Exploring nature profits: Development of novel and potent lipophilic antioxidants based on galloyl-cinnamic hybrids

Article abstract of DOI:10.1016/j.ejmech.2012.12.049

Phenolic acids are ubiquitous antioxidants accounting for approximately one third of the phenolic compounds in our diet. Their importance was supported by epidemiological studies that suggest an inverse relationship between dietary intake of phenolic antioxidants and the occurrence of diseases, such as cancer and neurodegenerative disorders. However, until now, most of natural antioxidants have limited therapeutic success a fact that could be related with their limited distribution throughout the body and with the inherent difficulties to attain the target sites. The development of phenolic antioxidants based on a hybrid concept and structurally based on natural hydroxybenzoic (gallic acid) and hydroxycinnamic (caffeic acid) scaffolds seems to be a suitable solution to surpass the mentioned drawbacks. Galloyl-cinnamic hybrids were synthesized and their antioxidant activity as well as partition coefficients and redox potentials evaluated. The structure-property-activity relationship (SPAR) study revealed the existence of a correlation between the redox potentials and antioxidant activity. The galloyl-cinnamic acid hybrid stands out as the best antioxidant supplementing the effect of a blend of gallic acid plus caffeic acid endorsing the hypothesis that the whole is greater than the sum of the parts. In addition, some hybrid compounds possess an appropriate lipophilicity allowing their application as chain-breaking antioxidant in biomembranes or other type of lipidic systems. Their predicted ADME properties are also in accordance with the general requirements for drug-like compounds. Accordingly, these phenolic hybrids can be seen as potential antioxidants for tackling the oxidative status linked to the neurodegenerative, inflammatory or cancer processes.

Full text of DOI:10.1016/j.ejmech.2012.12.049

European Journal of Medicinal Chemistry 62 (2013) 289e296
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Exploring nature profits: Development of novel and potent
lipophilic antioxidants based on galloylecinnamic hybrids
José Teixeira a, Tiago Silva a, Sofia Benfeito a, Alexandra Gaspar a,
,
,
,
*
E. Manuela Garrido a b, Jorge Garrido a b, Fernanda Borges a
a CIQ/Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto 4169-007, Portugal
b Departamento de Engenharia Química, Instituto Superior de Engenharia do Porto (ISEP), Porto 4200-072, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Phenolic acids are ubiquitous antioxidants accounting for approximately one third of the phenolic
compounds in our diet. Their importance was supported by epidemiological studies that suggest an
inverse relationship between dietary intake of phenolic antioxidants and the occurrence of diseases, such
as cancer and neurodegenerative disorders. However, until now, most of natural antioxidants have
limited therapeutic success a fact that could be related with their limited distribution throughout the
body and with the inherent difficulties to attain the target sites.
Received 17 September 2012
Received in revised form
14 December 2012
Accepted 31 December 2012
Available online 8 January 2013
The development of phenolic antioxidants based on a hybrid concept and structurally based on natural
hydroxybenzoic (gallic acid) and hydroxycinnamic (caffeic acid) scaffolds seems to be a suitable solution
to surpass the mentioned drawbacks. Galloylecinnamic hybrids were synthesized and their antioxidant
activity as well as partition coefficients and redox potentials evaluated. The structureepropertyeactivity
relationship (SPAR) study revealed the existence of a correlation between the redox potentials and
antioxidant activity. The galloylecinnamic acid hybrid stands out as the best antioxidant supplementing
the effect of a blend of gallic acid plus caffeic acid endorsing the hypothesis that the whole is greater than
the sum of the parts. In addition, some hybrid compounds possess an appropriate lipophilicity allowing
their application as chain-breaking antioxidant in biomembranes or other type of lipidic systems. Their
predicted ADME properties are also in accordance with the general requirements for drug-like com-
pounds. Accordingly, these phenolic hybrids can be seen as potential antioxidants for tackling the oxi-
dative status linked to the neurodegenerative, inflammatory or cancer processes.
Keywords:
Cinnamic acid
Gallic acid
Hybrid antioxidants
Antioxidant activity
Redox potential
SPAR
Drug-like properties
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
and neurodegenerative processes, mainly through its antioxidant
properties [8].
Research data have revealed that exogenous antioxidants are
intimately involved in the prevention of cellular damage and could
be appropriate for therapeutic purposes in several diseases related
with oxidative stress [1e3]. Indeed, exogenous antioxidants like
diet-associated phenolic compounds are often described as effec-
tive antioxidants that could play an important role in preventing
and/or minimizing oxidative processes [4e7].
Hydroxybenzoic acids (HBA), namely gallic acid (3,4,5-
trihydroxybenzoic acid) and its derivatives, are widely present in
the plant kingdom and represent a large family of plant secondary
phenolic metabolites [4]. Gallic acid has been shown to be involved
in numerous beneficial health effects in cancer, inflammation
Hydroxycinnamic acids (HCA), such as caffeic, ferulic and sinapic
acids, are a well-known group of natural compounds, that can occur
in fruits, vegetables, beverages, spices and herbs, and are present in
human diet in representative amounts [4e7]. Hydroxycinnamic
acids are of particular interest because of their potential biological
outline displaying a plethora of interesting activities namely, anti-
oxidant, antiallergic, anti-inflammatory, antimicrobial, antiviral,
anticarcinogenic and UV filter properties [9]. Hydroxycinnamic
acids have been recognized to be potent antioxidants, probably
through their radical-scavenging activity [10,11]. Some results evi-
dence that their antioxidant activity is strongly dependent on their
structural features and intrinsically related to the presence of hy-
droxyl function(s) in the aromatic structure [12e19].
Phenolic acids are ubiquitous antioxidants accounting for
approximately one third of the phenolic content in human diet [4e
7,20]. Their importance was supported by epidemiological studies
* Corresponding author.
E-mail address: fborges@fc.up.pt (F. Borges).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.049

290
J. Teixeira et al. / European Journal of Medicinal Chemistry 62 (2013) 289e296
that suggest an inverse relationship between dietary intake of phe-
nolic antioxidants and the occurrence of diseases such as cancer
and neurodegenerative disorders. However, the majority of natural
antioxidants have limited therapeutic success a fact that could be
related with their poor distribution and with the inherent difficulties
to attain the target sites [21,22]. Henceforth, suitable solutions must
be met to surpass the mentioned drawbacks enabling effective
exogenous antioxidant compounds that can efficiently operate in
both aqueous and lipid systems and, in that way supplement the
body’s endogenous antioxidant defence systems [23,24].
plates (Merck, Darmstadt, Germany) and spots were detected using
a UV lamp at 254 nm. Column chromatography purifications were
performed with silica gel 60A (Carlo Erba Reactifs e SDS, France).
2.2. Apparatus
1H and 13C NMR data were acquired, at room temperature, on
a
Brüker AMX 300 spectrometer operating at 300.13 and
75.47 MHz, respectively. CDCl3 and CD3OD were used as solvents;
chemical shifts are expressed in (ppm) values relative to tetra-
d
Several studies suggest that mixtures of different phenolic
antioxidants may have synergistic effects yielding a greater effi-
ciency than individual antioxidants [25]. Furthermore, the combi-
nation of two different and independently acting antioxidants into
one covalently linked (hybrid compound) can convey synergy from
the effects of both independently acting moieties [25,26]. The
outcome is expected to have greater antioxidant efficacy than that
of the sum of each individual moiety.
In this context, herein we report the rational design and syn-
thesis of hybrid phenolic antioxidants structurally based on natural
hydroxybenzoic (gallic acid) and hydroxycinnamic (caffeic acid)
scaffolds (Fig. 1, Schemes 1 and 2). Therefore, the aim of our work is
to synthesize phenolic acid derivatives, evaluate their antiradical
properties, and elucidate the relationship among their antioxidant
activity, chemical structure, and physicochemical parameters. To
increase the lipophilicity structural modifications were also per-
formed. Overall, the gathered information will allow the estab-
lishment of a significant correlation among the chemical structure,
physicochemical properties and the activity, herein called SPAR
(structureepropertyeactivity relationship).
methylsilane (TMS) as internal reference; coupling constants (J) are
given in Hz. Electron impact mass spectra (EI-MS) were carried out
on a VG AutoSpec instrument; the data are reported as m/z (% of
relative intensity of the most important fragments).
Microwave-assisted synthesis was executed in a BiotageÒ Ini-
tiator Microwave Synthesizer.
Voltammetric studies were performed using an Autolab PGSTAT
12 potentiostat/galvanostat (Eco-Chemie, Netherlands) and a one-
compartment glass electrochemical cell. Voltammetric curves
were recorded at room temperature using a three-electrode sys-
tem. A glassy carbon working electrode (GCE) (d ¼ 2 mm), a plati-
num wire counter electrode and an Ag/AgCl saturated KCl reference
electrode were used (Metrohm, Switzerland). A Crison pH-meter
with glass electrode was used for the pH measurements (Crison,
Spain).
The antioxidant assays were performed in a multiplate reader
(Powerwave XS Microplate Reader) of Bio-Tek instruments.
2.3. Synthesis
2.3.1. General synthetic microwave esterification procedure
Compounds 2 and 4 were synthesized by Fisher esterification
according the general procedure described in Silva et al. [27], but
the conventional reaction was replaced by a microwave-assisted
organic process.
2. Experimental section
2.1. General
Gallic acid (1), caffeic acid (3), malonic acid, mono-
ethylmalonate, diethylmalonate, DPPH and ABTS were purchased
from SigmaeAldrich Química S.A. (Sintra, Portugal). All other
reagents and solvents were pro analysis grade and were acquired
from Merck (Lisbon, Portugal) and used without additional purifi-
cation. Deionized water (conductivity < 0.1
throughout all the experiments. Thin layer chromatography (TLC)
analyses were performed on aluminium silica gel sheets 60 F254
Briefly, the acid (2 g) with the appropriate aromatic pattern,
ethanol (2.5 mL) and H2SO4 (3 drops) were put together in a glass
vial (2e5 mL) sealed with a cap and heated in the MW reactor
cavity under mechanical stirring at 100 ꢁC for 7 min. After cooling
to room temperature, the solvent was removed and the compounds
were purified by flash chromatography (silica gel; hexane with
increasing ethyl acetate gradient) and recrystallized from diethyl
ether/petroleum ether.
m
S cmꢀ1) was used
A
B
C
Fig. 1. Phenolic acids and derivatives under study.

J. Teixeira et al. / European Journal of Medicinal Chemistry 62 (2013) 289e296
291
Scheme 1. Synthetic strategy designed for the obtention of phenolic esters.
2.3.2. General synthetic procedure for KnoevenageleDoebner
condensation
(1H, s, H(
b
)), 9.20 (3H, s, OH); 13C NMR
d
: 13.8 (OCH2CH3), 14.1
(OCH2CH3), 61.1 (OCH2CH3), 61.3 (OCH2CH3), 109.4 (CH (2, 6)), 121.7
Compounds 5e8 were synthesized according to an adaptation
of the process described by Hubner et al. [14]. Accordingly, the
KnoevenageleDoebner condensation was performed between the
corresponding hydroxybenzaldehyde (1 g) and malonic acid (1 g)
(for compound 6), monoethylmalonate (1.2 mL) (for compound 7)
or diethylmalonate (1.6 mL) (for compounds 5 and 8), in pyridine
(5 mL) using piperidine (four drops) as catalyst. The reactions took
place under reflux at 50 ꢁC for 20 h. The mixtures were diluted with
ethyl acetate and washed twice with 2 N HCl and water. The organic
layers were then dried over anhydrous sodium sulphate, filtered,
and concentrated. The compounds were purified by flash chro-
matography (silica gel; ethyl acetate/n-hexane (7:3) for compounds
5 and 8 and dichloromethane/methanol (9:1) for compounds 6 and
7).
(C(1)), 122.3 (C(a)), 137.2 (CeOH), 142.0 (CH(b)), 146.0 (CeOH),
164.0 (C]O), 166.6 (C]O); EI-MS m/z (%): 296 (Mþ, 100), 251
(36), 222 (38), 205 (49), 178 (38), 150 (58); mp. 182e184 ꢁC (156 ꢁC
subl.).
2.4. Electrochemical measurements
Stock solutions of each phenolic compound (10 mM) were
prepared by dissolving an appropriate amount in ethanol. The
voltammetric working solutions were prepared, in the electro-
chemical cell, by diluting 0.1 mL of the stock solution in 10 mL of
supporting electrolyte in order to obtain a final concentration of
0.1 mM. The pH 7.3 supporting electrolyte was prepared by diluting
6.2 mL of 0.2 M dipotassium hydrogen phosphate and 43.8 mL of
0.2 M potassium dihydrogen phosphate to 100 mL [30].
The spectroscopic results obtained for the synthesized com-
pounds (2, 4, 5) are in accordance with the data described in the
literature [14,27e29].
2.5. Total antioxidant capacity (TAC) assays
2.3.2.1. trans-3-(3,4,5-Trihydroxyphenyl)-2-propenoic acid 6. Yield
Total antioxidant capacity assays were performed using ABTS
and DPPH as radicals.
35%; 1H NMR
d
: 6.09 (1H, d, J ¼ 15.8, H(
(1H, d, J ¼ 15.8, H(
)), 8.74 (1H, s, OH), 9.17 (2H, s, OH); 13C NMR
107.5 C(2, 6), 115.2 C(
a)), 6.57 (2H, s, H(2), H(6)), 7.32
b
d:
a
), 124.6 C(1), 136.3 C(4), 145.1 C(
b), 146.2 C(3, 5),
2.5.1. ABTS radical cation assay
ABTSꢂþ radical-scavenging activity was determined according to
the method of Re et al. [31]. The ABTSꢂþ radical cation solution was
pregenerated by reacting of 10 mL of 7 mM aqueous ABTS solution
168.0 (C]O); EI-MS m/z (%): 196 (Mþ, 100), 179 (22); 152 (57), 133
(27), 105 (16), 78 (51), 63 (57); mp 186e188 ꢁC.
2.3.2.2. trans-Ethyl-3-(3,4,5-tri-hydroxyphenyl)-2-propenoate
Yield 65%; 1H NMR
: 1.23 (3H, s, CH3), 4.15 (2H, q, CH2), 6.18 (1H, d,
J ¼ 15.8, H( )), 6.60 (2H, s, H(2), H(6)), 7.38 (1H, d, J ¼ 15.8, H( )),
8.80 (1H, s, OH), 9.18 (2H, s, OH); 13C NMR
: 14.4 (CH3), 59.8 (CH2),
), 146.2 C(3,
7.
and 163 mL of 150 mM (2.45 mM final concentration) potassium
d
persulfate solution. After storage in the dark at room temperature
for 16 h, the radical cation solution was diluted in ethanol until the
absorbance 0.72 ꢃ 0.01 was reached. Different ethanolic solutions
a
b
d
107.7 C(2, 6), 114.2 C(
a
), 124.5 C(1), 136.5 C(4), 145.5 C(
b
of each compound were prepared. Each solution (20 mL) was added
5), 166.6 (C]O); EI-MS m/z (%): 224 (Mþ, 100), 196 (17); 179 (86),
152 (48), 133 (40), 105 (21), 77 (32); mp 176e179 ꢁC.
to 180
734 nm was recorded for ABTSꢂþ after 15 min. The absorbance of
the blank (20 L ethanol and 180 L of radical) assay was set as
mL of radical solution (in triplicate) and absorbance at
m
m
2.3.2.3. Diethyl 2-(3,4,5-trihydroxyphenylmethylene)malonate 8.
100% radical. The IC50 values were calculated as the minimum
concentration of each sample required to inhibit 50% of the ABTS
radical.
Yield 70%; 1H NMR
d: 1.23 (6H, t, COOCH2CH3), 4.19 (2H, q,
COOCH2CH3), 4.30 (2H, q, COOCH2CH3), 6.48 (2H, s, H(2), H(6)), 7.37
Scheme 2. Synthetic strategy designed for the obtention of phenolic acids and diester derivatives.

292
J. Teixeira et al. / European Journal of Medicinal Chemistry 62 (2013) 289e296
2.5.2. DPPH radical assay
and/or an electron to a free radical can be predicted from standard
one-electron potentials. As a result, voltammetric methods have
been often applied to characterize a diversity of natural phenolic
antioxidants, including flavonoids, and synthetic antioxidants
mainly to get an insight on their mechanism [34,35].
To contribute to the understanding of the structureepropertye
activity relationship of phenolic acids and to comprehend the role
that redox potentials exert on this relationship and in the co-
operative effect of antioxidants (synergism), a comparative study of
the electrochemical properties of carboxylic acids and their syn-
thesized derivatives was accomplished. The oxidative behaviour of
the synthesized hydroxycinnamic and hydroxybenzoic derivatives
and galloylecinnamic hybrids was studied, at physiological pH 7.3,
by differential pulse and cyclic voltammetry, using a glassy carbon
working electrode.
A total antioxidant capacity assay was carried out using
DPPH as radical source [32]. Radical-scavenging activity was
spectrophotometrically evaluated by monitoring the disappearance
of 1,1-diphenyl-2-picrylhydrazyl radical (DPPHꢂ) at 515 nm. An
ethanolic radical solution (6.85 mM) was prepared and then diluted
in ethanol to reach the absorbance of 0.72 ꢃ 0.01. Increasing con-
centrations of each compound under study were prepared in
triplicate, also in ethanol. Each solution (20
of DPPH radical solution, and the absorbance was recorded every
minute for a 45 min period. The absorbance of the blank (20
ethanol and 180 L of DPPH radical solution) assay was set as 100%
mL) was added to 180 mL
mL
m
radical. The IC50 values were calculated as the minimum concen-
tration of each sample required to inhibit 50% of the DPPH radical.
Vitamin E and trolox, a water-soluble vitamin E analogue, were
used as reference standards in both assays.
The differential pulse voltammetric study of gallic acid (1) and
ethyl gallate (2) revealed the presence of two well-defined anodic
waves at physiological pH (Fig. 2A). The oxidation peaks at
2.6. Statistical analysis
0
0
Ep ¼ þ0.117 V, Ep ¼ þ0.552 V and Ep ¼ þ0.057 V, Ep ¼ þ0.541
(Table 1) obtained for 1 and 2, respectively, are related to the oxi-
dation of the pyrogallol unit present in their structure.
Each experiment was performed in triplicate. Statistical com-
parisons of both assays were done by one-way ANOVA followed by
the multiple Duncan test (P < 0.05).
2.7. Evaluation of drug-like properties
A
Calculation of partition coefficients (log P), number of rotatable
bonds (n-ROTB), number of hydrogen bond donors (n-OHNH) and
number of hydrogen acceptors (n-ON) was done using the
Molinspiration Calculation software.
1
µA
3. Results and discussion
3.1. Synthesis
Phenolic acids and their derivatives (Fig. 1) were synthesized
according to the strategies depicted in Schemes
1 and 2.
Generally, the ethyl esters derivatives (2 and 4) were synthesized
by Fischer esterification between the corresponding acids and
ethanol under acid catalysis [27], except for compound 7 that was
obtained by KnoevenageleDoebner condensation. Microwave-
assisted organic synthesis was used in this work since it pres-
ents several advantages in relation to the classic esterification
process, such as the reaction time (7 min) and the volume of
solvent/reagent [27]. Compounds 5e8 were obtained via
KnoevenageleDoebner condensation between the correspond-
ing hydroxybenzaldehyde and malonic acid (compound 6),
monoethylmalonate (compound 7) or diethylmalonate (com-
pounds 5 and 8) [14]. These reactions led to moderate yields of
the desired compounds, which were identified by spectroscopic
techniques: NMR (1H and 13C NMR) and MS-EI.
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
E / V vs Ag/AgCl
B
500 nA
3.2. Electrochemical measurements
Human antioxidant defences exist to protect biological systems
against reactive oxygen and nitrogen species (ROS/RNS), and a so-
phisticated cooperative array of antioxidant defence mechanisms is
found in biological systems [33]. Interventions aimed at enhancing
the endogenous antioxidant defences (e.g., dietary antioxidants)
may gain special interest.
The antioxidant effectiveness is influenced by the chemical
properties of the compound including hydrogen bond capability,
resonance delocalization, and susceptibility to autoxidation. The
ability of the primary antioxidant molecules to donate a hydrogen
atom to the free radical is the initial requirement. The ability of the
free radical interceptor (scavenger) to donate a hydrogen atom
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
E / V vs Ag/AgCl
Fig. 2. Differential pulse voltammograms for 0.1 mM solutions of (A) (d) gallic acid, (-
- -) ethyl gallate and (B) (d) caffeic acid, (- - -) ethyl caffeate, (ꢂꢂꢂ) diethyl 2-(3,4-
trihydroxyphenylmethylene)malonate, in physiological pH 7.3 supporting electrolyte.
Scan rate: 5 mV sꢀ1
.

J. Teixeira et al. / European Journal of Medicinal Chemistry 62 (2013) 289e296
293
The differential pulse voltammetric behaviour of caffeic acid (3)
and ethyl caffeate (4) shows the occurrence of only one anodic
A
peak at physiological pH (Fig. 2B). The redox potentials found
for these cinnamic compounds were Ep
¼
þ0.131
V and
2 µA
Ep ¼ þ0.162 V for 3 and 4, respectively (Table 1). The diethyl ester
((2-(3,4-dihydroxyphenylmethylene)malonate) (5)) also presents
an anodic peak at Ep ¼ þ0.193 V. For this compound, a second
oxidation peak is clearly distinguished at more anodic potentials,
0
Ep ¼ þ0.392 V. The anodic waves observed could be ascribed to
the oxidation of the catechol group existing in the molecule’s
structure. In fact, it was already suggested that this kind of phenolic
antioxidants are oxidized to quinone via semiquinone form
[13,23,36e38]. The occurrence of the second oxidation wave at
more anodic potentials is evident for most of the compounds
studied. However, for caffeic acid and its ethyl ester this peak is not
clearly distinguishable. This fact could result from the decrease in
the interface concentration of the semiquinone radical due to its
instability [37].
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
E / V (vs. Ag/AgCl)
Cyclic voltammograms were recorded at different sweep rates.
The cyclic voltammograms obtained for caffeic acid (3), ethyl caf-
feate (4) and the related diethyl ester (5) are characteristic of an
electrochemical reversible reaction showing only one anodic peak
and one cathodic peak on the reverse scan (Fig. 3B). Cyclic voltam-
metric experiments showed a single oxidation peak for gallic acid
(1) and ethyl gallate (2) without any distinct reduction wave on the
reverse sweep which shows that gallic acid and its derivative are
irreversibly oxidized at the glassy carbon electrode (Fig. 3A). These
results are in agreement with the literature data in that concerns the
oxidative behaviour of caffeic and gallic acids. Electrochemical
studies on the caffeic and gallic acid oxidation mechanisms have
shown that their oxidation involves two electrons per molecule that
likely correspond to the formation of the caffeic and gallic acids
ortho-quinone [24,30,37e39]. Nevertheless, results show that the
existence of an additional phenolic group in gallic acid and its de-
rivative (pyrogallol unit) seems to influence the stabilization of the
semiquinone intermediate. The gathered data suggest that the extra
hydroxyl group could promote the stabilization of the radical pro-
duced by oxidation, expressed by the lower redox potentials
attained, and have also an obvious influence on the oxidative
mechanism. Further studies are currently in progress to clarify this
subject.
B
1 µA
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
E / V (vs. Ag/AgCl)
Fig. 3. Cyclic voltammograms for 0.1 mM solutions of (A) (d) gallic acid, (- - -) ethyl
gallate and (B) (d) caffeic acid, (-
-) ethyl caffeate, (ꢂꢂꢂ) diethyl 2-(3,4-
trihydroxyphenylmethylene)malonate, in physiological pH 7.3 supporting electrolyte.
Scan rate: 50 mV sꢀ1
-
The differential pulse voltammetric study of the synthesized
galloylecinnamic hybrid, 3,4,5-trihydroxycinnamic acid (6),
revealed the existence of two well defined anodic waves at
physiological pH0 (Fig. 4A). The oxidation peaks seen at
Ep ¼ þ0.011 V, Ep ¼ þ0.384 V (Table 1) are related to the oxi-
dation of the catechol group existing in its structure. Similarly to
.
caffeic and gallic acids, it is reasonable to assume that this
galloylecinnamic hybrid is oxidized to quinone via semiquinone
form.
Table 1
Antioxidant efficacy (DPPH and ABTS), redox potentials (Ep) and drug-like properties for phenolic acids and their respective derivatives.
Compound
Mw (g molꢀ1
)
IC50
(
mM)
Log P
Ep (V)
n-ROTB
n-ON Acceptors
n-OHNH Donors
DPPH
ABTS
1
2
3
4
5
6
7
8
170.12
198.17
180.16
208.28
280.28
196.16
224.21
296.28
e
9.9
11.8
18.1
26.6
21.9
11.8
15.1
12.8
16.1
17.2
16.7
30.9
31.1
7.8
8.1
17.9
22.6
21.1
7.2
0.589
1.079
0.941
1.934
2.110
0.650
1.642
1.818
e
0.117; 0.552
0.057; 0.541
0.131
1
2
2
4
7
2
4
7
e
e
e
1
5
4
4
4
6
5
5
7
e
e
e
4
2
4
3
3
2
2
4
3
3
e
e
e
2
1
0.162
0.193; 0.392
0.011; 0.384
0.026; 0.133
0.043; 0.159
0.119; 0.928
e
9.0
7.7
1 þ 3
1 þ 4
1 þ 5
Troloxa
Vitamin E
12.0
13.0
12.8
26.3
26.8
e
e
e
e
e
250.29
430.71
3.071
9.043
0.099
e
12
a
For details see Ref. [36].

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J. Teixeira et al. / European Journal of Medicinal Chemistry 62 (2013) 289e296
one anodic wave is seen for 3,4,5-trihydroxycinnamic acid (6) (Fig. 5).
A
The anodic peaks appear to correspond to irreversible processes as
any distinct reduction wave is not seen on the cathodic sweep.
The gathered data obtained for the new galloylecinnamic hy-
brids are consistent with the oxidative profile obtained for caffeic
and gallic acids and its derivatives. The proposed mechanism in-
volves an overall two-electron process: one-electron transfer to the
semiquinone followed by a second electron transfer to give the
ortho-quinone.
500 nA
Although the voltammetric profile of the new galloylecinnamic
hybrids is comparable to that found for caffeic and gallic acids
counterparts, the redox potentials obtained for these new com-
pounds are significantly lower. In fact, a considerable decrease
on the peak potential, ca. 100 mV, is observed for 3,4,5-
trihydroxycinnamic acid relatively to caffeic and gallic acids
(Fig. 4B, Table 1). In order to effectively compare the antioxidant
capabilities of the new galloylecinnamic acid hybrids, the anti-
oxidant interactions and cooperative effects of equimolar blends of
caffeic and gallic acids were also investigated (Fig. 4B). The results
obtained show no significant differences on the redox potentials
when both compounds are combined with regard to them sepa-
rately (Table 1). These results suggest that galloylecinnamic hybrids
possess distinctive properties and could represent a very interesting
approach to the development of new synthetic antioxidants.
The voltammetric results reinforce the proposition previously
established by our group in previous reports [23,24,30] that states
that the electrochemical potential is closely correlated with the
number of hydroxyl substituents on the aromatic ring and that the
alkenyl chain connecting to the phenolic ring and the carboxylic
group may stabilize the radical formed during the oxidation. On the
contrary, the introduction of an ester/diester group in the conju-
gated side chain did not produce any noteworthy change in the
oxidation potentials, when compared to the lead compounds.
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
E / V vs Ag/AgCl
B
1 µA
3.3. Total antioxidant capacity (TAC)
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Total antioxidant capacity (TAC) assays have been often used to
determine the hierarchy of radical-scavenging abilities of potential
phenolic antioxidant compounds that work either through elec-
tron- or H-donating mechanisms [23,32]. Thus, to evaluate the
E / V (vs. Ag/AgCl)
Fig. 4. Differential pulse voltammograms for 0.1 mM solutions of (A) (d) 3,4,5-
trihydroxycinnamic acid, (-
-
-) ethyl 3,4,5-trihydroxycinnamate, (ꢂꢂꢂ) diethyl 2-
(3,4,5-trihydroxyphenylmethylene)malonate and (B) (d) 3,4,5-trihydroxycinnamic
acid, (- - -) caffeic acid, (ꢂꢂꢂ) gallic acid, (ꢂ-ꢂ-ꢂ) blend of caffeic and gallic acids, in
physiological pH 7.3 supporting electrolyte. Scan rate: 5 mV sꢀ1
.
For the 3,4,5-trihydroxycinnamic alkyl esters, ethyl 3,4,5-
trihydroxycinnamate (7) and diethyl 2-(3,4,5-trihydroxyphenyl-
methylene)malonate (8), two overlapped anodic waves were
observed at physiological pH using differential pulse voltammetry
100 nA
0
(Fig. 4A). The oxidation peaks Ep ¼ þ0.026 V, Ep ¼ þ0.133 V and
0
Ep ¼ þ0.043 V, Ep ¼ þ0.159 V (Table 1) obtained for 7 and 8,
respectively, are related to the oxidation of the catecholic group
present in their structure. The appearance of the two waves may be
interpreted by assuming that the electrochemical oxidation of
these compounds takes place on the glassy carbon electrode by
electron transfer from both free and adsorbed forms. The free form
corresponds to the first peak whereas the strongly adsorbed forms
get oxidized at a more anodic potential. The occurrence of an
adsorption peak has also been described in previous voltammetric
studies for related compounds [23,24,36]. The higher adsorption
propensity of these compounds when compared with 3,4,5-
trihydroxycinnamic acid could result from their utmost lipophilic
character (Table 1).
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
E / V (vs. Ag/AgCl)
Fig. 5. Cyclic voltammograms for 0.1 mM solutions of (d) 3,4,5-trihydroxycinnamic
acid, (-
-) ethyl 3,4,5-trihydroxycinnamate, (ꢂꢂꢂ) diethyl 2-(3,4,5-trihydroxy-
phenylmethylene)malonate, in physiological pH 7.3 supporting electrolyte. Scan rate:
50 mV sꢀ1
The cyclic voltammograms obtained for ethyl 3,4,5-trihydroxy-
cinnamate (7) and diethyl 2-(3,4,5-trihydroxyphenylmethylene)
malonate (8) also shows two overlapped anodic peaks, while only
-
.

J. Teixeira et al. / European Journal of Medicinal Chemistry 62 (2013) 289e296
295
radical-scavenging ability of the phenolic acids and their de-
3.5. Structureepropertyeactivity relationships (SPAR)
rivatives total antioxidant capacity assays (DPPH-2,20-diphenyl-1-
picrylhydrazyl radical); ABTS e 2-azino-bis(3-ethylbenzthiazo-
line-6-sulfonic acid) were used. These methods have the advant-
age of establishing an accurate ranking hierarchy of antioxidant
activity since some factors which interfere in other model systems,
such as metal chelation, partitioning abilities, are absent [11,27,40].
From the results obtained in the ABTS assay, it is clear that gallic
acid and its ester derivative (1 and 2) have higher antioxidant ac-
tivity towards ABTSꢂþ than caffeic acid and their respective ester
derivatives (3 and 4, 5) (Table 1) [11]. In both cases the esterification
process reduces the antioxidant potency to some extent [23,36].
The possibility of the existence of an extra radical stabilization due
to the presence of an additional hydroxyl group in position-5 in
pyrogallol vs catechol systems was previously mentioned to justify
its higher antioxidant activity [11].
For the galloylecinnamic acid hybrids (6, 7 and 8) an interesting
antioxidant outline was attained: the hybrid compounds exhibit
a better antioxidant activity than all the caffeic derivatives and
show a similar radical efficacy to that observed for gallic acid and its
ester derivative. Notice that the esterification of the carboxylic acid
group (ester/diester) in the conjugated side chain did not produce
any significant change in total antioxidant capacity.
To attest the benefit of the galloylecinnamic acid hybrids
approach, the antioxidant interactions and cooperative effect of
equimolar blends of caffeic and gallic acids were also evaluated. The
overall data allow concluding that each galloylecinnamic acid hy-
brids per se has better radical-scavenging activity than the systems
in which two antioxidants are combined (1 þ 3, 1 þ 4, 1 þ 5). In fact
although gallic acid (1) improves the antioxidant profile of caffeic
acid and derivatives (3, 4, and 5) in the mixture the overall activity
was lower than the observed for each hybrid per se.
The development of novel hybrid antioxidants structurally
based on the natural models gallic (1) and caffeic (3) acids herein
designated as galloylecinnamic hybrids was successfully per-
formed. In addition the improvement of their lipophilicity was
attained by introducing alkyl groups on the conjugated side chain
using an ester as spacer. In accordance with the aim of the work the
physicochemical properties as well as the antioxidant activities of
all compounds were examined to carry out structureepropertye
activity relationship studies as an upgrading tool for the rational
design of lipophilic antioxidants [12,13,26,27,30,36].
From the data depicted in Table 1 one can conclude that the
esterification process increases the lipophilicity of the galloyle
cinnamic acid hybrid (6, log P 0.65) without a significant loss of
activity (7 and 8, log P 1.64 and 1.82, respectively) (Table 1). Con-
sidering that compounds that have a log P ꢄ1, are not able to
effectively cross biological membranes, it appears that ester-
ification of the galloylecinnamic acid hybrid can improve its
membrane crossing ability. Furthermore, the compounds have
drug-like properties since no violations to the Lipinsky’s Rule of
Five were found. From the electrochemical results one can conclude
that the structural features governing the redox potentials of
galloylecinnamic hybrids were the phenolic moiety, preferentially
a pyrogallol unit, as well as the additional resonance-effective
substituents in the aromatic ring. These new compounds showed
unique redox potentials representing an interesting approach to
the development of new potent and effective synthetic antioxi-
dants. Moreover, a correlation was found between antioxidant ac-
tivity (DPPH and ABTS assays) and the redox data. This assumption
reinforces the idea that the structural principles governing the
oxidationereduction processes can be directly linked with the
antioxidant scavenging mechanisms and the structural features
present in the compounds [23].
The DPPH results follow the same tendency than those obtained
with the ABTS assay (Table 1) [11].
In general, all the compounds were found to be radical
scavengers in a dose-dependent manner and present better radical-
4. Conclusion
scavenging activity than the reference antioxidants tested (
a-
tocopherol and trolox).
The structureepropertyeactivity relationship studies allow
concluding that the phenolic aromatic pattern (pyrogallol vs cat-
echol) markedly influences the antioxidant profile of phenolic acids
and derivatives. In fact, all the pyrogallol derivatives display
a higher antioxidant activity compared to the catechol analogues
and even with the antioxidant used as reference (tocopherol or
trolox).
Galloylecinnamic acid hybrid reveals to be the best anti-
oxidant. This new antioxidant exceed, in terms of antioxidant
capacity, the effect of a blend of gallic acid plus caffeic acid con-
firming that the whole is greater than the sum of the parts. The
esterification of the carboxylic acid group (ester/diester) in the
conjugated side chain did not produce any significant change in
total antioxidant capacity but led to a significant improvement on
lipophilicity.
Galloylecinnamic acid hybrid antioxidants can be useful as
chain-breaking antioxidant in biomembranes or in other type of
lipidic systems and can have brain permeable properties. In addi-
tion, the ADME properties predicted for these antioxidants are in
accordance with the general requirements for potential drugs.
Notice that there are numerous advantages of employing hybrid
molecules over the traditional cocktail drugs in therapy. Compared
to the latter, hybrid drugs may be less expensive since, in principle,
the risks and costs involved may not be different from any other
single entity. Another advantage is the lower risk of drugedrug
adverse interactions, lower difference in the relative rates of
metabolism and a great control of pharmacokinetic/pharmacody-
namic parameters.
3.4. Evaluation of drug-like properties
The structural modifications introduced in the parent com-
pounds (hydroxycinnamic acids) were designed to obtain an
increment in lipophilicity while retaining or improving the anti-
oxidant radical-scavenging activity. In fact, it was previously shown
that a rational increment of lipophilicity lead to an improvement of
the biological activity of this type of compounds [12,23,24].
The Lipinski’s Rule of Five is a rule of thumb to evaluate drug
likeness or determine if a chemical compound with a certain
pharmacological or biological activity has the appropriate features
that would make it a likely orally active drug. The rule describes
molecular properties relevant for drug’s pharmacokinetics in the
human body, including their absorption, distribution, metabolism,
and excretion (ADME).
Thus, the evaluation of lipophilicity, expressed as the octanole
water partition coefficient, and drug-like properties of the com-
pounds under study were attained. The results are depicted in
Table 1.
Results show that either esterification (2, 4, 7) or diesterification
(5, 8) of the carboxylic function of the phenolic acids lead to a sig-
nificant increase in lipophilicity, attained by theoretical determi-
nation of the partition coefficient (log P), within the di- and
trihydroxylated series. The results are well correlated with their
structural features: the diester derivatives have higher partition
coefficients than the esters and acids.

296
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Acknowledgements
This study was supported by the Foundation for Science and
Technology (FCT), Portugal (project PTDC/QUI-QUI/113687/2009). J.
Teixeira (SFRH/BD/79658/2011), T. Silva (SFRH/BD/79671/2011) and
A. Gaspar (SFRH/BD/43531/2008) thank FCT grants.
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