Lipophilic phenolic antioxidants: Correlation between antioxidant profile, partition coefficients and redox properties

Article abstract of DOI:10.1016/j.bmc.2010.06.090

Lipophilic compounds structurally based on caffeic, hydrocaffeic, ferulic and hydroferulic acids were synthesized. Subsequently, their antioxidant activity was evaluated as well as their partition coefficients and redox potentials. The structure-property-

Full text of DOI:10.1016/j.bmc.2010.06.090

Bioorganic & Medicinal Chemistry 18 (2010) 5816–5825
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Lipophilic phenolic antioxidants: Correlation between antioxidant profile,
partition coefficients and redox properties
b
c
d
d
Fernanda M. F. Roleira a, , Christophe Siquet , Elizabeta Orrù , E. Manuela Garrido , Jorge Garrido ,
*
Nuno Milhazes e, Gianni Podda c, Fátima Paiva-Martins f, Salette Reis b, Rui A. Carvalho g,
Elisiário J. Tavares da Silva a, Fernanda Borges f,
*
a CEF/Laboratório de Química Farmacêutica, Faculdade de Farmácia, Universidade de Coimbra, Portugal
b REQUIMTE/Departamento de Química Física, Faculdade de Farmácia, Universidade do Porto, Portugal
c Departimento Farmaco Chimico Tecnologico, Università degli Studi di Cagliari, Italy
d CIQUP/Departamento de Engenharia Química, Instituto Superior de Engenharia, IPP, Porto, Portugal
e CIQUP/Instituto Superior de Ciências da Saúde-Norte, Gandra PRD, Portugal
f CIQUP/Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Portugal
g CNC/Departamento das Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipophilic compounds structurally based on caffeic, hydrocaffeic, ferulic and hydroferulic acids were syn-
thesized. Subsequently, their antioxidant activity was evaluated as well as their partition coefficients and
redox potentials. The structure–property–activity relationship (SPAR) results revealed the existence of a
clear correlation between the redox potentials and the antioxidant activity. In addition, some compounds
showed a proper lipophilicity to cross the blood–brain barrier. Their predicted ADME properties are also
in accordance with the general requirements for potential CNS drugs. Accordingly, one can propose these
phenolic compounds as potential antioxidants for tackling the oxidative status linked to the neurodegen-
erative processes.
Received 4 June 2010
Revised 25 June 2010
Accepted 29 June 2010
Available online 1 July 2010
Keywords:
Phenolic antioxidants
Lipid peroxidation
Redox potentials
Partition coefficients
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
tion of free radicals from environment to living system leads to
serious penalty and therefore to neuronal death.1 Neuronal system
Neurodegenerative diseases (ND) are a group of illnesses with di-
verse clinical importance and etiologies. ND include motor neuron
disease such as amyotrophic lateral sclerosis (ALS), cerebellar disor-
ders, Parkinson’s disease (PD), Huntington’s disease (HD), Alzhei-
mer’s disease (AD) and schizophrenia. Neurodegeneration appears
to be a multifactorial process where a complex set of toxic reactions,
including oxidative stress, leads to the demise of neurons.1
The molecular mechanisms of neuronal degeneration remain
largely unknown and effective therapies are not currently avail-
able. Yet, it is widely accepted that oxidative stress increases with
age, and that age is a major risk factor for several neurodegenera-
tive diseases.
Reactive oxygen species (ROS) are particularly active in the
brain and neuronal tissue and aggressive to glial cells and neurons.
In-built endogenous antioxidant system plays its decisive role in
prevention of any loss due to free radicals. However, imbalanced
defense mechanism of antioxidants, overproduction or incorpora-
is particularly susceptible to ROS owing to the high content of
unsaturated fatty acids that are more susceptible to oxidative dam-
age, namely lipid peroxidation. To note that brain consumes an
inordinate fraction (20%) of total oxygen for its relatively small
weight (2%) and it is not particularly enriched in endogenous anti-
oxidant defenses.1
Exogenous antioxidants are nowadays considered a promising
therapeutic approach in neurodegenerative diseases since they
could play an important role in preventing and/or minimizing neu-
ronal oxidative damage.1,2 Recent interest has been focused on die-
tary phenolic antioxidants, such as carotenoids, cinnamic acids and
flavonoids, as potentially useful agents in ND.2,3 However, few of
them have shown efficiency in animal models or in clinical studies,
a drawback that is usually related with their inability to cross the
blood–brain barrier (BBB) after systemic administration.4
In fact, BBB acts as a regulatory interface that selectively limits
drug delivery to the brain. From the positive and negative cross-
walks lipid solubility is believed to be a key feature in determining
the rate at which a drug passively crosses the BBB and thus
correspond to a major challenge in the rational design of novel
neuroprotective antioxidants. However, it is important to mention
* Corresponding authors. Tel.: +351 239 488 400; fax: +351 239 488 503
(F.M.F.R.), tel.: +351 22 2078900; fax: +351 22 0402560 (F.B.).
E-mail addresses: froleira@ff.uc.pt (F.M.F. Roleira), fborges@fc.up.pt (F. Borges).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.090

F. M. F. Roleira et al. / Bioorg. Med. Chem. 18 (2010) 5816–5825
5817
F
F
that other physicochemical drug properties such as solubility,
molecular weight, and pKa have also a noticeably influence on drug
permeability and hence in drug bioavailability.
F
F
F
O
R1
R2
COOH
R1
R2
DCC, PFP,
Dioxane/r.t.
In this context, this work reports the rational design and syn-
thesis of a new set of lipophilic phenolic antioxidants structurally
based on the natural antioxidants caffeic and ferulic acids, and
their saturated counterparts (Scheme 1). With this purpose, hex-
ylamides and hexylesters of cinnamic and hydrocinnamic acids
were synthesized in which the lipophilicity was essentially im-
proved by an introduction of an additional alkyl side chain
(Schemes 2 and 3). The antioxidant profile of the synthesized
amides and esters, as well as the corresponding acid precursors,
was determined by different methods, specifically throughout a
lipoperoxidation assay.5,6 The overall information obtained to-
gether with the assessment of the lipophilicity and redox poten-
tials of the compounds allow acquiring significant information to
establish a correlation between the physicochemical properties
and the activity, herein called SPAR (structure–property–activity
relationship). In addition, important data to predict the BBB per-
meation of the new chemical entities was attained using in silico
models relying on the physicochemical parameters (e.g., solubility,
lipophilicity, molecular size and hydrogen-bonding capacity).
O
5
6
R
1 = OCH3; R2 = OH
1 R1 = OCH3; R2 = OH
2 R1 = R2 = OH
R1 = R2 = OH
Hexanol, DCC,
DMAP, THF/r.t.
Hexylamine,
CHCl3/r.t.
O
O
R1
(CH2)5CH3
R1
(CH2)5CH3
O
N
H
R2
R2
11
R1 = OCH3; R2 = OH
7
8
R
1 = OCH3; R2 = OH
12 R1 = R2 = OH
R1 = R2 = OH
Scheme 2. Synthetic strategies used for the obtention of hydrocinnamic amides
and esters.
2. Results and discussion
2.1. Chemistry
DCC/DMAP (dimethylaminopyridine) method from the corre-
sponding parent hydrocinnamic acids (1 and 2).11 In contrast, com-
pounds 13 and 14 (Scheme 3) were prepared by base-catalyzed
alkylation from the corresponding dihydroxycinnamic acids (3
and 4) with in situ generation of the carboxylate anions and subse-
quent reaction, at room temperature, with hexylbromide.12 Efforts
were made to drive both reactions to a satisfactory yield by chang-
ing either the coupling reagent or the nature of the base. However,
fairly moderate yields were obtained in all cases.
The synthesis of hexylamides (7 and 8) (Schemes 1 and 2) starts
with the corresponding hydrocinnamic acids (1 and 2) which were
previously converted into the intermediates pentafluorophenyl es-
ters (5 and 6) by reaction with pentafluorophenol (PFP) and dicy-
clohexylcarbodiimide (DCC), in dioxane, at room temperature.7
Subsequent reaction of the resulting activated esters with hexyl-
amine, in chloroform and at room temperature, provided the re-
quired hexylamides (7 and 8).7 This method was useful for the
amidation of saturated acids containing phenolic groups.8,9
2.2. Effect of the phenolic derivatives on lipid peroxidation
Lipid peroxidation is an uncontrolled deleterious process that
occurs in cellular and subcellular membranes, causing or enhanc-
ing the formation of lipid hydroperoxides. These reactive species
are cytotoxic and capable of reacting with numerous cellular com-
ponents providing one of the mechanisms underlying the toxicity
of several drugs. The use of model membranes such as unilamellar
liposomes has lately been encouraged as a helpful tool for under-
standing the effect of drugs, such as antioxidants, in membrane
phospholipid bilayers. The advantage of using this type of systems,
In the synthesis of unsaturated hexylamides (9 and 10) (Scheme
3), the cinnamic acids (3 and 4) reacted straightforward with
hexylamine, in dimethylformamide (DMF), in the presence of the
coupling agent (benzotriazol-1-yloxy)tris(dimethylamino)phos-
phonium hexafluorophosphate (BOP), at room temperature. This
is a particularly suitable method for the direct amidation of
a
,b-unsaturated acids.10
All the adopted strategies for the preparation of phenolic esters
are outlined in Schemes 2 and 3. As the classic acid-catalyzed Fish-
er esterification is not appropriate when using long chain alkyl
alcohols, compounds 11 and 12 (Scheme 2) were obtained by using
including unilamellar versus multilamellar liposomes, and
a
water-soluble radical azo-generator (AAPH), is largely documented
in the literature.13 In AAPH induced peroxidation of unilamellar
R
COOH
HO
1 Hydroferulic acid
R=OCH3
R=OH
2 Hydrocaffeic acid
3 Ferulic acid
R=OCH3
R=OH
4 Caffeic acid
CONH(CH2)5CH3
R
R
COO(CH2)5CH3
HO
HO
7
8
9
Hydroferuloylhexylamide
Hydrocaffeoylhexylamide
Feruloylhexylamide
R=OCH3
R=OH
11 Hexylhydroferulate
R=OCH3
12 Hexylhydrocaffeate
13 Hexylferulate
14 Hexylcaffeate
R=OH
R=OCH3
R=OH
R=OCH3
R=OH
10 Caffeoylhexylamide
Scheme 1. Phenolic acids and their derivatives.

5818
F. M. F. Roleira et al. / Bioorg. Med. Chem. 18 (2010) 5816–5825
O
R1
COOH
BOP, Et3N, DMF,
CH2Cl2/r.t.
R1
R2
(CH2)5CH3
N
H
R2
9
10
R1 = OCH3; R2 = OH
R1 = R2 = OH
3
R
1 = OCH3; R2 = OH
4 R1 = R2 = OH
NaOH 5%, HMPA,
Bromohexane/r.t.
O
R1
(CH2)5CH3
O
R2
13
R1 = OCH3; R2 = OH
14 R1 = R2 = OH
Scheme 3. Synthetic strategies used for the obtention of cinnamic amides and esters.
liposomes the chain initiating radical is generated in the aqueous
phase and the chain-propagating lipid peroxyl radicals are located
within the membranes. In the assay, DPH-PA was used as fluores-
cence probe and Trolox as chain-breaking antioxidant standard.14
The data obtained for the phenolic acids and derivatives are
shown in Table 1. From the results one can conclude that hydroxy-
cinnamic acids are more effective than their saturated counterparts
(3 vs 1 and 4 vs 2) and that the catecholic moiety plays an important
role in the antioxidantactivity (4 and 2 vs 3 and 1). The observed ten-
dency is in accordance with data reported in the literature.15 In addi-
tion a ranking antioxidant order could be inferred from the data of
catecholic cinnamic derivatives: hexylcaffeate (14) presented high-
er antioxidant activity than caffeoylhexylamide (10) and this last
better than caffeic acid (4). An identical succession was observed
for the antioxidant profile of hydrogenated analogues (hexylhydro-
caffeate (12) > hydrocaffeoylhexylamide (8) > hydrocaffeic acid
(2)). From these results it was also possible to infer that the cinnamic
series present better antioxidant activity than the saturated ones.
In general, for catecholic systems it can be concluded that esters
(14 and 12) are better antioxidants than amides (10 and 8) and that
amides have a superior antioxidant efficacy than acids (4 and 2), a
fact that could be related with the intrinsic lipophilicity of each
system (Table 2).
tively (Table 1). As stated above it was also possible to infer that
the monohydroxylated cinnamic derivatives are more effective as
antioxidants than the saturated ones. In this series the monohydr-
oxylated amide derivatives (9 and 7) were slightly better antioxi-
dants than the correspondent esters (13 and 11).
In summary, taking into account the lipid peroxidation data one
can conclude that in general the catecholic derivatives are effective
lipophilic antioxidant candidates. The performed chemical modifi-
cations (introduction of an ester or amide spacer and an alkyl side
chain) do not diminish the antioxidant properties relatively to the
natural precursor acids but on contrary, in some cases, it led to a
huge activity enhancement. Dihydroxycinnamic hexylester and
its saturated analogue look like to be the greatest antioxidant
agents
2.3. Evaluation of the total antioxidant capacity (TAC) of the
catecholic derivatives
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. Classically, Trolox, a water-solu-
ble vitamin E analogue, is used as reference. The results of this type
of assays are usually expressed as trolox equivalent antioxidant
capacity (TEAC).16,17
Accordingly, the TAC assays (ABTS and DPPH) were applied to
assess the radical-scavenging ability of the most outstanding anti-
oxidant compounds (compounds 2, 4, 8, 10, 12 and 14 Scheme 1).
Regarding the monohydroxylated derivatives the following
antioxidant outlines were obtained for the cinnamic acid and
derivatives and for their saturated analogues: feruloylhexylamide
(9) > hexylferulate (13) > ferulic acid (3) and hydroferuloylhexyla-
mide (7) > hexylhydroferulate (11) > hydroferulic acid (1), respec-
Table 1
TEAC results for the lipoperoxidation, ABTS and DPPH assays of phenolic acids and derivatives
Phenolic compounds
Lipoperoxidation DPH-DA
ABTS
DPPH
5 min
20 min
Caffeic acid (4)
Hexylcaffeate (14)
Caffeoylhexylamide (10)
2.23 0.05
3.55 0.05
2.41 0.07
1.14 0.02
0.91 0.02
1.04 0.04
1.27 0.01
0.93 0.01
1.05 0.01
1.29 0.05
0.97 0.05
1.11 0.07
Hydrocaffeic acid (2)
Hexylhydrocaffeate (12)
Hydrocaffeoylhexylamide (8)
1.75 0.04
3.49 0.04
2.08 0.05
1.16 0.03
0.96 0.02
1.01 0.02
1.41 0.01
0.96 0.01
1.02 0.01
2.05 0.06
0.99 0.05
1.00 0.03
Ferulic acid (3)
Hexylferulate (13)
Feruloylhexylamide (9)
0.79 0.01
1.13 0.03
1.22 0.03
—
—
—
—
—
—
—
—
—
Hydroferulic acid (1)
Hexylhydroferulate (11)
Hydroferuloylhexylamide (7)
0.22 0.02
0.57 0.01
0.68 0.02
—
—
—
—
—
—
—
—
—

F. M. F. Roleira et al. / Bioorg. Med. Chem. 18 (2010) 5816–5825
5819
Table 2
Partition coefficients and redox potentials of the phenolic acids and derivatives
Phenolic compounds
miLog P/Log D (Theor., pH 5)a
Log P (Theor.)b
Log P (Exp., pH 4.7)
Ep
Caffeic acid (4)
Hexylcaffeate (14)
Caffeoylhexylamide (10)
1.53/0.16
4.12
3.16
1.15
3.49
2.81
—
0.183
0.175
0.162
3.91 0.08c
3.61 0.02d
Hydrocaffeic acid (2)
Hexylhydrocaffeate (12)
Hydrocaffeoylhexylamide (8)
1.45/0.26
3.81
3.08
1.17
3.51
2.83
_
0.139
0.125
0.125
3.79 0.06c
2.62 0.14d
Ferulic acid (3)
Hexylferulate (13)
Feruloylhexylamide (9)
1.67/0.42
4.27
3.30
1.42
3.76
3.08
—
0.350
0.328
0.322
4.05 0.04c
3.58 0.04d
Hydroferulic acid (1)
Hexylhydroferulate (11)
Hydroferuloylhexylamide (7)
1.59/0.51
3.95
3.22
1.44
3.78
3.10
—
0.410
0.434
0.388
3.60 0.08c
2.83 0.17d
a
b
c
Determined with Molinspiration Calculation software.31
Determined with ChemDraw software.30
Determined in liposomes.
d
Determined in micelles.
The levels of ABTSꢀ+ were recorded after 5 and 20 min of reaction,
in order to determine the kinetics of each compound in the neu-
tralization step of the radical (data not shown).
of antioxidants in a certain system but SAR cannot be readily
inferred.6
The data obtained is depicted in Table 1. From the results ob-
tained in the ABTS assay one can infer that, in general, the parent
acids (2 and 4) have a higher antioxidant activity towards ABTSꢀ+
than the corresponding amides (8 and 10) and esters (12 and
14), particularly after 20 min of reaction. From this assay, and in
opposition to what was observed in the lipid peroxidation assay,
the saturated cinnamic acids (2) showed a higher antioxidant
activity (after 20 min) than the unsaturated ones (4). To note that
the phenolic acids are still reacting after the first 5 min of reaction.
All the lipophilic compounds (8, 10, 12 and 14) exhibit approxi-
mately the same antioxidant performance, reaching their maxi-
mum antioxidant capacity after 5 min. The presence of a double
bond (compounds 4, 10 and 14) does not seem to markedly influ-
ence the antioxidant capacity of the esters and amides when com-
pared to their saturated homologues (2, 8 and 12). The results are
in accordance with the data obtained with similar systems.15
Concerning the DPPH assay (Table 1) it was observed that, in
general, phenolic acids (2 and 4) have higher antioxidant activity
than the corresponding amides (8 and 10) and esters (12 and
14). The saturated acid (2) showed a higher antioxidant activity
than the unsaturated one (4). In these experimental conditions
the phenolic derivatives reveal to be less active than their parent
compounds.
The DPPH results follow the same tendency than those obtained
with the ABTS assay. However, a larger dissimilarity in the antiox-
idant potency was observed between the acids and their deriva-
tives (esters and amides), a fact that could be related with to
steric hindrance caused by the bulkiness of alkyl groups.17
The radical-scavenging ability data obtained for hydroxycin-
namic acids and derivatives against DPPH and ABTS radicals were
in good agreement with the expected activities of this type of phe-
nolic systems: higher when a catechol group is present (caffeic or
hydrocaffeic series) and lower when the meta-hydroxyl function is
substituted by a methoxyl group (ferulic and hydroferulic series)
(see Table 1).
2.4. Evaluation of the redox properties of the phenolic
derivatives
Many antioxidants are proven to undergo an electron-transfer
mechanism upon exerting their antioxidant functions. As a result,
electrochemistry has been employed to evaluate antioxidant activ-
ity since it can provide direct and detailed information on the oxi-
dation of these compounds. Quantitative correlations have been
already established between the antioxidant activities and redox
potentials suggesting that this physicochemical parameter could
be considered a good measure of the antioxidant activity.18–20
Hence, it was found important to study the electrochemical prop-
erties of the synthesized caffeic and ferulic acid and their deriva-
tives (Scheme 1). Thus, the redox properties of the synthesized
phenolic compounds were studied, at physiological pH 7.3, by dif-
ferential pulse and cyclic voltammetry, using a glassy carbon work-
ing electrode.
The differential pulse voltammograms of dihydroxylated cin-
namic series, caffeic and hydrocaffeic acid derivatives (Scheme 1),
present only one well-defined anodic peak at physiological pH
(Fig. 1A and B). The oxidation peaks observed for these cinnamic
compounds are intrinsically related with the oxidation of catechol
group. Cyclic voltammograms were also recorded at different sweep
rates. The cyclic voltammograms obtained are characteristic of an
electrochemical reversible reaction showing only one anodic peak
and one cathodic peak on the reverse scan (Fig. 2A and B). Linear
plots of peak current (Ip) as a function of the square root of scan rate
(m) were obtained indicating that the oxidation processes are diffu-
sion controlled.21 The ratio of the anodic to cathodic peak heights
raise gradually with increasing potential scan rate from 10 to
100 mVsꢀ1 and the calculated current functions (Ipꢁ
m
-1/2) diminish
gradually with the potential scan rate, showing the classical behav-
iour of an oxidation process coupled with a slow subsequent chem-
ical reaction. The results are in agreement with the literature in
which concerns the oxidative behaviour of the parent com-
pounds.22–25 Electrochemical studies on the caffeic acid (4) oxida-
tion mechanism have shown that the first oxidation step involves
two electrons per molecule which likely correspond to the formation
of the caffeic acid ortho-quinone, an intermediate that is quickly
decomposed at pH higher than 7.4.22–24
From the results obtained one must conclude that the data ob-
tained from TAC assays could be used only for a qualitative and ra-
pid antioxidant screen. In fact, as demonstrated herein quantitative
data must be carefully analysed mainly if the compounds under
analysis present either intrinsic steric hindrance or lipophilic prop-
erties. The results are in accordance with previous studies that con-
cluded that the TAC assays may give an indication for the presence
The differential pulse voltammetric study of ferulic acid (3)
(Scheme 1), revealed the presence of two convolved anodic peaks

5820
F. M. F. Roleira et al. / Bioorg. Med. Chem. 18 (2010) 5816–5825
ion followed by one irreversible dimerisation process due to a rad-
A
ical–radical coupling reaction between two phenoxyl radicals.22,26
The voltammetric results showed that the higher the number of
hydroxyl substituents on the aromatic ring, the lower the electro-
chemical potential (Table 2). Redox potentials of caffeic and hydro-
caffeic acid derivatives (8, 10, 12 and 14) are lower than those of
ferulic and hydroferulic acid derivatives (7, 9, 11 and 13), a fact
that could be related with the presence of the cathecol moiety.
The methoxylation of the meta-hydroxy group of the cinnamic
derivatives (ferulic and hydroferulic acid derivatives) shifted the
peak potentials toward more positive values (Table 2).
1 µA
Extension of the conjugation via the side chain also affects con-
siderably the redox potentials of the cinnamic acids and deriva-
tives studied. This effect is more noticeable in caffeic acid and its
derivatives, due to an increase of the resonance effects on the sta-
bility of the intermediate radical.27–29
-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
The introduction of an alkylamide or an alkylester groups in the
conjugated side chain did not significantly change the oxidation
potentials, when compared to the parent compounds. The data ob-
tained strongly suggest that the structural modifications per-
formed result in modest or even no effect on the electron density
of the phenol or catechol ring. Hence, the introduction of these
groups did not significantly influence the energetic of the electron
transfer as can be ascribed from the similarity of the Ep values for
these derivatives.
B
1 µA
From the electrochemical results one can conclude that the
structural principles governing the redox potentials of the cin-
namic acids and its derivatives under study were found to be the
presence of a phenolic group, preferentially a catechol.
2.5. Partition coefficient measurements
-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
In view of better correlate the overall properties of the antioxi-
dant compounds the lipophilicity, expressed as the octanol–water
partition coefficient and herein called log P was calculated either
theoretically (according to Broto’s fragmentation method and
using Molinspiration property calculation program) or by deriva-
tive spectrophotometry in two mimetic systems (liposomes and
in micelles) (see Table 2).30–33 As expected the log P (exp), at pH
4.7, could not be determined for the acids (1–4) because at this
pH they are ionized, a fact that is in accordance with the theoretical
values (see Table 2). From the data obtained, one can notice that
the studied acids (1–4) are not able to cross membranes effectively,
once they have a log P 6 1. On the contrary, the synthesised amides
(7–10) and esters (11–14), possessing an additional lipophilic alkyl
chain showed a superior lipophilicity, with log P values compatible
with those required to cross membranes, particularly the blood–
brain barrier (log P between 1 and 3).34
In general, the experimental values of log P for the esters and
amides studied are in accordance with the theoretical ones, and es-
ters are more lipophilic than amides. The hexylesters (13, 14) are
more lipophilic than the correspondent hexylhydroesters (11,
12). The same ranking order is observed for the hexylamides (9,
10) versus hexylhydroamides (7, 8). From all of compounds stud-
ied, hexylferulate (13) (log P = 4.05) is the most lipophilic which
is in agreement with the predicted theoretical values. The hexylhy-
drocaffeic amide (8) and the hexylhydroferulic amide (7), with a
log P of 2.62 and 2.83, respectively, seem to have the ideal lipophil-
icity to cross the blood–brain barrier.34 In addition, the theoretical
prediction of ADME properties of all compounds was carried out
(see Tables 2 and 3). From the data obtained it can be observed that
no violations of Lipinski’s rule (molecular weight, log P, number of
hydrogen donors and acceptors, and number of rotatable bonds)
were found making the cinnamic derivatives promising antioxi-
dant agents. Topological polar surface area (TPSA), described to
E/V vs Ag/AgCl
Figure 1. Differential pulse voltammograms for 0.1 mM solutions of (A) (—) caffeic
acid, (——) caffeoylhexylamide, (ꢂꢂꢂꢂ) hexylcaffeate and (B) (—) hydrocaffeic acid,
(——) hydrocaffeoylhexylamide, (ꢂꢂꢂꢂ) hexylhydrocaffeate, in physiological pH 7.3
supporting electrolyte. Scan rate: 5 mV sꢀ1
.
at physiological pH (Fig. 3A). The oxidation peaks at Ep = +0.335 V
and E0p = +0.443 V are related with the oxidation of the phenolic
group present in the structure. The results may be interpreted by
assuming that cinnamic acids oxidation takes place by electron
transfer for both free and adsorbed forms. The free form corre-
sponds to the first peak, Ep, while the strongly adsorbed form,
which is consequently stabilized, is oxidized at a more anodic po-
tential, E0p. The appearance of an adsorption peak, E0p, has been de-
scribed in literature in electrochemical studies involving ferulic
acid analogues.25,26
For ferulic (3) and hydroferulic acid (1) derivatives (Scheme 1),
only one anodic wave was observed at physiological pH using dif-
ferential pulse voltammetry (Fig. 3A and B). The oxidation peaks
observed are also related with the oxidation of the phenolic group
present in the structure. The large wave shape observed could be a
sign of a higher superimposition of the peaks from both free and
adsorbed forms or might indicate a lesser adsorption propensity
of this molecule. The cyclic voltammograms obtained for ferulic
acid have also shown two convolved anodic peaks. The anodic
peaks obtained for ferulic and hydroferulic series appear to corre-
spond to irreversible processes (Fig. 2C and D). Plots of peak cur-
rent (Ip) as a function of the square root of scan rate (m) gave a
straight line indicating that the oxidation processes are diffusion
controlled.21 The assembled data corroborate the results found in
literature regarding ferulic acid oxidative behaviour. The proposed
mechanism involves a one-electron transfer from the phenolate

F. M. F. Roleira et al. / Bioorg. Med. Chem. 18 (2010) 5816–5825
5821
A
B
200 nA
200 nA
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
-0.2 0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
E/V vs Ag/AgCl
E/V vs Ag/AgCl
C
D
200 nA
200 nA
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
E/V vs Ag/AgCl
E/V vs Ag/AgCl
Figure 2. Cyclic voltammograms for 0.1 mM solutions of (—) acids, (——) hexylamides and (ꢂꢂꢂꢂ) hexylesters for (A) caffeic, (B) hydrocaffeic, (C) ferulic and (D) hydroferulic
series, in physiological pH 7.3 supporting electrolyte. Scan rate: 50 mV sꢀ1
.
be a predictive indicator of blood–brain barrier penetration, is also
found to be positive for these potential CNS drugs.35–37
The overall structure–property–activity outcome allow to con-
clude that the best antioxidant candidates are the dihydroxycin-
namic hexylester (14) and its saturated analogue (12) since they
possess a suitable redox potential (Ep 0.175–0.125) supplemented
by a proper lipophilicity (log P 3.91–3.79) (Table 2).
2.6. Structure–property–activity relationships (SPAR)
The development of novel lipophilic antioxidants structurally
based on the natural models—ferulic (3) and caffeic (4) acids and
their corresponding saturated counterparts (1 and 2, respectively)
was performed by introducing alkyl groups in the side chain and a
diverse spacer either of amide or ester type. In accordance with the
aim of the work the physicochemical properties as well as antiox-
idant activity of the compounds were examined in order to carry
out structure–property–activity relationship studies as an upgrad-
ing tool for the rational design of lipophilic antioxidants.5,6,25
The antioxidant efficacy of the phenolic derivatives is higher
when the redox potential is lower (as seen in the caffeic and hydro-
caffeic series). The presence of a catechol group leads to an increase
of the activity due mainly to resonance stabilization of the phenoxyl
radical intermediate with subsequent ortho-quinone formation.6,15
A redox potential increase and a decrease of the antioxidant activity
was obtained in monohydroxylated compounds (ferulic and hydrof-
erulic series). The higher lipophilicity of the alkylamides and alky-
lesters in comparison with their parent acids lead to an increase of
activity since they have the ability to interact with the polar head
groups of the membrane and attain a local concentration at the
water–lipid interface.
3. Conclusions
From the results obtained it is possible to infer that the synthe-
sized antioxidants of amide or ester type possess an amplified
lipophilicity, increasing the antioxidant activity relatively to the
precursor acids. The esters revealed to be more active than the
homologous amides. The hexylamides (10 and 8) and hexylesters
(14 and 12) of caffeic and hydrocaffeic acids showed to be the most
active of all the phenolic compounds investigated. The hexylesters
(14 and 12) were found to be the most promising compounds, a fact
that can be related with their lipophilicity and redox behaviour.
From the electrochemical results one can conclude that the
structural features governing the redox potentials of the cinnamic
acids and derivatives were found to be a phenolic group, preferen-
tially a catechol, as well as the additional resonance-effective
substituents in the aromatic ring. Furthermore, the determined
partition coefficients have shown the desired increased lipophilic-
ity of the synthesized amides and esters relatively to the precursor
acids, giving special emphasis on their brain permeable properties.
In addition, the ADME properties predicted for these antioxidants

5822
F. M. F. Roleira et al. / Bioorg. Med. Chem. 18 (2010) 5816–5825
4. Experimental
A
4.1. Chemicals
250 nA
2,20-Azobis(2-amidinopropane)dihydrochloride (AAPH), 2,20-
azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-di-
phenyl-1-picrylhydrazyl radical (DPPH), 4-(2-hydroxyethyl)piper-
azine-1-ethanesulfonic acid hemisodium salt (Hepes), egg L-a-
phosphatidylcholine (EPC), 2-carboxy-2,5,7,8-tetramethyl-6-chro-
manol (Trolox) and 1-hexadecylphosphorylcholine (HDPC) were
obtained from Sigma. Diphenylhexatriene propionic acid (DPH-
PA) was obtained from Molecular Probes. All the other chemicals
were purchased from Aldrich, Fluka and Merck and used as sup-
plied by the manufacturers. Flash column chromatography was
performed with Silica Gel 60—particle size: 0.040–0.063 mm.
-0.2 0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
E/V vs Ag/AgCl
4.2. Apparatus
B
Melting-points were determined on a Reichert Thermopan or
Thermovar hot block apparatus and were not corrected. IR spectra
were recorded on a Jasco 420FT/IR spectrometer. The 1H and 13C
NMR spectra were recorded at 300.13 and 75.6 MHz, respectively,
on a Bruker-AMX 300 spectrometer using deuterated dimethyl
sulfoxide (DMSO-d6) as a solvent. The 1H and 13C NMR spectra of
compound 9 were recorded at 599.7 and 150.8 MHz, respectively,
on a Varian 600 MHz VNMRX spectrometer. Chemical shifts were
recorded in d (ppm) values downfield from TMS as internal stan-
dard for 1H and 13C NMR. Coupling constants (J) are given in Hertz.
Assignments were also made from DEPT (distortionless enhance-
ment by polarization transfer) (underlined values). Electron impact
mass spectra (EI-MS) were carried out on a VG AutoSpec instru-
ment; the data are reported as m/z (% of relative intensity of the
most important fragments).
200 nA
-0.2 0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
UV/vis and fluorescence measurements were carried out on a
Bio-Tek Synergy HT multiplate reader) in flat-bottomed 96-well
microplates (Orange Scientific).
Liposomes extrusion was performed at room temperature with
an extruder Lipex (Lipex Biomembranes, Vancouver, Canada) un-
der high pressure of nitrogen (N2).
E/V vs Ag/AgCl
Figure 3. Differential pulse voltammograms for 0.1 mM solutions of (A) (—) ferulic
acid, (——) feruloylhexylamide, (ꢂꢂꢂꢂ) hexylferulate and (B) (—) hydroferulic acid,
(——) hydroferuloylhexylamide, (ꢂꢂꢂꢂ) hexylhydroferulate, in physiological pH 7.3
supporting electrolyte. Scan rate: 5 mV sꢀ1
.
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 system. A
glassy carbon working electrode (GCE) (d = 2 mm), a platinum wire
counter electrode and an Ag/AgCl saturated KCl reference electrode
were used. A Crison pH-meter with glass electrode was used for
the pH measurements (Crison, Spain).
are in accordance with the general requirements for potential CNS
drugs.
The results gathered along this work provide a rational ap-
proach to the design of membrane-target antioxidants that could
be effective candidates for preventing or reducing the oxidative
status associated with the neurodegenerative processes.
Table 3
Structural properties of the phenolic acids and derivativesa
Phenolic compounds
Molecular weight
n-ROTB
n-ON acceptors
n-ONNH donors
TPSA
Caffeic acid (4)
Hexylcaffeate (14)
Caffeoylhexylamide (10)
180.16
264.32
263.34
2
8
7
4
4
4
3
2
3
77.76
66.76
69.55
Hydrocaffeic acid (2)
Hexylhydrocaffeate (12)
Hydrocaffeoylhexylamide (8)
182.18
266.34
265.35
3
9
8
4
4
4
3
2
3
77.76
66.76
69.55
Ferulic acid (3)
Hexylferulate (13)
Feruloylhexylamide (9)
194.19
278.35
277.36
3
9
8
4
4
4
2
1
2
66.76
55.76
58.56
Hydroferulic acid (1)
Hexylhydroferulate (11)
Hydroferuloylhexylamide (7)
196.20
280.36
279.38
4
10
9
4
4
4
2
1
2
66.76
55.77
58.56
a
n-ROTB, number of rotatable bonds; n-OHNH, number of hydrogen bond donors; n-ON, number of hydrogen acceptors; TPSA, topological polar surface area. Determined
with Molinspiration Calculation software.31

F. M. F. Roleira et al. / Bioorg. Med. Chem. 18 (2010) 5816–5825
5823
4.3. Synthesis of phenolic compounds
ken to dryness and the remaining residue was purified by flash col-
umn chromatography.
4.3.1. General procedure to obtain the pentafluorophenyl esters
(5–6)
4.3.4.1. Hexyl (E)-3-(4-hydroxy-3-methoxyphenyl)propanoate
(11). Chromatographic solvent: chloroform/methanol (9:1); yield:
23% (oil); 1H NMR d: 0.86 (3H, t, J = 6.4, CH3), 1.26 (6H, m,
According previous descriptions by the authors.7
4.3.2. General procedure to obtain the hydrocinnamic
hexylamides (7–8) from the pentafluorophenyl esters (5–6)
According previous descriptions by the authors.7
3 ꢃ CH2(30–50)), 1.53 (2H, m, CH2(20)), 2.58 (2H, t, J = 7.4, CH2(
a)),
2.74 (2H, t, J = 7.4, CH2(b)), 3.74 (3H, s, OCH3), 3.99 (2H, t, J = 6.6,
CH2(10)), 6.58 (1H, dd, J = 8.0; 1.8, CH(6)), 6.67 (1H, d, J = 8.0,
CH(5)), 6.78 (1H, d, J = 1.8, CH(2)), 8.72 (1H, s, OH); 13C NMR d:
4.3.3. General procedure to obtain the cinnamic hexylamides
(9–10)
13.9 CH3, 22.0, 25.1, 28.2, 30.9 C(20)–C(50), 28.2 C(b), 35.6 C(
a),
55.5 OCH3, 63.7 C(10), 112.4 C(2), 115.3 C(5), 120.2 C(6), 131.3
C(1), 144.8 C(4), 147.4 C(3), 172.4 C@O; EI-MS m/z (%): 280 (M+ꢀ,
5), 178 (21), 151 (14), 137 (100), 122 (9), 107 (14), 91 (18), 77
(12), 65 (8). For structural data comparison, see Ref. 40.
In order to synthesize the unsaturated amides, cinnamic acids
(3 and 4) (5.0 mmol) were dissolved in 10 mL of DMF containing
0.7 mL of triethylamine. The solution was then cooled in an ice-
water bath and hexylamine (0.67 mL, 5.0 mmol) was added, fol-
lowed by a solution of BOP (2.21 g, 5.0 mmol) in 10 mL of dichloro-
methane (CH2Cl2). The mixture was stirred at 0 °C for 30 min and
at room temperature for additional 4 h. Dichloromethane was re-
moved under reduced pressure and the remaining solution was di-
luted with 100 mL of water. The mixture was then extracted with
2 ꢃ 100 mL of ethyl acetate. The extracts were washed with 1 N
HCl (2 ꢃ 100 mL), water (2 ꢃ 100 mL), NaHCO3 5% (3 ꢃ 100 mL),
and finally with water (2 ꢃ 100 mL), dried over MgSO4, filtered
and concentrated. The obtained residues were purified by flash col-
umn chromatography to provide the corresponding hexylamides
(9 and 10).
4.3.4.2. Hexyl (E)-3-(3,4-dihydroxyphenyl)propanoate (12).
Chromatographic solvent: diethyl ether; yield: 22%; 1H NMR d:
0.85 (3H, t, J = 6.5, CH3), 1.25 (6H, m, 3 ꢃ CH2(30–50)), 1.51 (2H, m,
CH2(20)), 2.49 (2H, t, J = 7.3, CH2(
a)), 2.65 (2H, t, J = 7.3; CH2(b)),
3.97 (2H, t, J = 6.6, CH2(10)), 6.42 (1H, dd, J = 8.0; 2.0, CH(6)), 6.56
(1H, d, J = 2.0, CH(2)), 6.60 (1H, d, J = 8.0, CH(5)), 8.64 and 8.71
(2H, s, OH(3) and (4)); 13C NMR d: 13.9 CH3, 22.0, 25.0, 28.1, 30.9
C(20)–C(50), 29.8 C(b), 35.6 C( ), 63.7 C(10), 115.4 C(5), 115.6 C(2),
a
118.7 C(6), 131.2 C(1), 143.5 C(4), 145.0 C(3), 172.4 C@O; EI-MS
m/z (%): 266 (M+ꢀ, 55), 182 (44), 138 (41), 136 (79), 123 (100).
(lit.29 mp 67–68 °C). For structural data comparison, see Ref. 29.
4.3.3.1.
N-Hexyl-3-(4-hydroxy-3-methoxyphenyl)-2-propena-
4.3.5. General procedure to obtain the cinnamic hexylesters
esters (13–14)
mide (9). Chromatographic solvent: petroleum ether/ethyl ace-
tate (from 9:1 to 5:5); yield: 70% (as oil); IR mmax (NaCl plates,
cmꢀ1): 3426 (O–H), 3284 (N–H stretch), 1620 (C@O); 1H NMR d:
0.86 (3H, t, J = 6.8, CH3), 1.26 (6H, m, 3 ꢃ CH2(30–50)), 1.42 (2H, m,
CH2(20)), 3.14 (2H, m, CH2(10)), 3.80 (3H, s, OCH3), 6.43 (1H, d,
Cinnamic acids (11.0 mmol), 25 mL of hexamethylphosphora-
mide (HMPA) and 2.28 mL of aqueous NaOH 5% were placed in a
100 mL one-neck round bottom flask. After vigorous stirring for
about 1 h, a solution of bromohexane (6.18 mL, 44 mmol) in HMPA
(10 mL) was added dropwise. The solution was stirred for addi-
tional 2 h. After quenching the reaction with addition of water/
ice mixture, it was extracted with diethylether (2 ꢃ 50 mL). The or-
ganic layer was washed twice with HCl 1 M and water, dried over
anhydrous magnesium sulphate, filtered and finally evaporated to
dryness. The remaining residue was purified by flash column
chromatography.
J = 15.7, CH(a)), 6.78 (1H, d, J = 8.2, CH(5)), 6.97 (1H, dd, J = 8.2;
1.9, CH(6)), 7.10 (1H, d, J = 1.9, CH(2)), 7.30 (1H, d, J = 15.7,
CH(b)), 7.92 (1H, t, J = 5.6, NH), 9.40 (1H, s, OH); 13C NMR d: 14.0
CH3, 22.1, 26.2, 29.2, 31.0 C(20)–C(50), 55.5 OCH3, 38.6 C(10), 110.6
C(2), 115.6 C(5), 119.1, 121.5 C(6) and C(a), 126.4 C(1), 138.7
C(b), 147.8 C(4), 148.2 C(3), 165.2 C@O. EI-MS m/z: 277 (M+ꢀ). For
structural data comparison, see Ref. 38.
4.3.3.2. N-Hexyl-3-(3,4-dihydroxyphenyl)-2-propenamide (10).
Chromatographic solvent: petroleum ether/ethyl acetate (from 8:2
to 5:5); yield: 65%; IR mmax (ATR, cmꢀ1): 3324 (O–H and N–H
stretch), 1608 (C@O); 1H NMR d: 0.87 (3H, t, J = 6.7, CH3), 1.36
(8H, m, 4 ꢃ CH2(20–50)), 3.14 (2H, m, CH2(10)), 6.32 (1H, d,
4.3.5.1. Hexyl (E)-3-(4-hydroxy-3-methoxyphenyl)-2-propeno-
ate (13). Chromatographic solvent: chloroform/methanol (8:2);
yield: 27% (oil); 1H NMR d: 0.85 (3H, t, J = 6.4, CH3), 1.28 (6H, m,
3 ꢃ CH2(30–50)), 1.58 (2H, m, CH2(20)), 3.82 (3H, s, OCH3), 4.10
(2H, t, J = 6.6, CH2(10)), 6.46 (1H, d, J = 15.9, CH(
a)), 6.79 (1H, d,
J = 15.7, CH(
a
)), 6.74 (1H, d, J = 8.1, CH(5)), 6.83 (1H, dd, J = 8.1;
J = 8.1, CH(5)), 7.10 (1H, dd, J = 8.2; 1.9, CH(6)), 7.32 (1H, d,
J = 1.9, CH(2)), 7.54 (1H, d, J = 15.9, CH(b)), 9.59 (1H, br s, OH). 13C
NMR d: 13.9 CH3, 22.1, 25.2, 28.3, 31.0 C(20)–C(50), 55.6 OCH3,
1.8, CH(6)), 6.93 (1H, d, J = 1.8, CH(2)), 7.22 (1H, d, J = 15.7,
CH(b)), 7.96 (1H, t, J = 5.5, NH), 9.14 (1H, s, OH(3)), 9.37 (1H, s,
13
OH(4)); C NMR d: 14.1 CH3, 22.2, 26.3, 29.3, 31.1 C(20)–C(50),
63.7 C(10), 111.1 C(2), 114.5 C(5), 115.5 C(
a), 123.2 C(6), 125.6
38.6 C(10), 113.9 C(2), 115.9 C(5), 118.8, 120.5 C(6) and C(
a
),
C(1), 145.0 C(b), 147.9 C(4), 149.3 C(3), 166.7 C@O; EI-MS m/z
(%): 278 (M+ꢀ, 70), 194 (100), 177 (47), 150 (30), 145 (16), 137
(13), 117 (10), 89 (11), 77 (7), 55 (8). The compound was also pre-
viously reported by Murakami et al.41
126.6 C(1), 139.0 C(b), 145.7, 147.4 C(3) and C(4), 165.4 C@O; EI-
MS m/z (%): 263 (M+ꢀ, 5), 178 (36), 163 (100), 145 (14), 134 (14),
117 (12), 89 (18), 77 (10); mp 130–133 °C. For structural data com-
parison, see Ref. 39.
4.3.5.2. Hexyl (E)-3-(3,4-dihydroxyphenyl)-2-propenoate (14).
Chromatographic solvent: chloroform/methanol (9:1); yield: 8%;
1H NMR d: 0.88 (3H, t, J = 6.5, CH3), 1.33 (6H, m, 3 ꢃ CH2(30–50)),
1.62 (2H, m, CH2(20)), 4.11 (2H, t, J = 6.6, CH2(10), 6.25 (1H, d,
4.3.4. General procedure to obtain the hydrocinnamic
hexylesters (11–12)
The hydrocinnamic acids (5.0 mmol), 1-hexanol (0.627 mL,
5.0 mmol), N,N0-dicyclohexylcarbodiimide (DCC) (1.03 g, 5.0 mmol),
4-(dimethylamino)pyridine (DMAP) (1.22 g, 10.0 mmol) and 30 mL
of anhydrous tetrahydrofuran (THF) were stirred in a one-neck
round bottom flask at room temperature for 24 h. The mixture was
then cooled to 0 °C, and the solid side product, dicyclohexylurea,
was separated and removedby vacuum filtration. The filtrate was ta-
J = 15.9, CH(a)), 6.75 (1H, d, J = 8.1, CH(5)), 7.00 (1H, dd, J = 8.2;
2.0, CH(6)), 7.05 (1H, d, J = 2.0, CH(2)), 7.47 (1H, d, J = 15.9,
13
CH(b)); C NMR d: 13.9 CH3, 22.0, 25.1, 28.2, 30.9 C(20)–C(50),
63.7 C(10), 113.8 C(2), 114.7 C(5), 115.7 C(
a), 121.4 C(6), 125.3
C(1), 145.1 C(b), 145.7 C(3), 148.7 C(4), 166.7 C@O; EI-MS m/z
(%): 264 (M+ꢀ, 45), 181 (11), 180 (100), 163 (55), 136 (15), 134

5824
F. M. F. Roleira et al. / Bioorg. Med. Chem. 18 (2010) 5816–5825
(12),117 (7), 89 (10). mp 129–132 °C. For structural data compari-
order to get absorbance values of 0.45 0.01 at 734 nm and 30 °C,
son, see Ref. 29.
and 0.38 0.01 at 515 nm and 25 °C, when 180 lL samples were
placed in the plate reader.
4.4. Determination of antioxidant activity
Six different ethanolic solutions of each polyphenol (with con-
centrations ranging from 1.5 ꢃ 10ꢀ5 to 15 ꢃ 10ꢀ5 M) were pre-
4.4.1. Lipoperoxidation assay
pared in duplicate. Each solution (20 lL) was added to 180 lL of
Antioxidant activity against lipoperoxidation was estimated in
liposomes of EPC, containing a radical sensitive fluorescent probe,
according to the procedure described by Arora et al.13 Peroxyl rad-
icals were generated as a consequence of thermal decomposition of
AAPH.14 The experimental method was adapted in order to use a
multiplate reader.
radical solution (in quadruplicate) and absorbances were recorded:
for ABTSꢀ+, every 5 min for a 20 min period; for DPPHꢀ every minute
for a 10 min period, followed by every 5 min for the next 50 min.
The absorbance of a blank control (20 lL ethanol plus 180 lL of
radical) was set as 100% of radical (0% bleaching). Trolox was used
as a reference antioxidant.
4.4.1.1. Liposomes preparation. EPC (15.75 mg, 2.25 ꢃ 10ꢀ5 mol)
4.4.2.3. Data analysis. The radical concentrations (both ABTSꢀ+
and DPPHꢀ) were plotted as a function of the concentration of the
phenolic compounds, for 5 and 20 min of reaction time for ABTSꢀ+,
and for 60 min of reaction time for DPPHꢀ. Second degree polyno-
mial regressions of the experimental points were generated with
a y-axis intercept at 100% of radical. The TEAC value was consid-
ered as the ratio between the Trolox concentration corresponding
to a 50% bleach of the radical (IC50) and the concentration of phenol
needed to achieve the same effect (for the different reaction times
considered).
and DPH-PA (38 lL of a 60% (m/v) methanolic solution) were dis-
solved in a 50 mL round flask containing 10 mL of a CHCl3/CH3OH
(3:1) mixture. The solvent was evaporated on a rotavapor at 30 °C,
under a nitrogen flow in a light-protected environment, leaving a
homogeneous lipidic film on the flask wall. The film was kept in
a dessicator, under vacuum and protected from light, until further
use. Before the measurements, the film was vigorously shaken for
20 min in a vortex mixer with 15 mL of a Hepes solution (5 mM)/
NaCl (0.1 M), in order to obtain a suspension of Multilamellar Ves-
icles (MLVs). This suspension was extruded ten times through a
100 nm pore polycarbonate filter (Nucleopore, Whatman), yielding
a suspension of Large Unilamellar Vesicles (LUVs) containing the
DPH-PA fluorescent probe.
IC50ðtroloxÞ
TEAC ¼
IC50ðcompoundÞ
4.5. Determination of redox potentials
4.4.1.2. Liposomes oxidation. Polyphenol solutions (6.42, 32.14
and 64.28
6.5% of ethanol. Reagents were introduced in the 96-wells plate
as follows: 160 L of the LUVs suspension, 70 L of polyphenol
solution under study and 70 L of a AAPH solution. The final con-
centrations were: 0.80 mM of LUVs, 1.50, 7.50 and 15.00 M of pol-
yphenol, 1.5% of ethanol and 15.00 mM of AAPH. Each assay was
conducted in duplicate. Before the addition of the radical initiator,
the LUV/polyphenol mixtures were shaken for 10 min at 37 °C in
the multiplate reader. The maximum of fluorescence emission
(kex: 360/40 nm, kem: 460/40 nm) was set to 100% (0% of oxida-
tion). AAPH was added and the fluorescence decay over time was
recorded at 37 °C, at regular intervals, for 3 h. Trolox was used in
the experiment as reference antioxidant.
lM) were prepared in Hepes/NaCl solution, containing
Solutions used in the electrochemical determinations were ob-
tained as follows: 10 mM stock solutions of the studied com-
pounds were prepared by dissolving them in an appropriate
amount in ethanol. The voltammetric working solutions were pre-
pared, in the electrochemical cell, by diluting 100 lL of the stock
solution in 10 mL of the supporting electrolyte in order to get a fi-
l
l
l
l
nal 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.
Deionised water with conductivity less than 0.1 l
S cmꢀ1 was
used throughout. Buffer solutions employed were 0.2 M in the
pH range 1.2–12.2.15
4.4.1.3. Data analysis. The area under the curve of a control assay
(without polyphenol) was subtracted from the area obtained for
the polyphenol and Trolox assays. For a given concentration, the
area obtained for the polyphenol was divided by the one obtained
for Trolox yielding TEAC values:
4.6. Determination of partition coefficients
Partition coefficients were determined at pH 7.4 and 25 °C,
using liposomes of EPC or micelles of HDPC at pH 2, as previously
described.32,33
AREAðcompoundÞ ꢀ AREAðcontrolÞ
TEAC ¼
AREAðtroloxÞ ꢀ AREAðcontrolÞ
4.7. Statistical analysis
4.4.2. Total antioxidant capacity (TAC) assays
Each experiment was performed at least three times. Results
were expressed as the mean SEM. Data were analyzed by a
one-way analysis of variance (ANOVA). Differences were consid-
ered significant where p <0.05.
Total antioxidant capacity assays were performed using ABTS
and DPPH as radicals. The experimental procedures were adapted
from the literature,16,17,42 in order to use a multiplate reader.
4.4.2.1. ABTS solution. An aqueous solution (25 mL) of ABTS
(96.02 mg, 1.75 ꢃ 10ꢀ4 mol) and potassium persulfate (16.55 mg,
6.12 ꢃ 10ꢀ5 mol) was left standing overnight allowing to develop
the deep blue–green colour of ABTSꢀ+.
Acknowledgements
The authors are grateful to FCT (Fundação para a Ciência e Tecn-
ologia) for financial support of this research.
4.4.2.2. DPPH solution. An ethanolic solution (25 mL) of DPPH
(19.13 mg, 4.85 ꢃ 10ꢀ5 mol) was prepared yielding a deep purple
solution of DPPHꢀ.
Supplementary data
Prior to the measurements, the concentration of the ABTSꢀ+ and
DPPHꢀ solutions were adjusted with different volumes of ethanol in
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.090. These data

F. M. F. Roleira et al. / Bioorg. Med. Chem. 18 (2010) 5816–5825
5825
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