inhibition of DPPH
radical.
A 150 ꢀL of a DPPH ethanolic solution of 0.05 mM were
added to 50 ꢀL of ethanol solution of resveratrol analogs at
concentrations from 0.97 to 250 ꢀg /mL in 96-well plates.
Resveratrol was used as
a
standard at the same
concentrations. Reactions elapsed at room temperature for 30
minutes in the dark and then the absorbance was read in a
spectrophotometer (UV mini 1240, Shimadzu®, Japan) (ꢁ=
510 nm). The concentration of DPPH radical inhibition
(IC50) was calculated using the equation: IC50(%) = 100 x (A0
– As) / Ao, being Ao absorbance as the negative control and As
a absorbance of test samples. The IC50 value was calculated
from the line in the equation of the linear dispersion graph.
All tests were performed in triplicate.
STATISTICAL ANALYSIS
Although the analog C has shown the IC50 value
statistically different to resveratrol, its antioxidant activity
was considered satisfactory. It is important to study the
Descriptive statistics and analysis of variance (ANOVA)
followed by Tukey’s post hoc test were carried out using
Table 1. Spectral Data of Resveratrol Analogs
Analogs
ꢀ
Yield
CH=N
Melting Point (ºC)
(%)
C=N
C=N
A
B
C
D
E
8.51(s,1H)
8.43(s,1H)
8.89(s,1H)
9.66(s,1H)
9.78(s,1H)
161.5
160.2
160.2
172.0
172.2
1609
1607
1616
1605
1602
189.0
203.7
141.4
191.5
182.4
61
53
55
59
57

678 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 7
Calil et al.
Table 2. Radical Scavenging Activities of Resveratrol and Analogs
Sample
Chemical Structure
IC50 (ꢀg/mL)*
HO
N
A
24.79 ± 0.40 a
OMe
HO
N
B
7.41 ± 0.11 b
OH
HO
N
HO
C
55.78 ± 1.51a,b,c
HO
D
E
442.80 ± 15.63 a,b,c
HO2C
N
NMe2
HO
539.41 ± 6.21a,b,c
8.53 ± 0.12 c
HO2C
N
OH
OH
HO
Resveratrol
OH
*Results expressed as mean ± standard deviation (n=3). Means followed by same letter differ by ANOVA followed by Tukey’s post hoc test (a,b,cp<0.001 for comparison of average
values of IC50 among samples). IC50 = concentration of drugs that inhibits 50% of DPPH radicals.
hydroxyl group elsewhere in this structure in order to find
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