Synthesis and structure/antioxidant activity relationship of novel catecholic antioxidant structural analogues to hydroxytyrosol and its lipophilic esters

Article abstract of DOI:10.1021/jf301131a

A large panel of novel catecholic antioxidants and their fatty acid or methyl carbonate esters has been synthesized in satisfactory to good yields through a 2-iodoxybenzoic acid (IBX)-mediated aromatic hydroxylation as the key step. The new catechols are

Full text of DOI:10.1021/jf301131a

Article
pubs.acs.org/JAFC
Synthesis and Structure/Antioxidant Activity Relationship of Novel
Catecholic Antioxidant Structural Analogues to Hydroxytyrosol and
Its Lipophilic Esters
Roberta Bernini,*,§ Fernanda Crisante,§ Maurizio Barontini,§ Daniela Tofani,*,† Valentina Balducci,#
and Augusto Gambacorta†
§Department of Agriculture, Forests, Nature and Energy (DAFNE), University of Tuscia, Via S. Camillo De Lellis, 01100 Viterbo,
Italy
†Department of Mechanical and Industrial Engineering, University “Roma Tre”, Via della Vasca Navale 79, 00146 Rome, Italy; and
Centro Interdipartimentale di Servizi per la Didattica Chimica (CISDiC), Via della Vasca Navale 79, 00146 Roma, Italy
#Department of Biology, University “Roma Tre”, Viale G. Marconi 446, 00146 Rome, Italy
S
* Supporting Information
ABSTRACT: A large panel of novel catecholic antioxidants and their fatty acid or methyl carbonate esters has been synthesized
in satisfactory to good yields through a 2-iodoxybenzoic acid (IBX)-mediated aromatic hydroxylation as the key step. The new
catechols are structural analogues of naturally occurring hydroxytyrosol (3,4-DHE). To evaluate structure/activity relationships,
the antioxidant properties of all catecholic compounds were evaluated in vitro by ABTS assay and on whole cells by DCF
fluorometric assay and compared with that of the corresponding already known hydroxytyrosyl derivatives. Results outline that all
of the new catechols show antioxidant capacity in vitro higher than that of the corresponding hydroxytyrosyl derivatives. Less
evident positive effects have been detected in whole cells experiments. Cytotoxicity experiments, using MTT assay, on a
representative set of compounds evidenced no influence in cell survival.
KEYWORDS: catechols, aromatic hydroxylation, lipophilic antioxidants, ABTS assay, cell culture DCF assays,
structure/antioxidant activity relationship
INTRODUCTION
■
In the human body, reactive oxygen species (ROS) are
continuously produced during metabolic processes, and their
action is controlled by endogenous antioxidants that suppress
or minimize free radicals and their chain reactions. When there
is an imbalance in the redox equilibrium, oxidative stress is
Figure 1. Hydroxytyrosol and its lipophilic derivatives.
activated. These conditions stimulate aging pathogenesis and
degenerative or chronic diseases and favor cell apoptosis and
cancer.1 Several epidemiological studies have provided support
Therefore, in view of the above remarkable biological
for a protective effect of the consumption of fresh fruits,
vegetables, and virgin olive oil, the integral ingredients of the
Mediterranean diet. In particular, the protective effect of virgin
olive oil has been attributed to the presence of bioactive
polyphenols2 and their antioxidant activity, notably high in
catechols because of their lower O−H bond dissociation
enthalpy (77.7−80.1 kcal/mol) when compared to phenols
(85.1−88.0 kcal/mol).3 Among the polyphenols present in
virgin olive oil, hydroxytyrosol [2-(3,4-dihydroxyphenyl)-
ethanol, referred to as 3,4-DHE, Figure 1] has been shown to
scavenge hydrogen peroxide4 and to inhibit the hypochlorous
acid-derived radicals. In addition, it exhibits several biological
activities reducing the risk of coronary heart disease and
atherosclerosis5 and showing antimicrobial,6 antiproliferative,
and apoptotic activities.7−9
properties, many 3,4-DHE derivatives have been prepared in
the past few years, and their structure/antioxidant activity
relationships (SAR) have been studied. Namely, SAR studies
on 3,4-DHE derivatives have evidenced the key role of the free
catechol function as stated by the low level of antioxidant
activity shown by 3,4-DHE derivatives carrying an ester
function at the catechol hydroxyls.10 On the contrary,
chemoselective acylation or alkylation at the 3,4-DHE alcohol
function with long saturated and unsaturated alkyl chains gives
rise to lipophilic 3,4-DHE esters10−16 or ethers17 (Figure 1). As
a consequence, the importance of the chemoselective
esterification/etherification at the alcoholic hydroxyl has been
outlined. Moreover, lipophilic 3,4-DHE derivatives overcome
Received: March 19, 2012
Revised: July 10, 2012
Accepted: July 11, 2012
Published: July 11, 2012
Having amphiphilic character (log P = 0.08), 3,4-DHE has
only limited applications in hydrophobic/lipidic media or as a
preservative in food technology and the cosmetic industry.
© 2012 American Chemical Society
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the above solubility limitations and are easily incorporated in
cosmetic and food preparations.18 In the literature, most of the
studies on lipophilic antioxidants reported that the longer the
alkyl chain length, the more active the antioxidant; some other
contributions showed, in contrast, a nonlinear trend of the
chain length in emulsified,19−21 liposomal,22,23 and cellular
systems.14,24,25 In these works, the antioxidant activity increases
more or less when the hydrophobicity is increased, whereas
after a threshold is reached for medium chain length, the
antioxidant activity collapses to a near zero value.
As to the effect on the antioxidant activity of the relative
position of the two phenolic hydroxyls in these systems,
previous SAR studies have shown that the best results are
obtained when the two hydroxyls are adjacent (catechols). This
is related both to the above-mentioned lower dissociation
enthalpy3 and to the formation of an intramolecular H-bond.
However, nothing is known about the effect of moving the
catechol function to the 2,3-position with respect to the alcohol
side chain. Furthermore, a previous study, carried out on a few
3-(3,4-dihydroxyphenyl)propyl fatty acid esters (3,4-DHP
esters), reported an increase of antioxidant activity with respect
to the corresponding 3,4-DHE derivatives.10
ethylbenzothiazoline-6-sulfonic)diammonium salt (ABTS)
assay28 and on whole cells by dichlorodihydrofluorescein
(DCF) fluorometric assay.29 Structure/antioxidant activity
relationships of all new compounds are discussed by
comparison with that of hydroxytyrosol 1, methyl carbonate
2,30 and its already known esters.14
MATERIALS AND METHODS
■
Safety. IBX was reported to be explosive under impact or heating
to >200 °C.31 Dess and Martin suggested that the explosive properties
of some samples of IBX were due to the presence of impurities of
potassium bromate utilized during its preparation, that is, the oxidation
of 2-iodobenzoic acid in acidic medium.32 A safe and convenient
preparation of IBX involves the utilization of oxone in water.33
Reagents. All chemicals used were of analytical grade. Solvents and
starting phenols 3 and 4 (Figure 3) were purchased from Sigma
Aldrich (Milan, Italy). 2-Iodoxybenzoic acid (IBX) was prepared
according to the safe procedure reported in the literature.33 IBX−
polystyrene was furnished by Novabiochem (loading factor = 1.1
mmol/g). 3,4-DHE 1, hydroxytyrosyl methyl carbonate 2,26,34 and 3-
(2-hydroxyphenyl)propanol 535 were synthesized as already described
in the literature. Silica gel 60 F254 plates and silica gel 60 were
purchased from Merck (Milan, Italy). 2′,7′-Dichlorodihydrofluorescein
diacetate (DCFH2-DA) was obtained from Molecular Probes (Eugene,
OR, USA). L6 cells from rat skeletal muscle were from the American
Type Culture Collection (Rockville, MD, USA). Dulbecco’s modified
Eagle’s medium (DMEM), antibiotics, and sterile plastic ware for cell
culture were from Flow Laboratory (Irvine, CA, USA). Fetal bovine
serum was from GIBCO (Grand Island, NY, USA).
On these bases and as a part of our research devoted to the
preparation of biologically active compounds inspired by 3,4-
DHE,14,26,27 it seemed interesting to explore SAR in novel
catechols. Therefore, we planned a study focused on the
synthesis and evaluation of the antioxidant activities of a large
panel of novel catecholic compounds and their lipophilic
derivatives (Figure 2). Namely, with respect to 3,4-DHE 1, the
Instrumental Analyses. HPLC analyses were performed on a
Thermo Spectra series P200 apparatus equipped with a Thermo
Hypersil BDS C18 column (250 × 4.6 mm, 5 μm) and an UV detector
selected at λ = 280 nm. Elutions were carried out at 1 mL/min flow
rate by using a 10 min gradient from an H2O/CH3CN mixture (90:10,
v/v) to pure CH3CN. GC-MS analyses were performed on a
Shimadzu VG 70/250S apparatus equipped with a Supelco SLB-5
ms column (30 m, 0.25 mm, and 0.25 μm film thickness). The
analyses were performed using an isothermal temperature profile of
100 °C for 2 min, followed by a 10 °C/min temperature gradient until
280 °C for 15 min. The injector temperature was 280 °C. High-
resolution mass spectrometry (HRMS) analyses were recorded with
1
Micromass Q-TOF micro mass spectrometer (Waters). H and 13C
NMR spectra were recorded in CDCl3 (99.8% in deuterium) and in
CD3OD (99.8% in deuterium) using a 200 MHz nuclear resonance
spectrometer from Bruker. All chemical shifts are expressed in parts
per million (δ scale) and referenced to either the residual protons or
carbon of the solvent. Spectral data of new compounds are reported in
the Supporting Information.
Synthesis of Catechols 6−8. To a solution of the appropriate
substrate 3, 4, or 5 (0.5 mmol) in dimethyl carbonate (DMC, 5.0 mL)
was added IBX−polystyrene (1.2 mmol). The mixture was kept at
room temperature for 1−2 h under magnetic stirring. The reaction was
monitored by thin layer chromatography (TLC) and by GC-MS
analysis. When the substrate disappeared, the heterogeneous reagent
was recovered by filtration and the resulting solution was treated with
sodium dithionite for several minutes. After evaporation of DMC
under reduced pressure, the corresponding catecholic products were
recovered as colorless oils (6, 80%; 7, 68%; 8, 65%).
Synthesis of Methyl Carbonate Catecholic Derivatives 12−
14. Step a. The appropriate substrate 3, 4, or 5 (0.5 mmol) was
dissolved in DMC (5.0 mL); then, 1,8-diazabicicylo[5.4.0]undec-7-ene
(DBU, 0.6 mmol) was added, and the mixture was heated to reflux
temperature for 6−8 h. The reaction was monitored by TLC and GC-
MS. After the disappearance of the substrate, the reaction mixture was
cooled to room temperature and DMC was evaporated under vacuum.
The residue was dissolved in ethyl acetate and washed with 1 N HCl
and saturated NaCl solution. The organic phase was dried over
Na2SO4 and then filtered and concentrated under vacuum. Methyl
Figure 2. Catechol derivatives projected as antioxidants together with
hydroxytyrosol 1 and its methyl carbonate 2.
3-(3,4-dihydroxyphenyl)propanol (3,4-DHP) derivatives 6, 12,
and 18 are higher homologues; 2-(2,3-dihydroxyphenyl)-
ethanol (2,3-DHE) derivatives 7, 13, and 19 show a different
substitution pattern of the catechol moiety; and 3-(2,3-
dihydroxyphenyl)propanol (2,3-DHP) derivatives 8, 14, and
20 exhibit a combination of the two previous structural
modifications.
We describe here a convenient and practical procedure useful
to synthesize the novel catechols reported in Figure 2 from
commercially inexpensive phenols. The antioxidant properties
of all new catechols were evaluated in vitro by 2,2′-azinobis(3-
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Figure 3. Synthesis of catechols 6, 7, and 8; the corresponding methyl carbonates 12, 13, and 14; and fatty acid esters 18a−k, 19a−k, and 20a−k.
carbonates 9, 10, and 11 were isolated as colorless oils in quantitative
yield.
and treated with a saturated solution of NaHCO3. The aqueous phase
was extracted with ethyl acetate. The organic phases were washed with
a saturated solution of NaCl and dried over Na2SO4. After the workup,
esters 18a−k, 19a−k, and 20a−k were obtained in 52−95% yields.
Partition Coefficients Values (Log P). Log P values, simulating
partitioning of catechol derivatives in an n-octanol/water system, have
been calculated by Advance Chemistry Development Chem Sketch
Software V 12.01 (1994−2009 ACD/Lab).
Step b. The appropriate methyl carbonate 9, 10, or 11 (0.5 mmol)
was dissolved in methanol (2.5 mL), and then IBX (0.6 mmol) was
added. The solution was stirred at 0 °C until disappearance of the
substrate. At the end, water and sodium dithionite were added. After
the workup, catecholic compounds 12, 13, and 14 were obtained in 75,
64, and 54% yields.
Hydrophilic−Lipophilic Balance (HLB). HLB has been calcu-
lated using Griffin’s method: HLB = 20 × Mh/MW (where Mh =
molecular mass of hydrophilic portion and MW = molecular weight).
Evaluation of Antioxidant Capacity by ABTS Assay. The
antioxidant capacity of all compounds was measured according to the
method of Pellegrini et al.17 as the quenching capacity toward the
ABTS radical cation, but the analyses were run at room temperature
instead of 30 °C. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (trolox) was used as reference antioxidant. The analyses were
performed in ethanol (0.2% of water) by measuring the absorbance at
λ = 734 nm using a Perkin-Elmer Lambda 14P spectrophotometer.
Each sample (1 mL) was obtained by dilution of the ABTS•+ aqueous
mother solution with ethanol to an absorbance of 0.70 0.20 and
then adding 10 μL aliquots of the ethanol solutions of antioxidants. All
catecholic derivatives were analyzed at four different final concen-
trations ranging from 1 to 10 μM. The extent of color fading was
Synthesis of Lipophilic Esters 18a−k, 19a−k, and 20a−k.
Step a. The appropriate catechol 3, 4, or 5 (0.5 mmol) was dissolved
in DMC (5.0 mL), and then the suitable acyl chloride (0.6 mmol) was
added. The solution was stirred at room temperature for 24 h until
disappearance of the substrate. DMC was evaporated under vacuum to
afford a mixture that was solubilized with ethyl acetate, washed with
saturated NaCl solution, and dried over Na2SO4. Purification on silica
gel of the mixture by eluting with hexane/ethyl acetate gave the
corresponding esters 15a−k, 16a−k, and 17a−k in satisfactory to
good yields (60−90%).
Step b. The appropriate esters 15a−k, 16a−k, or 17a−k (0.1 mmol)
were dissolved in CH3OH (2 mL), and then IBX (0.1 mmol) was
added. The solution was stirred at 0 °C until disappearance of the
substrate. At the end, water and Na2S2O4 were added and the solution
was stirred for 5 min at room temperature. After evaporation of the
solvent under vacuum, the residue was solubilized with ethyl acetate
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measured after 2 min, and four measures were recorded for each
concentration. Solvent blanks were also run. Collected data showed
standard deviation below 5%. The dose−response curves were
expressed as the percentage of absorbance decrease (% ABTS
inhibition) against the amount of antioxidant concentration. Linear
regression was elaborated using Microcal Origin 5.0 software. The
antioxidant capacities were extrapolated at 10 μM concentration and
reported as trolox equivalent antioxidant capacity (TEAC), defined as
the concentration (mmol/L) of trolox having the antioxidant capacity
equivalent to that of a 1.0 mmol/L solution of the substance under
investigation. Results are expressed as the mean standard deviation
and reported as numerical data in the Supporting Information and
plotted in Figures 4 and 5. Statistical analyses were performed by
applying Student’s test. The level of significance was p < 0.005 for all
data.
and ethanol (6%). Student's t test evidenced that none of the data
were statistically different from control. Results are reported in Figure
9.
RESULTS AND DISCUSSION
■
Synthesis of Catecholic Compounds. The synthesis of
hydroxytyrosol-like catechols and their lipophilic esters suffers
from two difficulties. The first problem arises from the intrinsic
high antioxidant activity of the catechol function that results in
quick oxidation in the air, particularly on silica gel and in
alkaline medium, to afford black polymeric matter. Therefore,
the usual synthetic approaches to hydroxytyrosol-like catechols
rule out the oxidative introduction of hydroxyls into the
aromatic ring while favoring the reduction of an appropriate
acyl side chain in a preformed catechol.38 The second problem
concerns the simultaneous presence of alcoholic and catecholic
groups that gives rise to chemoselectivity problems during the
esterification reactions. Reported solutions for chemoselective
acylation at the alcohol function stand either on trans-
esterification,12 lipase-catalyzed,11,13 or catecholic hydroxyl
protection/deprotection procedures.14
Interatomic Distance Calculations. The distances between the
acyl oxygen and the nearest catechol hydroxyl in hydroxytyrosyl
acetate and acetates 18a, 19a, and 20a were calculated after MM2
minimizations using CS Chem3D Ultra, 2001 Cambridge Soft Corp.,
and are reported in Figure 6.
Evaluation of Antioxidant Activity in L6 Myoblast Cells by
DCF Assay. The antioxidant activities in cell culture of all catecholic
derivatives were analyzed using L6 cells derived from rat skeletal
muscle, following the procedure described by Pedersen at al.29 Trolox
was used as reference antioxidant. Incubation with the probe DCFH2−
DA at a final concentration of 10 μM (from a stock solution of 10 mM
in DMSO) was carried out for 30 min in the dark at 37 °C, as reported
by Pallottini et al.36 The assay was carried out in 3 mL of final buffer
containing 200 μL of cell suspension. Intracellular fluorescence was
measured under continuous gentle magnetic stirring at 37 °C using a
Perkin-Elmer (Norwalk, CT, USA) LS 50B luminescence spectrom-
eter. Excitation and emission wavelengths were set at λ = 498 and 530
nm, respectively, using 5 and 10 nm slits for the two light paths.
Cumene hydroperoxide in DMSO was used as radical generator (final
concentration = 300 μM); DMSO at the concentrations used did not
affect the fluorescence signal. Cells were incubated with compounds at
the final concentration of 10 μM for 10 min at 37 °C before the
addition of cumene hydroperoxide; none of the tested compounds
gave rise to fluorescence by itself. The antioxidant activities of all
catechol derivatives were determined by the decrease in the
intracellular DCF fluorescence, reported as ΔF/10 min, and were
calculated relative to the fluorescence change induced by 300 μM
cumene hydroperoxide alone (100%). Data are reported as the mean
standard deviation of at least n = 4 different experiments. Statistical
analyses were carried out by applying Student’s test. Statistical
significance with respect to cumene hydroperoxide (considered 100%)
is always below 0.005% except for compounds 18i−k, 19g, 19j, 19k,
and 20i−k (p < 0.05). Results are expressed as the mean standard
deviation and reported as numerical data in the Supporting
Information and plotted in Figures 7 and 8.
Cell Survival Assessment. Cell survival was assessed by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay.37 Five millimolar stock solutions of catechols 1, 2, 6−8, 12−
14, 18d, 19d, 20d, 18h, 19h, and 20h were prepared and subsequently
diluted in DMEM supplemented with 10% fetal bovine serum, 100
μg/mL streptomycin, and 100 units/mL penicillin to final
concentrations of 50, 20, and 10 μM. L6 cells were plated at 5000/
well in a 96-well plate in 200 μL of complete medium. After 24 h,
DMEM was discarded from the wells, and 100 μL of the test solutions
was added. After 24 h of incubation, 10 μL of MTT solution (5 mg/
mL) was added to each well and further incubated for 4 h. Lysis buffer
was prepared by dissolving 40% (w/v) sodium dodecyl sulfate (SDS)
in deionized water, mixing an equal volume of N,N-dimethylforma-
mide with the SDS solution, and adjusting the pH to 4.7. After
incubation with MTT, 100 μL of lysis buffer was added to each well
and the absorbance read at λ = 570 nm, using 630 nm as the reference
wavelength, on a microplate reader (Camberra Packard, Schwadorf,
Austria). Each compound was tested in triplicate; medium without
antioxidant and medium with 3% EtOH were added in control wells.
Standard deviations were always below 5% except for control (10%)
However, we have recently reported that both 2-
iodoxybenzoic acid (1-hydroxy-1-oxo-1H-1λ5-benz[d][1,2]-
iodoxol-3-one, IBX)39−41 and its supported recyclable mod-
ification (IBX−polystyrene)42 are able to chemo- and
regioselectively introduce a second hydroxyl on tyrosyl esters
[2-(4-hydroxyphenyl)ethanol esters], giving rise to 3,4-DHE
esters.26,27,34 This efficient aromatic hydroxylation occurs
without any further oxidation of the produced catechol function
and takes place under mild and not dry conditions. In addition,
we have also found that the use of DMC as solvent in the
acylation of tyrosol derivatives emphasizes the higher
nucleophilicity of the alcoholic hydroxyl with respect to the
phenolic ones and results in chemoselective esterification at the
alcoholic side chain.26,30
On these bases and in view of the environmentally benign
properties of both IBX and DMC, we planned the synthesis of
the novel catechols 6−8 and their respective methyl carbonates
12−14 and fatty acid esters 18−20 (Figure 2) as depicted in
Figure 3.
Namely, 3-(3,4-dihydroxyphenyl)-1-propanol 6 (3,4-DHP),
2-(2,3-dihydroxyphenyl)-1-ethanol 7 (2,3-DHE), and 3-(2,3-
dihydroxyphenyl)-1-propanol 8 (2,3-DHP) were prepared by
chemoselective IBX−polystyrene aromatic hydroxylation in
DMC from the parent commercial phenols 3, 4, and 5. Methyl
carbonate derivatives 12, 13, and 14 were prepared according
to a two-step procedure (Figure 3). A selective protection of
the alcoholic group of 3, 4, and 5 by DMC/DBU system gave
the intermediate methyl carbonates 9, 10, and 11.30 The
subsequent IBX-mediated hydroxylation afforded the corre-
sponding catecholic methyl carbonates 12, 13, and 14. In the
same manner, acylation of phenols 3, 4, and 5, carried out with
the appropriate acyl chloride in DMC, was highly chemo-
selective at the alcoholic side chain and gave esters 15a−k,
16a−k, and 17a−k. Finally, esters 15a−k, 16a−k, and 17a−k
underwent IBX-mediated hydroxylation to afford the lipophilic
catecholic derivatives 18a−k, 19a−k, and 20a−k. To the best
of our knowledge only compounds 18b, 18h, and 18i are
reported in the literature.10,11 Spectroscopic properties of all
new products are described in the Supporting Information.
Lipophilicity Measurement. To evaluate the effect of
structural modifications on the lipophilicity of all catecholic
compounds, partition coefficients (log P) and HLB values were
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Table 1. Partition Coefficient Values (Log P) and Hydrophilic−Lipophilic Balance (HLB) of Catechols 6, 7, and 8, Methyl
Carbonates 12, 13, and 14, and Fatty Acid Esters 18a−k, 19a−k, and 20a−k
a
b
a
b
a
b
compd
log P
HLB
compd
log P
HLB
compd
log P
HLB
6
1.2500
2.1927
1.8430
2.7550
3.6670
4.5790
5.4910
6.0166
6.8512
7.6858
8.5204
8.2012
7.8820
7
0.7940
1.7580
1.3870
2.2990
3.2110
4.1230
5.0350
5.9470
6.4339
7.2685
8.1031
7.7839
7.4647
8
1.2500
2.1927
1.8430
2.7550
3.6670
4.5790
5.4910
6.0166
6.8512
7.6858
8.5204
8.2012
7.8820
12
13.4
14.4
12.7
11.4
10.3
9.7
13
12.9
14.0
12.2
10.9
9.8
14
13.4
14.4
12.7
11.4
10.3
9.7
18a
18b
18c
18d
18e
18f
18g
18h
18i
18j
18k
19a
19b
19c
19d
19e
19f
19g
19h
19i
19j
19k
20a
20b
20c
20d
20e
20f
20g
20h
20i
20j
20k
8.9
8.6
8.2
8.6
8.0
7.5
8.0
7.4
6.9
7.4
7.0
6.5
7.0
7.0
6.6
7.0
7.0
6.6
7.0
a
b
Calculated by ACD Chem Sketch Software V12.01 (1994−2009 ACD/Lab). Griffin’s method: HLB = 20 × Mh/MW, where Mh = molecular
mass of hydrophilic portion and MW = molecular weight.
calculated. As shown in Table 1 and as expected, methyl
carbonate derivatives 12, 13, and 14 exhibit a higher
lipophilicity than the corresponding catechols 6, 7, and 8 and
even slightly higher than the acetates 18a, 19a, and 20a,
confirming that the introduction of the methyl carbonate
moiety induces an increase of the lipophilicity. A similar trend
is observed for hydroxytyrosol 1 (log P = 0.7940, see Materials
and Methods) and its methyl carbonate derivative 2 (log P =
1.758, HLB = 12.9). Finally, the lipophilicity of catechol
derivatives 18, 19, and 20 increases as the alkyl chain length
increases as well, and this is in agreement with previous data
about hydroxytyrosyl esters.
Figure 4. Radical-scavenging capacity of hydroxytyrosol 1, catechols 6,
7, 8, and the corresponding methyl carbonates 2, 12, 13, and 14
evaluated by the ABTS assay in EtOH. Results are expressed as
millimoles of trolox equivalents. Reported data show standard
deviation below 5%. Statistical significance has p values always below
0.005%.
Antioxidant Capacity by ABTS Assay. The antioxidant
capacity of polyphenolic antioxidants having different degrees
of lipophilicity has been deeply investigated in the past few
years.19,24,43 Porter’s polar paradox hypothesis, which claims
that “polar antioxidants are more effective in non-polar lipids
whereas non-polar antioxidants are more active in water/oil
emulsions”,44 has been proved not to be always valid to explain
the complex behavior of antioxidants having amphiphilic
properties, such as fatty acid esters of catechols and related
compounds. Recent works have demonstrated that the
antioxidant activity of lipophilic catechols is not linearly
correlated with hydrophobicity and is often connected with
the antioxidant location in the biphasic environ-
ment.19,25,43,45−47 The ABTS assay is a widely applied method
for measuring the radical-scavenging ability of antioxidants in a
polar environment. In this context, this assay, performed in
ethanol, in which all catecholic compounds are soluble in the
range of concentrations used, can give interesting insight into
the real effect of growing lipophilicity, avoiding the problems
connected with solubility or micelle formation. The antioxidant
capacities of all synthesized compounds have been measured in
ethanol solution within a concentration range of 1−10 μM.
Linear regression calculations of dose−response curves of the
antioxidant capacity versus concentration of the sample were
performed. Trolox was utilized as reference. The antioxidant
capacity of catechol derivatives was measured and used to
calculate the TEAC. Experimental values for catechols 6−8, the
corresponding methyl carbonates 2 and 12−14, and fatty acid
esters 18−20 are listed in the Supporting Information and are
graphically reported in Figures 4 and 5, respectively.
Figure 5. Radical-scavenging capacity evaluated by the ABTS assay in
EtOH for catechols 6, 7, 8 and the corresponding fatty acid esters
18a−k, 19a−k, and 20a−k. Results are expressed as millimoles of
trolox equivalents. Reported data show standard deviation below 5%.
Statistical significance has p values always below 0.005%.
As depicted in Figure 4, both catechols 6, 7, and 8 and their
methyl carbonates 12, 13, and 14 show a good antioxidant
capacity with TEAC values ranging between 0.6 and 1.1 mM.
As above-discussed, methyl carbonates 12−14 display a higher
lipophilicity than the corresponding catechols 6−8, and the
antioxidant properties are maintained or slightly enhanced by
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the presence of the methyl carbonate moiety. In particular, the
relative position of the two phenolic groups (2,3 or 3,4) does
not affect the antioxidant activities, whereas side-chain
homologation, irrespective of the phenolic position, results in
a small increase of activity.
Antioxidant data of fatty acids esters 18a−k, 19a−k, and
20a−k (Figure 5) can be discussed by taking into account three
parameters: (1) the length (C2−C18) of the acyl chain; (2) the
length (C2 or C3) of the alcohol chain; and (3) the position
(2,3- or 3,4-) of the phenolic hydroxyls.
As to the first point, irrespective of both the acyl chain length
and the catechol position, all tested esters show good
antioxidant capacity, comparable to or higher than that of the
parent catechols 6−8 (TEAC ranging between 0.5 and 1.2 vs
between 0.6 and 0.8). The highest values are shown by
medium-sized chain esters (C8−C14), whereas C16−C18
saturated fatty acid ester derivatives exhibit the lowest values.
Furthermore, esters of unsaturated fatty acids (18j−k, 19j−k,
and 20j−k) display a better radical-scavenging activity than the
corresponding saturated esters 18i, 19i, and 20i.
generally show better TEAC values than the latter. In analogy,
2,3-DHE esters 19a−k exhibit a better antioxidant capacity
than the previously reported hydroxytyrosyl analogues (3,4-
DHE esters). Therefore, both the homologation of the
alcoholic chain and the 2,3-position of catecholic hydroxyls
produce an increase of TEAC values by acting either alone or
together.
The increased antioxidant activity in 2,3-dihydroxy deriva-
tives could be related to the easier formation of intramolecular
hydrogen bonds between the C(2′)−OH and the acyl oxygen
of the ester chain. Indeed, molecular mechanics MM2
minimizations showed (Figure 6) that only in 2,3-dihydroxy
Any attempt to correlate antioxidant capacity with lip-
ophilicity (see log P and HLB calculation, Table 1) failed,
confirming that antioxidant properties do not depend only on
this parameter. These findings are generally coherent with
recent literature,19,20,46 where the antioxidant activity of
catecholic esters or ethers seems to follow a general parabolic
trend with a maximum for medium-chain esters. This behavior,
named “cutoff effect” and previously known only in a biological
environment,48 has been only recently observed in in vitro
antioxidant capacity experiments of amphiphilic antioxidants in
heterogeneous systems such as emulsions19−21 and lip-
osomes.22,23 The qualified hypothesis is that the surfactant
effect of polyphenolic fatty acid esters, which depends on the
length of the fatty acyl chain, could affect the antioxidant
capacity through a different placement of the antioxidant at the
solution/oil droplet interface.19,47
Figure 6. Calculated distances between the acyl oxygen and the
nearest catechol hydroxyl after MM2 minimizations in 3,4-DHE
acetate 18a, 19a, and 20a.
However, this hypothesis cannot be taken into account in the
case of the ABTS assay carried out in a monophasic system,
where the formation of micelles can be ruled out at the used
concentrations. In this case, the drop of antioxidant capacity for
longer acyl chains could be explained in terms of increased
conformational freedom of the ester chain resulting in folded
structures in which catechol hydroxyls are shielded. A similar
behavior has been already reported for hydroxytyrosyl esters,
and the hypothesis of long-chain folding has been supported by
molecular dynamics simulations for hydroxytyrosyl stearate.14
Accordingly, the higher antioxidant capacity measured for oleic
and linoleic esters (18j−k,19j−k, and 20j−k) can derive from
lowering of the acyl chain conformational freedom as a
consequence of the double-bond rigidity.
derivatives 19a and 20a is the distance between the C(2′)−OH
and the acyl oxygen of the ester chain suitable for intra-
molecular H-bonding, whereas, due to the presence of the
proton at C(2′), this is impossible in both 3,4-DHE and 3,4-
DHP acetates.
Therefore, both homologation and hydroxyl position
cooperate to make effective intramolecular H-bonding in either
2,3-DHE esters 19a−k or 2,3-DHP esters 20a−k. Intra-
molecular H-bonding could weaken the phenolic O−H bond,
thus resulting in both an easier hydrogen extraction and better
radical-scavenging properties. On the other hand, these positive
effects can be reduced by the above-mentioned shielding effect
of folded conformations. This could account for the lower
antioxidant activity observed in longer chain esters (>C14).
Antioxidant Activity in Cell Culture. The determination
of the radical-scavenging activity of all catechol derivatives was
performed using a DCF standard assay, based on the
production of dichlorofluorescein radical (DCF), as fluorescent
probe, by oxidation of dichlorofluorescin (DCFH2) in cells. L6
rat muscle cells were used due to their sensitivity to ROS
level.49 Cumene hydroperoxide was used as H2O2 generator to
produce oxidative stress. The experimental data for catechols
6−8, the corresponding methyl carbonates 2, 12−14, and fatty
acid esters 18−20 are available in the Supporting Information
and graphically summarized in Figures 7 and 8, respectively.
The percentage of DCF fluorescence inhibition is reported
versus each tested antioxidant at 10 μM final concentration.
In addition to the above acyl chain-dependent effects, the
consequences of both the alcoholic chain length and the
position of catechol hydroxyls lead to some interesting
structure/activity evaluation. Elongation of the alcoholic side
chain from two to three carbons seems to positively affect the
antioxidant capacity. Indeed, excluding the case of the 2,3-DHE
octanoate 19d, TEAC values of 3,4-DHP esters 18a−k and 2,3-
DHP esters 20a−k are always comparable or higher than those
of both the corresponding previously reported hydroxytyrosyl
esters (3,4-DHE esters)14 and 2,3-DHE esters 19a−k.
The position of the phenolic groups seems to positively affect
the radical-scavenging activity as well. When 2,3-DHP esters
20a−k are compared with 3,4-DHP esters 18a−k, the former
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ascorbyl,24 hydroxytyrosyl,14 and chlorogenate esters.25 For
short to medium (C2−C8) acyl chains (esters 18a−d, 19a−d,
and 20a−d) the antioxidant activity displays values generally
higher than or comparable with that of free catechols 6−8, with
a maximum for C6−C8 derivatives. However, irrespective of
both the alcohol chain length and the phenol hydroxyl position,
esters with chains longer than eight carbons show a cutoff effect
that results in a progressive activity drop from C10 to C12 acyl
chains (esters 18e,f, 19e,f, and 20e,f) and a sharp decrease for
longer chains. At variance with the TEAC measurements, the
decreasing trend is not reverted in the esters of unsaturated
fatty acids (18j,k, 19j,k, and 20j,k).
The causes of the biological “cutoff effect” are still a matter of
study. As already suggested for hydroxytyrosyl esters, it is
possible to suppose that, at a certain level of lipophilicity, the
easy diffusion of esters into the cells could be balanced (C10)
or even made unproductive (C12−C18) by the entrapment
into the plasma membrane caused by the higher affinity of long
acyl chains with the phospholipids or hydrophobic proteins
inside the bilayer. This hypothesis might be confirmed by
recent experiments on membrane fluidity in daidzein alkoxy
derivatives.23
Figure 7. Percentage of DCF fluorescence inhibition in cell culture
experiments for hydroxytyrosol 1, catechols 6, 7, 8, and the
corresponding methyl carbonates 2, 12, 13, and 14. Final
concentration is 10 μM. Standard deviations are below 14%. Statistical
significance with respect to cumene hydroperoxide (considered 100%)
is always below 0.005%.
Cell Survival Assessment. Considering the novelty of the
compounds synthesized, to both rule out any influence of their
cytotoxicity on the antioxidant activity observed and make
possible their use as preservatives in food technology and the
cosmetic industry, MTT measurements were carried out.
Catechols 6, 7, 8, the corresponding methyl carbonates 12,
13, 14, and fatty acid esters 18d, 19d, and 20d (C8 acyl chain)
or 18h, 19h, and 20h (C18 acyl chain) were chosen as
representative compounds to measure cell survival in L6 rat
skeletal muscle cells. Results were compared with those of 3,4-
DHE 1 and its methyl carbonate 2. Cells were treated with
different doses of compounds (10, 20, and 50 μM) for 48 h.
The extent of cell survival was measured by MTT assay (Figure
9). The percentage of cell survival remained high (>90%) at all
doses analyzed, confirming the low cytotoxicity of all of the
compounds examined. Furthermore, the similarity of data
obtained for medium and long acyl chain esters rules out any
effect of cytotoxicity on the decrease of antioxidant activity
observed in long-chain fatty acid esters.
Figure 8. Percentage of DCF fluorescence inhibition in cell culture
experiments for catechols 6, 7, 8 and fatty acid esters 18a−k, 19a−k,
and 20a−k. Final concentration of esters is 10 μM. Standard
deviations are always below 14%. Statistical significance with respect
to cumene hydroperoxide (considered 100%) is below 0.005%. ∗,
compounds 18i−k, 19g, 19j, 19k, and 20i−k have p < 0.05. Under the
same conditions, trolox gives a value of DCF inhibition of 84%.
Hydroxytyrosol 1, catechols 6, 7, and 8, and the
corresponding derivatives 2, 12, 13, and 14 (Figure 7) show
a decrease of fluorescence in comparison with the experiments
with cumene hydroperoxide alone. These data suggest the
penetration of all compounds into cells and the subsequent
quenching of peroxide radicals. The antioxidant activity trend is
similar to that obtained in in vitro experiments (see Figures 4
and 5). Both in catechols 1, 6, 7, and 8 and in the
corresponding carboxymethylated derivatives 2, 12, 13, and
14, the relative position of the catechol hydroxyls seems not to
be related to the antioxidant activity, whereas a small favorable
role is played by elongation of the alcohol chain.
Esters 18a−k, 19a−k, and 20a−k penetrate into the cells as
shown by a sharp decrease of measured fluorescence (Figure 8),
but the structural changes in the different series of esters seem
to have a lower influence in cell culture experiments if
compared with the corresponding in vitro ones. However, also
in the biological environment, 2,3-DHP derivatives 20a−k
show an antioxidant activity similar to or better than that of
both their shorter homologues 2,3-DHE derivatives 19a−k and
the 3,4-DHP derivatives 18a−k.
Figure 9. Effect of hydroxytyrosol 1, catechols 6, 7, and 8, and the
corresponding methyl carbonate esters 2, 12, 13, and 14 and fatty acid
ester derivatives 18d, 19d, 20d (C8 acyl chain) or 18h, 19h, and 20h
(C18 acyl chain) on L6 skeletal muscle cell survival. Cells were
subjected to MTT assay, as detailed under Materials and Methods.
The data are the averages of three experiments. Standard deviations
are always below 5% except for control (10%) and ethanol (6%). None
of the data were statistically different from control.
On the contrary, the relationship between acyl chain length
and antioxidant activity for compounds 18−20 follows a
general and distinctive sigmoid curve, already noted for
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In conclusion, a convenient and efficient procedure has been
developed to synthesize a large panel of novel catechol-based
compounds, structurally related to naturally occurring hydroxy-
tyrosol and its fatty acid esters. The procedure is practical and
efficient and requires neither harsh reaction conditions nor
anhydrous solvents.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR data and analytical data of all new
compounds; TEAC and DCF assay values. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(R.B.) Phone: +39 0761 357452. Fax: +39 0761 357242. E-
mail: berninir@unitus.it (D.T.) Phone: +39 06 57333371. Fax:
+39 06 57333327. E-mail: tofani@uniroma3.it.
■
Funding
We gratefully acknowledge the Italian Ministry for Education,
University and Research, General Management for the
Internationalization of Scientific Research and University of
Tuscia for financial support.
Notes
The authors declare no competing financial interest.
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