Synthesis and evaluation of the antioxidant activity of lipophilic phenethyl trifluoroacetate esters by in vitro ABTS, DPPH and in cell-culture DCF assays

Article abstract of DOI:10.3390/molecules23010208

Polyphenols are natural compounds showing a variety of health-promoting effects. Unfortunately, due to low lipid solubility, their applications in the pharmaceutical, food, and cosmetic industries are limited. With the aim of obtaining novel lipophilic derivatives, the present study reports the synthesis of a series of phenethyl trifluoroacetate esters containing up to two hydroxyl groups in the aromatic ring. Experimental logP values confirmed a greater lipophilicity of the novel compounds compared to the parent compounds. The radical scavenging capacity of all phenethyl trifluoroacetate esters was evaluated by in vitro assays (ABTS, DPPH) and in cultured cells (L6 myoblasts and THP-1 leukemic monocytes) using 2,7-dichlorodihydrofluorescein diacetate. These data revealed that the esters showed a good antioxidant effect that was strictly dependent on the grade of hydroxylation of the phenyl ring. The lack of toxicity, evaluated by the MTT assay and proliferation curves, makes these trifluoroacetates attractive derivatives for pharmaceutical, food, and cosmetic applications.

Full text of DOI:10.3390/molecules23010208

molecules
Article
Synthesis and Evaluation of the Antioxidant Activity
of Lipophilic Phenethyl Trifluoroacetate Esters by In
Vitro ABTS, DPPH and in Cell-Culture DCF Assays
Roberta Bernini 1,
*
*
ID , Maurizio Barontini 1, Valentina Cis 1, Isabella Carastro 1,
ID , Rosa Anna Chiodo 2, Paolo Lupattelli 4 and Sandra Incerpi 2
Daniela Tofani 2,3,
ID
1
Department of Agricultural and Forestry Sciences (DAFNE), University of Tuscia, Via S. Camillo de Lellis,
01100 Viterbo, Italy; barontinimaurizio@gmail.com (M.B.); r_vale87@libero.it (V.C.); isabella109@alice.it (I.C.)
Department of Sciences, University Roma Tre, Viale G. Marconi 446, 00146 Rome, Italy;
rosa.anna.chiodo@gmail.com (R.A.C.); sandra.incerpi@uniroma3.it (S.I.)
Centro Interdipartimentale di Servizi per la Didattica della Chimica (CIDSiC), University Roma Tre,
Via della Vasca Navale 79, 00146 Rome, Italy
2
3
4
Department of Sciences, University of Basilicata, Via dell’Ateneo Lucano 10, 85100 Potenza, Italy;
paolo.lupattelli@unibas.it
*
Correspondence: berninir@unitus.it (R.B.); daniela.tofani@uniroma3.it (D.T.);
Tel.: +39-0761-357452 (R.B.); +39-06-57333371 (D.T.)
Received: 29 December 2017; Accepted: 16 January 2018; Published: 19 January 2018
Abstract: Polyphenols are natural compounds showing a variety of health-promoting effects.
Unfortunately, due to low lipid solubility, their applications in the pharmaceutical, food, and cosmetic
industries are limited. With the aim of obtaining novel lipophilic derivatives, the present study reports
the synthesis of a series of phenethyl trifluoroacetate esters containing up to two hydroxyl groups in
the aromatic ring. Experimental logP values confirmed a greater lipophilicity of the novel compounds
compared to the parent compounds. The radical scavenging capacity of all phenethyl trifluoroacetate
esters was evaluated by in vitro assays (ABTS, DPPH) and in cultured cells (L6 myoblasts and THP-1
leukemic monocytes) using 20,70-dichlorodihydrofluorescein diacetate. These data revealed that the
esters showed a good antioxidant effect that was strictly dependent on the grade of hydroxylation of
the phenyl ring. The lack of toxicity, evaluated by the MTT assay and proliferation curves, makes
these trifluoroacetates attractive derivatives for pharmaceutical, food, and cosmetic applications.
Keywords: phenethyl trifluoroacetate esters; tyrosol trifluoroacetate; hydroxytyrosol trifluoroacetate;
lipophilic antioxidants; ABTS assay; DPPH assay; cell culture DCF assay
1. Introduction
In recent years, antioxidants have become of increasing interest for food, cosmetic, and
pharmaceutical applications [1]. In this respect, polyphenols, a large family of compounds found in
plants and plant-derived food and beverages, play an important role due to their well-recognized
health-promoting effects [2,3]. In particular, they act as protective agents against oxidative attacks by
blocking reactive oxygen species (ROS) and reactive nitrogen species (RNS) responsible for oxidative
modifications of relevant biomolecules including polyunsaturated fatty acids and nucleic acids [
Such structural changes have important implications as they can lead to cell membrane modifications,
DNA damage [ ], and the development of serious diseases (cancer, diabetes, atherosclerosis, and
4].
5
neurodegenerative and cardiovascular disorders) [6].
Unfortunately, as phenolic compounds show poor lipid solubility, their applications in
non-aqueous media (oils, fats, and emulsions) are limited, and their bioavailability in the cellular
Molecules 2018, 23, 208; doi:10.3390/molecules23010208
www.mdpi.com/journal/molecules

Molecules 2018, 23, 208
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environment is generally low. These physicochemical properties can be conveniently modified by
increasing the lipophilicity of polyphenols, namely introducing a hydrophobic moiety into the molecule
without changing the phenolic or catecholic group responsible for the antioxidant activity. As an
example, lipophilic polyphenols have been obtained by simple esterification [7–11] or etherification
reactions [12] introducing long alkyl chains. As an alternative, in the literature, it is reported that the
incorporation of fluorine atoms and trifluoromethyl groups into a molecule increases its lipophilicity
and modulates the binding affinity in protein-ligand complexes, thus improving metabolic stability
and exerting a relevant effect on the conformation of the molecule [13]. A significant number of
drugs and bioactive compounds including phenols and flavonoids are halogenated structures [14–17].
For these reasons, in medicinal chemistry, fluorination represents a useful strategy to obtain lipophilic
derivatives endowed of interesting physicochemical and pharmacodynamic properties [18,19].
Based on these literature data and our expertise on the synthesis of phenolic antioxidants [8–11,20,21],
we obtained a series of novel trifluoroacetate esters from phenethyl alcohols containing up to two
phenolic groups in the aromatic ring. Following evaluation of the lipophilicity of all compounds, we
tested their antioxidant capacity by in vitro spectrophotometric analyses (ABTS, DPPH assays) and in
cultured cells (L6 myoblasts from rat skeletal muscle and THP-1 human leukemic monocytes) after
induction of oxidative stress by the radical generator cumene hydroperoxide using the intracellular
fluorescent probe 20,70-dichlorodihydrofluorescein diacetate (DCF). Using the same cell lines, we also
evaluated the capability of the novel compounds to modulate proliferation and cytotoxicity by the
MTT assay. Finally, we estimated cell proliferation by cell counts.
2. Results and Discussion
2.1. Synthesis of Phenethyl Trifluoroacetate Esters
Biologically active compounds such as phenethyl alcohol
1, 2-hydroxyphenethyl alcohol
2,
3-hydroxyphenethyl alcohol , 4-hydroxyphenethyl alcohol (tyrosol)
3
4, 4-hydroxy-3-methoxyphenethyl
alcohol (homovanillyl alcohol)
starting materials (Figure 1).
5, and 3,4-dihydroxyphenethyl alcohol (hydroxytyrosol) 6 were used as
Figure 1. Phenethyl alcohols 1–6.
Phenethyl alcohol
1 is a flavoring molecule found in numerous foods and beverages, in particular
beer and wine [22]. It is also an active ingredient of fragrance and antimicrobial preservatives in
cosmetic formulations, including make-up and skin care products, shampoos, perfumes, and colognes.
The safety of phenethyl alcohol has recently been assessed by the Cosmetic Ingredient Review (CIR)
Expert Panel and the Food and Drug Administration (FDA) [23], which included it in the list of
substances Generally Recognized As Safe (GRAS) [24]. In addition, it shows antimicrobial [25],
antiviral [26] and antitumor effects [27]. The hydroxylated derivatives tyrosol
, and hydroxytyrosol are low-weight molecular phenols present in extra-virgin olive oil and olive
oil by-products [28], well known for their beneficial health effects. Hydroxytyrosol is the most
4, homovanillyl alcohol
5
6
6
relevant compound, characterized by a potent anticancer and antioxidant activity [29]. It has been
widely studied in our laboratories and used for many applications ranging from the synthesis of

Molecules 2018, 23, 208
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novel bioactive compounds [30
applications [32,33].
,31] to its inclusion in PVA-based films for active food packaging
In this study, we aimed to increase the lipophilicity of phenethyl alcohols 1–6 by derivatizing
the alcoholic group with the introduction of a fluorinated moiety. The reactions were performed in
the presence of trifluoroacetic acid used as both solvent and reagent at reflux temperature for 2 h.
The corresponding trifluoroacetate esters 7–12 were obtained in satisfactory to excellent yields and
filtered on silica gel to afford pure samples. This synthetic route was original and, as previously
observed by us in the carboxymethylation and acetylation reactions of tyrosol and hydroxytyrosol [34],
only the alcoholic chain of substrates 1–6 was esterified (Scheme 1). However, extending the reaction
time to 24 h, trifluoroacetylation also occurred on the phenolic groups.
O
O
CF3
OH
1, 7: R1=R2=R3=H
2 8
,
: R1=OH, R2=R3=H
3, 9: R1=R3=H, R2=OH
4 10
R1
R2
R1
R2
CF3COOH
,
: R1=R2=H, R3=OH
T= 70 °C, 2 h
5, 11: R1=H, R2=OCH3, R3=OH
6, 12: R1=H, R2=R3=OH
R3
7 12
R3
-
1-6
Scheme 1. Trifluoroacetylation reaction of phenethyl alcohols 1–6.
2.2. LogP Determination
Lipophilicity is a relevant property to estimate the bioavailability and dispersion of a molecule in
non-aqueous media (oils, fats, and emulsions) and in the cellular environment. Generally, it can be
related to logP, a parameter that evaluates the partition of a molecule between two immiscible solvents
(water and 1-octanol). The experimental logP values of all compounds were measured by UV-Vis
spectrophotometry [35] and are reported in Table 1. As expected, among substrates
alcohol was characterized by the highest lipophilic character, hydroxytyrosol by the lowest value
followed by homovanillyl alcohol , while 2-hydroxyphenethyl alcohol , 3-hydroxyphenethyl alcohol
, and tyrosol showed similar values. As already reported in the literature [35], the lipophilic
1–6, phenethyl
1
6
5
2
3
4
character increased from hydroxytyrosol 6 to homovanillyl alcohol 5 and tyrosol 4.
Table 1. LogP values of compounds 1–12.
Compound
LogP ± SD a
1
2
3
4
5
6
7
8
9
1.18 ± 0.08
0.72 ± 0.07
0.66 ± 0.06
0.69 ± 0.05
0.46 ± 0.02
0.10 ± 0.01
2.38 ± 0.18
2.05 ± 0.16
1.96 ± 0.14
1.98 ± 0.14
1.90 ± 0.12
1.62 ± 0.12
10
11
12
a
Mean of triplicate determinations.

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A similar behavior was observed comparing the logP values of trifluoroacetyl esters
in all cases resulted in greater values than those of the parent compounds
showed the highest logP values, while compounds 11 showed comparable values. The increase of
7–
12, which
1–6. In particular, esters 7 and
8
9–
the logP value of hydroxytyrosol trifluoroacetate 12 compared to hydroxytyrosol 6 was notable.
2.3. ABTS and DPPH Spectrophotometric Assays
Generally, the aim of in vitro radical scavenging experiments is to provide a preliminary appraisal
of the antioxidant capacity of compounds and a premise of the structure/antioxidant activity
relationship. As reported in Figure 2, the ABTS assay disclosed a very low antiradical activity of
phenethyl alcohol
esters 11 12 showed a good antioxidant capacity. Interestingly, in the phenolic derivatives
the aromatic ring position of the hydroxyl group appears to heavily influence the radical scavenging
ability. Indeed, the ortho hydroxyl derivative proved to be more antioxidant than the meta and para
isomers and . A similar behavior was observed regarding the trifluoroacetate ester derivative
which showed a higher antioxidant activity than its isomers and 10. This peculiar effect could be
1 and its trifluoroacetate ester 7. Phenolic derivatives 2, 5, 6 and their corresponding
8,
,
2–4,
2
3
4
8,
9
due to the formation of a hydrogen bond between the phenolic proton and the oxygen atom present
in the alkyl chain, which may promote the generation of the radical species in accordance to already
reported by us [9]. In the catecholic structures, the high hydrogen bond stabilization between the two
phenol groups caused the alcohol hydrogen bond stabilization to be insignificant.
In the DPPH assay, IC50 values were measured and the anti-radical activities (ARA) were
calculated (Table SI1). Comparing with Trolox (IC50 0.221
capacity of phenethyl derivatives and 10 (ARA from 8
and emphasized by the greater differences in ARA data between these compounds and catechols
and 11 12 (ARA from 1.99 to 8.1
−1). Specifically, TEAC values in the ABTS assay almost doubled
µ
M, ARA 4.5
µM
−1), the low antioxidant
M
10−2 µ −1) was confirmed
1
–4
7–
×
10−4 to 4.5
×
5, 6
,
µM
going from mono- to dihydroxyl derivatives, whereas ARA values of catechols were at least 40 times
higher than monohydroxylated compounds in the DPPH assay. Even though no significant increase
of antioxidant capacity was observed in the trifluoroacetate esters, the maintenance of antioxidant
activity after esterification was another important affirmation from the in vitro data of ABTS/DPPH
experiments (Figure 2, Table SI1).
Figure 2. Trolox Equivalent Antioxidant Capacity (TEAC) values, expressed as Trolox mM equivalent,
of phenethyl alcohols 1–6 and the corresponding trifluoroacetyl esters 7–12. Errors were calculated
using Student’s t test. Statistical analysis was performed with one-way ANOVA and Bonferroni
post-test, p < 0.05.

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2.4. ROS Determination
The fluorometric DCF assay is a useful technique to evaluate the antioxidant activity of compounds
in cells exposed to oxidative stress induced by cumene hydroperoxide. The analyses were carried out
in two different cell lines particularly sensitive to radical production: L6 myoblasts from rat skeletal
muscle and THP-1 human leukemic monocytes [8,9].
None of the tested compounds gave rise to fluorescence by itself, but all compounds showed
an antioxidant activity higher than 70% and between 80 and 90% for hydroxytyrosol
corresponding ester 12 (Figure 3, Table SI2). The high level of radical scavenging activity of
and their trifluoroacetate esters 10 12 was relatively easy to predict considering the general trend
observed in the chemical experiments. However, the good antioxidant activity of phenethyl alcohol
and its ester was unexpected. As stated earlier, compound has been extensively studied for its
6 and its
4–6
–
1
7
1
antimicrobial, antiviral, and antitumor activity, which is probably related to a physicochemical action
on cell membrane permeability. However, to the best of our knowledge, its antioxidant activity has
not previously been investigated until now. In this distinctive molecule, the lack of phenolic groups
and maintenance of an antioxidant activity supports the notion that the role of antioxidants in cells
may be related not only to radical scavenging capacity but also to activation of specific metabolic
pathways. Compound
simple neurotransmitter monoamines like dopamine. Therefore, the good antioxidant activity of
and its trifluoroacetyl ester may be derived from its capacity to interact with specific enzymes or
1 is structurally similar to tyrosol 4 and hydroxytyrosol 6, but also to other
1
7
modulators of the antioxidant metabolic pathway, even with just a phenyl ring and alcohol group
conveniently located two carbons away from the aromatic [36]. Further biological investigations in
this area could be of great interest.
120
*
*
100
80
60
40
20
0
L-6
THP-1
* p< 0.001vs all
° p< 0.001 vs 4 L-6
#p< 0.05 vs 6 THP-1
•p< 0.0001 12 THP-1
∇p< 0.05 vs all
∇
°
#
•
Figure 3. Determination of intracellular ROS in L6 and THP-1 cell lines after stimulation of DCF
fluorescence with 200 M cumene hydroperoxide (CH) alone or in presence of phenethyl alcohols
or the corresponding trifluoroacetate esters 12 at 10 M concentration. Data are reported as mean
values SD of five experiments carried out in triplicate. Statistical analysis performed with one-way
ANOVA and Bonferroni post-test, except for 4, 6, and 12 where Student’s t test was used.
µ
1–6
7–
µ
±

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2.5. Compound Toxicity and Cell Proliferation
Considering the good antioxidant activity observed for compounds
5
,
6
and their corresponding
trifluoroacetate derivatives 11 and 12, it was essential to check the potential toxicity on cell lines
and/or their effect on proliferation for cosmetic, food and pharmaceutical applications. Furthermore,
based on the unexpected cellular antioxidant activity of compounds
were also included in the analyses. Employing the MTT colorimetric assay, toxicity curves were
constructed using L6 myoblasts and a range of antioxidant concentrations from 10 to 40 M (Figure 4).
The results revealed a low toxicity for all compounds analyzed, with cell vitality barely diminished
at concentrations higher than 20 M. The comparable behavior of the trifluoroacetate esters and
1 and 7, these two compounds
µ
µ
their respective alcoholic precursors suggests that, at the concentrations used, trifluoroacetic acid
produced by hydrolysis does not cause any remarkable effects in the cells. Cell proliferation
experiments were performed in both cell lines (L6 and THP-1). The results demonstrated a low
interference in cell proliferation by all antioxidants under study, either in the presence or absence of
cumene hydroperoxide. As a representative example, the cell performances for compound
1 and its
corresponding trifluoroacetate derivative are shown in Figure 5. Homovanillyl alcohol showed a
7
5
remarkable peculiarity; it promoted L6 myoblast proliferation but inhibited THP-1 tumor cell growth
(Figures SI1 and SI2).
#
100
75
50
25
0
° °
# p<0.001 vs Ctrl
° p<0.01 at least, vs 1/7 40 μM
Compound 1
Compound 7
•
•
°
100
75
50
25
0
#
#
#
Compound 5
Compound 11
# p< 0.01 ,at least vs Ctrl
° p< 0.05 vs 5 (10μM)
• p< 0.01 at least, vs 5/11 (40μM)
Figure 4. Cont.

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#
100
75
50
25
0
*
°
*
°
°
°
°
Compound 6
Compound 12
*p<0.001 vs 6/12 20μM + CH
# p< 0.001 vs 6 40μM
° p< 0.01 at least, vs CH
Figure 4. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) spectrophotometric
assay of compounds 11 12 at 10 to 40 M concentration in L6 cells in presence or absence
of cumene hydroperoxide (CH, 27.5 M). Data are reported as mean values SD of each compound
1,
5,
6
,
7,
,
µ
µ
±
tested in quintuplicate. Statistical analysis performed with one-way analysis of variance (ANOVA) and
Bonferroni post-test. The analysis was carried out using the Prism 4 statistics program. Differences
were considered significant at p < 0.05.
Compound 1 in THP-1 cells
Compound 1 in L-6 cells
1.0×106
7.5×105
5.0×105
2.5×105
0
1.3×10 6
1.0×10 6
7.5×10 5
5.0×10 5
2.5×10 5
0
Ctrl
Ctrl
CH (200 μM)
1 (10μM)
CH (40μM)
1 (10μM)
°
#
1 (10μM )+ CH (200μM)
1 (10μM) + CH (200 μM)
*
*
°
*
-24
0
24
48
72
-24
0
24
48
72
hours
hours
* p<0.05 at least, vs 1+CH e CH (from24h to 48h)
#P<0.001 vs all
* p< 0.05 vs cumene 24h
°p< 0.01 at least, vs all ( from 48h to 72h)
Compound 7 in THP-1 cells
Compound 7 in L6 cells
1.0×106
7.5×105
5.0×105
2.5×105
0
1.3×10 6
1.0×10 6
7.5×10 5
5.0×10 5
2.5×10 5
0
Ctrl
Ctrl
CH (200 μM)
CH (40μM)
*
#
7 (10μM)
7 (10μM)
7 (10μM) + CH (200 μM)
7 (10μM) +CH (200 μM)
*
*
*
°
-24
0
24
48
72
-24
0
24
48
72
hours
hours
* p< 0.01 at least, vs 7+ CH e CH ( from24h to 48h)
#p< 0.001 at least, vs all
* p< 0.01 at least , vs all (from 48h to 72h)
Figure 5. Effect of compound
1
and its ester 7 (10 µM) on the proliferation of L6 and THP-1 cells in
presence or absence of cumene hydroperoxide (CH, 40
µM in L6 and 200
µM in THP-1 cells). Cell counting
was done with a Neubauer chamber. Data are reported as mean values
duplicate. Statistical analysis performed with one-way ANOVA and Bonferroni post-test.
±
SD of each compound tested in

Molecules 2018, 23, 208
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3. Materials and Methods
3.1. Chemicals
All chemicals (analytical grade), silica gel 60 F254 plates, and silica gel 60 were purchased from
0
0
Sigma-Aldrich (Milan, Italy). 2 ,7 -Dichlorodihydrofluorescein diacetate (DCFH2-DA) was supplied by
Molecular Probes (Eugene, OR, USA). Fresh pure hydroxytyrosol was synthetized in our laboratory as
previously reported [37]. L6 myoblasts from rat skeletal muscle and THP-1 human leukemic monocytes
were purchased from the American Type Culture Collection (Rockville, MD, USA). DMEM (Dulbecco’s
modified Eagle’s medium), RPMI 1640, streptomycin (100 mg/mL), penicillin (100 U/mL), D-glucose,
and sterile plasticware for cell culture were from Falcon (San Diego, CA, USA). Fetal bovine serum
was from GIBCO (Grand Island, NY, USA).
3.2. Instruments
1H, 13C, and 19F NMR spectra were recorded in deuterated solvents (CDCl3, CD3OD 99.8% in
deuterium) using a 400 MHz nuclear resonance spectrometer (Bruker, Munich, Germany). All chemical
shifts were expressed in parts per million (
carbon of the solvent. Coupling constants, J, were reported in Hz. GC-MS analyses were carried out
on a Shimadzu VG 70/250S apparatus equipped with a Supelco SLB column (30 m, 0.25 mm and
δ scale) and referenced to either the residual protons or
™
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. The fluorometric analyses were performed using a VICTOR 3V Perkin Elmer
spectrofluorometer (Milan, Italy).
3.3. Synthesis of Phenethyl Trifluoroacetate Esters: General Procedure
Phenethyl alcohol (0.5 mmol) was dissolved in trifluoroacetic acid at room temperature (2.0 mL).
The solution was kept under stirring at T = 70 ◦C. The reaction was monitored by thin layer
chromatography (eluent: hexane/ethyl acetate = 70/30), and after 2 h, the substrate disappeared.
Then, a saturated solution of NaHCO3 was added until neutral pH. After extraction with ethyl acetate
(3
×
10 mL), the reunited organic phases were washed with a saturated solution of NaCl (2.0 mL),
dried over Na2SO4, and filtered. Finally, the solvent was evaporated by distillation under reduced
pressure. The oily residue was purified by silica gel chromatographic column using hexane/ethyl
acetate = 70/30 as eluent and characterized by NMR and GC-MS analysis.
Phenethyl trifluoroacetate
7
[
38]. Yield: 98%; yellow oil; 1H-NMR (400 MHz, CDCl3)
δ
: 7.40–7.26 (5H, m,
: 156.9
Ph-H), 4.58 (2H, t, J = 8.0 Hz, OCH2), 3.09 (2H, t, J = 8.0 Hz, CH2); 13C-NMR (100 MHz, CDCl3)
δ
(JC-F = 42.0 Hz), 135.7, 128.4, 128.3, 126.6, 114.0 (JC-F = 284.0 Hz), 67.8, 34.1; 19F-NMR (376 MHz, CDCl3)
δ: −75.11; GC-MS: 218 (M+), 120, 107, 91, 77, 69, 51.
2-Hydroxyphenethyl trifluoroacetate
(2H, m, Ph-H), 6.94–6.91 (1H, t, J = 8.0 Hz, Ph-H), 6.78 (1H, d, J = 8.0 Hz, Ph-H), 4.61 (2H, t, J = 8.0 Hz,
8 δ: 7.19–7.15
. Yield: 96%; yellow oil; 1H-NMR (400 MHz, CDCl3)
OCH2), 3.11 (2H, t, J = 8.0 Hz, CH2); 13C-NMR (100 MHz, CDCl3)
δ: 156.9 (JC-F = 42.0 Hz), 153.3, 131.0,
127.9, 122.1, 120.5, 116,4, 114.0 (JC-F = 284.0 Hz), 66.9, 28.4; 19F-NMR (376 MHz, CDCl3)
δ:
−
75.10.
GC-MS: 234 (M+), 120, 107, 91, 77, 69, 51.
3-Hydroxyphenethyl trifluoroacetate
t, J = 8.0 Hz, Ph-H), 6.83–6.74 (3H, m, Ph-H), 4.54 (2H, t, J = 8.0 Hz, OCH2), 3.01 (2H, t, J = 8.0 Hz,
9 δ: 7.22 (2H,
. Yield: 98%; yellow oil; 1H-NMR (400 MHz, CDCl3)
CH2); 13C-NMR (100 MHz, CDCl3)
δ: 157.0 (JC-F = 42.0 Hz), 156.6, 137.8, 129.8, 120.5, 115.8, 114.7, 114.0
(JC-F = 284.0 Hz), 68.3, 34.4; 19F-NMR (376 MHz, CDCl3)
91, 77, 69, 51.
δ:
−
75.09; GC-MS (M+): 234 (M+), 120, 107,
4-Hydroxyphenethyl trifluoroacetate (tyrosol trifluoroacetate) 10. Yield: 98%; yellow oil; 1H-NMR (400 MHz,
CDCl3) : 7.11 (2H, t, J = 8.0 Hz, Ph-H), 6.82 (2H, d, J = 8.0 Hz, Ph-H), 4.52 (2H, t, J = 4.0 Hz, OCH2),
δ

Molecules 2018, 23, 208
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3.00 (3H, t, J = 4.0 Hz, CH2); 13C-NMR (100 MHz, CDCl3)
δ
: 157.0 (JC-F = 42.0 Hz), 154.1, 129.6, 127.9,
115.1, 114.0 (JC-F = 284.0 Hz), 68.1, 33.2; 19F-NMR (376 MHz, CDCl3)
δ:
−
75.10; GC-MS: 234 (M+), 107,
91, 77, 69, 51.
4-Hydroxy-3-methoxyphenethyl trifluoroacetate 11. Yield: 88%; yellow oil; 1H-NMR (400 MHz, CDCl3)
: 6.89 (1H, d, J = 8.0 Hz, Ph-H), 6.73–6.70 (2H, m, Ph-H), 4.53 (2H, t, J = 8.0 Hz, OCH2), 3.90 (3H, s,
δ
OCH3), 3.00 (2H, t, J = 8.0 Hz, CH2); 13C-NMR (100 MHz, CDCl3)
δ: 156.9 (JC-F = 42.0 Hz), 146.6, 144.7,
128.0, 121.6, 114.6, 114.0 (JC-F = 284.0 Hz), 111.4, 68.2, 55.8, 34.3; 19F-NMR (376 MHz, CDCl3)
δ:
−
75.10.
GC-MS: 264 (M+), 150, 137, 122, 107, 91, 77, 69, 51.
3,4-Dihydroxyphenethyl trifluoroacetate (hydroxytyrosol trifluoroacetate) 12. Yield. 85%; yellow oil; 1H-NMR
(400 MHz, CD3COCD3) : 6.83 (1H, d, J = 8.0 Hz, Ph-H), 6.78–6.65 (2H, m, Ph-H), 4.49 (2H, t, J = 8.0 Hz,
δ
OCH2), 2.93 (2H, t, J = 8.0 Hz, CH2); 13C-NMR (100 MHz, CD3COCD3)
δ: 157.0 (JC-F = 42.0 Hz), 143.8,
142.6, 129.1, 121.4, 115.8, 115.6, 114.0 (JC-F = 284.0 Hz), 68.5, 33.9; 19F-NMR (376 MHz, CD3COCD3)
δ: −75.09; GC-MS: 250 (M+), 136, 123, 107, 91, 77, 69, 51.
3.4. Measurement of Partition Coefficient (logP)
The logP value of all compounds was determined as reported in the literature by UV-Vis
spectrophotometry [35]. The partition coefficient (logP) was calculated according to the following
relationship: logP = logAx/(A0
−
Ax). A0 was the maximum of absorbance of a solution 0.15 mM
of each compound dissolved in 1-octanol; Ax is the maximum of absorbance of the organic solution
containing the compound after separation from the phosphate buffer (0.1 M, pH 7.4) solution.
A solution of 1-octanol saturated with the buffer was used as a blank. Experimental logP values
are reported in Table 1.
3.5. ABTS Assay
The antioxidant capacity of compounds
1–
12 was measured according to the method of Pellegrini
et al. [39] but working at room temperature [8,9]. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox) was used as reference. The analyses were carried out in ethanol (0.2% of water) at room
temperature and the absorbance was measured at
= 734 nm. Each sample (1.0 mL) of ABTS+• was
obtained from the aqueous mother solution by dilution with ethanol to an absorbance of 0.70 0.20,
and 10 L of the ethanol solution of the antioxidant was added. All compounds were analyzed at
four different final concentrations ranging from 1 to 10 M. The absorbance was measured after 2 min.
λ
±
µ
µ
Four measurements were recorded for each concentration. Each time blanks of pure solvent were
run. The standard deviations of data were all below 5%. The dose-response curves were expressed as
the percentage of absorbance decrease (ABTS inhibition expressed as percentage) against the amount
of antioxidant concentration. Linear regression was conducted using GraphPad Prism 4.01 (2004)
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 mean
Student’s t test. The level of significance was p < 0.05 for all data.
±
standard deviation. Statistical analyses were performed using the
3.6. DPPH Assay
DPPH assay was performed following the method described by Brand-Williams et al. [40] with
some modifications. A 75
and analyzed at three different final concentrations ranging from 1 to 10
sample solution was added to 0.950 mL of DPPH solution and left in the dark. After 30 min, the
absorbance of samples was measured at = 517 nm. For the blank, 50 L of pure ethanol was used.
µM solution of DPPH in methanol was prepared. Samples were diluted
µ
M. A 50 L volume of each
µ
λ
µ
Four measurements were recorded for each sample. Data collected showed a standard deviation below
5%. Percentages of DPPH inhibition versus antioxidant concentrations were plotted. Linear regressions

Molecules 2018, 23, 208
10 of 14
were conducted using GraphPad Prism 4 software. IC50 values were extrapolated from each graph
as the concentration of sample that decreases by 50% DPPH radical absorbance. Statistical analyses
were performed by the Student’s t test and one-way analysis of variance (ANOVA). Lastly, anti-radical
activity (ARA), as the inverse of IC50, was also calculated.
3.7. Cells in Culture
L6 myoblasts were seeded in 75-cm2 tissue culture flasks and grown in DMEM containing 4.5 g/L
glucose, supplemented with 10% heat-inactivated fetal bovine serum, 100
100 U/mL penicillin, in an atmosphere of 5% CO2 at 37 ◦C. Cells reached confluency after five days
(approximately 6.0
106 cells/flask) and were kept in culture as myoblasts by continuous passages
at preconfluent stages as previously reported [41]. THP-1 human leukemic monocytes were grown
µg/mL streptomycin, and
×
in suspension in RPMI 1640 medium with 10% fetal bovine serum, 100◦
µg/mL streptomycin, and
100 U/mL penicillin, in a humidified atmosphere with 5% CO2 at 37 C [42]. THP-1 monocytes
were passaged twice a week by 1:4 dilutions and re-seeded; cells from passages 7–23 were used for
the experiments.
3.8. Intracellular ROS Determination
The antioxidant activity of each compound was measured by a standard intracellular fluorescent
probe (DCF) assay [8,9,41,42] using L6 myoblasts and THP-1 monocytes exposed to oxidative stress
by the addition of cumene hydroperoxide, a radical generator. Fluorescence was measured over a
10-min period, and the ability of the compounds to eliminate the increased ROS production was
determined. For L6 myoblasts, growing attached to the bottom of the flask, the medium was discarded
and cells were washed twice with 5 mL phosphate buffered saline (PBS) containing 5.0 mM glucose
(PBS-glucose) at 37 ◦C. Cells were gently scraped off with PBS-glucose at 37 ◦C and centrifuged at
1200 rpm for 5 min (approximately 100
in PBS-glucose. Incubation with the probe DCFH2-DA at a final concentration of 10
×
g). The supernatant was discarded and the pellet resuspended
µM (from a stock
◦
solution of 10 mM in DMSO) was carried out for 30 min at 37 C in the dark. At the end of the
incubation, cells were washed twice, centrifuged at 1200 rpm for 5 min, and the final cell pellet was
resuspended in PBS-glucose. Before the experiment was initiated, cells were allowed to recover for
1 h at room temperature in the dark. The THP-1 monocytes, growing in suspension, were centrifuged
at 1200 rpm for 10 min. The supernatant was discarded, and cells were washed twice with 5 mL
PBS-glucose at 37 ◦C to remove the serum. The pellet was successively resuspended in PBS-glucose,
and the probe DCFH2-DA was added at 10
protocols for the two cell types were identical. Intracellular fluorescence was measured at 37 C using
µ
M final concentration. From that point onwards, the
◦
a Perkin Elmer VICTOR 3V spectrofluorometer (excitation at
λ
= 498 nm and emission at
λ = 530 nm).
Cumene hydroperoxide in DMSO was used as the radical generator (200
µM final concentration);
DMSO did not affect the fluorescence signal at the concentrations used. Cells were incubated with
compounds at the final designated concentration for 10 min at 37 ◦C before addition of the radical
generator. The decrease in the intracellular DCF fluorescence relative to the fluorescence change
induced by 200
µM cumene hydroperoxide alone (100%), reported as
∆F/10 min, determined the
antioxidant activities. Data are reported as the mean
experiments carried out in triplicate. One-way ANOVA test (non-parametric) and Bonferroni post-test
(comparing all pairs of columns) were used for the statistical analyses.
±
standard deviation of at least four different
3.9. MTT Assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) spectrophotometric
assay assessed proliferation/cytotoxicity. Compounds
and diluted in either DMEM or RPMI, supplemented as detailed above for the two cell types, to
final concentrations of 10, 20, and 40 M. L6 cells treated with and without cumene hydroperoxide
(27.5 M) [41] were seeded at a density of 10,000/well in 200 L of complete medium in a 96-well plate.
1, 5–7, 11, and 12 (1.0 mM) were tested
µ
µ
µ

Molecules 2018, 23, 208
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After 24 h, the DMEM was discarded and 100
µL of the test solutions was added. Following 24 h of
incubation, 10 µL of MTT solution (5 mg/mL) was added to each well and incubated for a further 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-dimethylformamide with the SDS solution, and adjusting the pH to
4.7 [42]. After incubation with MTT, 100
µL of lysis buffer was added to each well and the absorbance
read at = 590 nm in a microplate reader (Perkin Elmer VICTOR 3V spectrometer). Each compound
λ
was tested in quintuplicate.
3.10. Proliferation Curves
L6 cells were seeded in 60
×
15 mm Petri dishes in 4 mL of the complete growth medium at a
density of approximately 105 cells/well. The following day, the cells were exposed to the selected
compounds
1,
5–
7,
11, and 12 at a final concentration of 10
µM in the absence and presence of cumene
hydroperoxide (40
µM). Cell counts using a Neubauer chamber were made every 24 h up to confluence.
Each time the L6 myoblasts had to be counted, they first underwent a mild trypsinization. THP-1 cells
were seeded in 24-well plates in 1 mL of the growth medium. After 24 h, the cells were exposed to
10 µM of the same compounds in the absence and presence of cumene hydroperoxide (200 µM) and
incubated for an additional 24 h. Cell counts using a Neubauer chamber were made every 24 h up to
confluence. The results are given as the mean ± standard deviation of two different experiments.
3.11. Statistical Analysis
All data obtained from cultured cells were analyzed by one-way ANOVA and Bonferroni post-test.
In some cases, the Student’s t test was applied. Analyses were carried out using the GraphPad Prism
4.1 statistics program. Differences were considered significant at p < 0.05.
4. Conclusions
Novel trifluoroacetate esters
7–12 were prepared in high yield by a simple and efficient procedure
from phenethyl alcohols in the presence of trifluoroacetic acid. Following lipophilicity evaluation,
1–6
the antioxidant activity of all compounds was tested in vitro by ABTS, DPPH assays and in cell lines
by the DFC assay. Cell toxicity and proliferation were also assessed. The experimental data confirmed
the direct correlation between the number of phenolic groups on the aromatic ring and the radical
scavenging ability. In particular, in in vitro experiments, all trifluoroacetate esters showed a similar
antioxidant activity to their corresponding alcoholic precursor, while in cellular experiments, they
demonstrated a weakly enhanced performance due to their higher lipophilicity. The rationale for
the good antioxidant activity of phenethyl alcohol
1 and its trifluoroacetate ester 7 in the cellular
environment lies beyond these considerations and may only be explained in the context of a more
general analysis of the metabolic activity of this class of compounds. Specifically, their effectiveness in
the cellular environment could be due to the attachment of the hydroxyl groups by cytochrome P450,
as previously reported for other molecules [43]. This interesting concept will be the subject of future
work in our laboratories.
Supplementary Materials: The following are available online. Table SI1. DPPH assay of phenethyl alcohols
and trifluoroacetyl esters 12. Table SI2. DCF assay: determination of intracellular ROS after stimulation with
cumene hydroperoxide (CH) in presence of phenethyl alcohols and their trifluoroacetyl esters 12 at 10
concentration on both cell lines L6 and THP-1. Data are reported as mean values SD of five experiments.
Figure SI1. Effect of and 11 (10 M) on the proliferation of L6 and THP-1 cells in the absence and presence
of cumene hydroperoxide (CH: 40 M in L6 and 200 M in THP-1 cells, respectively). Cell counting was done
with a Neubauer Chamber. Figure SI2. Effect of and 12 (10 M) on the proliferation of L6 and THP-1 cells in
M in L6 and 200 M in THP-1 cells, respectively).
1–6
7–
1–
6
7–
µM
±
5
µ
µ
µ
6
µ
µ
the absence and presence of cumene hydroperoxide (CH: 40
Cell counting was done with a Neubauer Chamber.
µ
Author Contributions: R.B. designed the research study and contributed to the data interpretation and to the
manuscript drafting and revisions. M.B., V.C., and I.C. performed the synthesis and characterization of novel
compounds. R.A.C. carried out the antioxidant assays. P.L. and S.I. analyzed the experimental data. D.T. was
involved in the conception of the research study, analyzed the data, and contributed to writing the manuscript.

Molecules 2018, 23, 208
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Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).