Synthesis and antioxidant of activity of alkyl nitroderivatives of hydroxytyrosol

Article abstract of DOI:10.3390/molecules21050656

A series of alkyl nitrohydroxytyrosyl ether derivatives has been synthesized from free hydroxytyrosol (HT), the natural olive oil phenol, in order to increase the assortment of compounds with potential neuroprotective activity in Parkinson's disease. In t

Full text of DOI:10.3390/molecules21050656

molecules
Article
Synthesis and Antioxidant Activity of Alkyl
Nitroderivatives of Hydroxytyrosol
Elena Gallardo 1,2, Rocío Palma-Valdés 1, Beatriz Sarriá 2, Irene Gallardo 1, José P. de la Cruz 3,
Laura Bravo 2, Raquel Mateos 2,* and José L. Espartero 1,
*
1
Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Seville,
41012 Seville, Spain; elegm@us.es (E.G.); xiopalma@yahoo.es (R.P.-V.); irenegalmar@hotmail.com (I.G.)
Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN),
Spanish National Research Council (CSIC), 28040 Madrid, Spain; beasarria@ictan.csic.es (B.S.);
lbravo@ictan.csic.es (L.B.)
2
3
Department of Pharmacology, Faculty of Medicine, University of Malaga, 29071 Malaga, Spain;
jpcruz@uma.es
*
Correspondence: raquel.mateos@ictan.csic.es (R.M.); jles@us.es (J.L.E.);
Tel.: +34-915-445-607 (R.M.); +34-954-556-544 (J.L.E.)
Academic Editor: Jean Jacques Vanden Eynde
Received: 17 March 2016; Accepted: 10 May 2016; Published: 18 May 2016
Abstract: A series of alkyl nitrohydroxytyrosyl ether derivatives has been synthesized from free
hydroxytyrosol (HT), the natural olive oil phenol, in order to increase the assortment of compounds
with potential neuroprotective activity in Parkinson’s disease. In this work, the antioxidant activity
of these novel compounds has been evaluated using Ferric Reducing Antioxidant Power (FRAP),
1
2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and Oxygen Radical
Scavenging Capacity (ORAC) assays compared to that of nitrohydroxytyrosol (NO2HT) and free
HT. New compounds showed variable antioxidant activity depending on the alkyl side chain length;
compounds with short chains (2–4 carbon atoms) maintained or even improved the antioxidant
activity compared to NO2HT and/or HT, whereas those with longer side chains (6–8 carbon atoms)
showed lower activity than NO2HT but higher than HT.
Keywords: nitrocatechol; alkyl nitrohydroxytyrosyl derivatives; hydroxytyrosol; antioxidant activity;
Parkinson’s disease
1. Introduction
Adherence to the Mediterranean Diet has been associated with reduced risk of suffering from
several pathological conditions including cardiovascular, cerebrovascular and neurodegenerative
diseases [
compounds, for these effects due mainly to their potent antioxidant activities [
compounds in olive oil are free hydroxytyrosol (HT, ) (Figure 1) and its secoiridoid derivatives. In this
1
]. Many studies conclude that polyphenols present in olive oil are responsible, among other
2–4]. The main phenolic
1
sense, the European Food Safety Authority (EFSA) has recently released a health claim concerning
phenolic compounds in olive oil and their ability to protect low-density lipoprotein from oxidative
damage [5].
Figure 1. Chemical structures of parent compounds (1) and (2).
Molecules 2016, 21, 656; doi:10.3390/molecules21050656
www.mdpi.com/journal/molecules

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A wide range of associated biological activities have been described for HT, such as the capacity to
scavenge free radical species [ ] as well as to regulate antioxidant enzyme activity [ ]. These properties
6
7
have in common their role against oxidative stress, which underlies neurodegenerative diseases such
as Parkinson’s disease (PD).
However, the highly polar nature of HT reduces its solubility in lipids, and, thus, efforts have
focused on the syntheses of HT derivatives with an enhanced hydrophilic/lipophilic balance [
fact, alkylation of HT with side chains of variable length has given rise to alkyl lipophilic HT ethers [
which have shown enhanced antioxidant activity in comparison to their precursor, if the aliphatic chain
length was up to 6–8 carbon atoms [ 10]. This result was in good accordance with the well-known
8
]. In
9
],
9,
cut-off effect, previously described in other lipophilic derivatives of polyphenols [11,12].
Most efforts in the treatment of PD have been directed at developing novel drugs with protective
activity on the central nervous system. For years, in the clinical treatment of PD, nitrocatechols have
been used in combination with levodopa [13,14], as the nitrocatechol ring seems to play an essential
role in the effective catechol orto-methyl transferase (COMT) inhibition. Moreover, it is known that
reactive oxygen species are closely related to neurodegeneration; therefore, if, in addition, the novel
compounds present antioxidant activity, their therapeutic potential is larger [15].
In view of all the above, new lipophilic nitroderivative compounds derived from HT, acyl
nitrohydroxytyrosyl derivatives, have been synthetized via nitrohydroxytyrosol (NO2HT, 2), and their
antioxidant potential has been tested [16]. Some compounds of this series have been studied, having
shown a significant effect on the COMT activity in both striatal tissue [17] and extracellular dialysate
levels in rats [18].
In the present work, the synthesis of alkyl ethers nitroderivatives of HT (6a–e), with side chain
lengths from one to eight carbon atoms (Scheme 1) is presented, as a second family of lipophilic
nitroderivatives of HT, and their antioxidant activity is evaluated using Reducing Antioxidant Power
(FRAP), 2,21-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and Oxygen
Radical Scavenging Capacity (ORAC) assays.
Scheme 1. Synthetic route for the preparation of the hydroxytyrosol (HT) nitroderivatives (6a–e).
2. Results
In the present work, the synthesis of certain alkyl NO2HT ethers with variable side chain lengths
(6b–e) is presented together with the evaluation of their antioxidant activity, which has been measured
using complementary methods (FRAP, ABTS, and ORAC) and comparatively studied against results
previously published for NO2HT [16] and HT [19].
2.1. Preparation and Characterization of Alkyl Nitrohydroxytyrosyl Ethers (6a–e)
The new compounds were obtained starting from HT recovered from olive oil wastewater
(OOWW) following a four-step process (Scheme 1). Pure HT (
dibenzyl derivative ( ) [20] by reaction with benzyl bromide/potassium carbonate in acetone.
Alkylation of the free alcoholic group with the corresponding alkyl iodides and further hydrogenolytic
cleavage of the protecting Bn groups yielded the alkyl hydroxytyrosyl ethers (5a ), as previously
1) was transformed into its known
3
–
e

Molecules 2016, 21, 656
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described [
9]. The desired alkyl NO2HT ethers (6a
–
e) were finally obtained in good yields by
nitration using sodium nitrite in acetate buffer (Scheme 1) and subsequent purification by column
chromatography. It is noteworthy that initially in this purification step a great loss of product was
observed, possibly due to its retention by the stationary phase used (silica gel), greatly reducing the
chemical yield of the whole process. In order to overcome this point, other inert substances were tested
as a column stationary phase (alumina, C18) without success. However, the addition of a small amount
of formic acid (1%) to the eluent used in each case (different hexane/diethyl ether mixtures) partially
solved this drawback and the yield was increased.
Synthetic NO2HT ethers (6a–e) were characterized by Nuclear Magnetic Resonance (NMR)
and High-Resolution Mass Spectrometry (HR-MS). 1H- and 13C-NMR chemical shifts (Tables 1 and 2
respectively) were unequivocally assigned for each derivative by 2D heteronuclear correlation
experiments (Hetero Single Quanta Correlation -HSQC- and Hetero Multiple Bond Correlation
-HMBC- spectra) and were in good agreement with the proposed structures. In the aromatic proton
region of all the new synthesized compounds (6a
be easily assigned to H4 and H7, by analogy with free NO2HT (
constant in their chemical shift values, confirming that the nitro group has been well incorporated in
–e
) (Table 1), only two signals are observed and could
2) [16]. These signals remained virtually
all compounds at position 8. This pattern contrasts with that of HT (
observed in the aromatic region, corresponding to H8.
1), in which a third resonance is
Table 1. 1H-NMR Data (500.13 MHz, hexadeuterated dimethyl sulfoxide DMSO-d6, 303 K) for
compounds 1, 2 and 6a–e a.
Position
1
2
6a
6b
6c
6d
6e
Phenethyl Unit
3.49 (t)
(J1,2 = 7.2)
3.56 (t)
(J1,2 = 6.8)
3.48 (t)
(J1,2 = 6.6)
3.51 (t)
(J1,2 = 6.7)
3.51 (t)
(J1,2 = 6.7)
3.50 (t)
(J1,2 = 6.5)
3.50 (t)
(J1,2 = 6.7)
1
2
4
2.52 (t)
2.90 (t)
2.99 (t)
2.99 (t)
2.99 (t)
2.99 (t)
2.99 (t)
6.57 (d)
(J4,8 = 2.0)
6.75 (s)
6.75 (s)
6.76 (s)
6.75 (s)
6.75 (s)
6.75 (s)
6.60 (d)
(J7,8 = 8.0)
7
7.43 (s)
7.45 (s)
3.21 (s)
7.44 (s)
7.44 (s)
7.44 (s)
7.44 (s)
8
6.42 (dd)
Alkyl Chain
3.39 (q)
3.33 (t)
3.32 (t)
3.22 (t)
11
(3J = 7.0)
(3J = 6.5)
(3J = 6.6)
(3J = 6.6)
21
31
1.06 (t)
1.42 (m)
1.26 (m)
1.43 (m)
1.22 (m)
1.43 (m)
1.22 (m)
0.84 (t)
41
51
61
71
(3J = 7.0)
0.83 (t)
(3J = 7.0)
0.84 (t)
81
(3J = 7.0)
a
Chemical shifts (δ, ppm) and coupling constants (J, Hz). Symbols: s, singlet; d, doublet; t, triplet; q, quartet;
dd, double doublet; m, multiplet.

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Table 2. 13C-NMR Chemical Shifts (ppm) (125.76 MHz, DMSO-d6, 303 K) for compounds
1
,
2
and 6a–e.
Position
1
2
6a
6b
6c
6d
6e
Phenethyl Unit
1
2
3
4
5
6
7
8
62.5
38.4
61.0
36.0
71.7
32.5
69.6
32.8
69.8
32.8
69.9
32.7
69.9
32.7
130.1
116.2
144.8
143.2
115.3
119.3
127.8
118.5
150.9
143.7
112.0
139.7
127.3
118.4
151.0
143.9
112.0
139.6
127.3
118.3
151.0
143.9
112.0
139.8
127.4
118.4
151.0
143.8
112.0
139.8
127.4
118.4
151.0
143.8
112.0
139.8
127.4
118.3
151.0
143.8
112.0
139.7
Alkyl Chain
11
21
31
41
51
61
71
81
57.8
65.2
15.0
69.6
31.2
18.8
13.7
69.8
29.1
25.3
31.0
22.0
13.8
69.8
29.1
25.6
28.7
28.6
31.2
22.0
13.9
These data are corroborated in Table 2, which shows a shift downfield (∆δ « +9–10 ppm) in the C1
signal of compounds 6a as compared with , due to the presence of the alkyl chain in this position. In
addition, a slight modification of C2 resonance, due to the -shielding effect produced by the presence
of such substituent in compounds 6a e, is also observed. Finally, it is noteworthy that the value for
the signal assigned to C8, which carries the nitro group, is almost constant throughout the series of
–
e
2
β
–
δ
synthesized derivatives ethers, and well away (∆δ « +20 ppm) from that of
1, that does not present the
NO2 group in its structure.
In addition, the study of the molecular ion for compounds 6a–e by HRMS spectrometry allowed
for confirmation of the calculated molecular masses and elemental compositions for each compound,
with a mass deviation ranging between 0.8 and 2.5 ppm (see Materials and Methods).
Lipophilicity of alkyl NO2HT ethers 6a
coefficients and compared to those of their precursors (
highest polar nature and HT ( ) was the second most hydrophilic compound amongst all the evaluated
compounds. Amongst new synthesized compounds (6a ), the lipophilicity was progressively
increasing with the length of the alkylic chain in a linear way.
–e
was expressed by the theoretical (LogPtheor) partition
1
and ) (Table 3). NO2HT ( ) showed the
2
2
1
–
e
Table 3. Log Ptheor, reducing power, evaluated using the FRAP assay, and radical scavenging activity,
determined using ABTS and ORAC assays, of hydroxytyrosol (1), nitrohydroxytyrosol (2), and alkyl
nitrohydroxytyrosyl ethers (6b´e).
Compound
Log Ptheor
FRAP Assay (mM) ABTS Assay (mM)
ORAC Assay (mM)
1.92 ˘ 0.04 f
2.48 ˘ 0.04 b
2.60 ˘ 0.05 a
2.36 ˘ 0.04 c
2.14 ˘ 0.03 d
2.01 ˘ 0.03 e
1 *
2 *
6b
6c
6d
6e
0.96
0.75
1.84
2.75
3.66
4.57
1.39 ˘ 0.05 c
2.51 ˘ 0.04 b
2.68 ˘ 0.03 a
2.52 ˘ 0.04 b
1.37 ˘ 0.05 c
1.01 ˘ 0.03 d
0.84 ˘ 0.02 e
2.13 ˘ 0.07 a
2.13 ˘ 0.03 a
2.00 ˘ 0.04 b
1.44 ˘ 0.04 c
1.26 ˘ 0.05 d
The antioxidant data represent the mean
assays and four determinations for ORAC assay. Results are expressed as mM Trolox equivalent (TEAC, mM).
All values within a column with different letters are significantly different, p < 0.05. FRAP and ABTS values of
HT (1) [21] and NO2HT (2) [16] have been previously published and are included for comparative purposes.
˘
standard deviation for three determinations for FRAP and ABTS
*

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2.2. Antioxidant Activity Evaluation of Alkyl NO2HT Ethers (6b–e)
Results of the reducing power of new compounds (6b
radical scavenging activity using ABTS and ORAC assays in comparison to free HT (
are summarized in Table 3 and Figure 2. Data are expressed as millimolar TEAC (Trolox equivalent).
–
e) obtained using the FRAP assay and the
1) and NO2HT (2)
Figure 2. Antioxidant activity of from HT (
obtained using the FRAP, ABTS, and ORAC assays.
1
), NO2HT (
2
), and alkyl nitrohydroxytyrosyl ethers (6b´e
)
2.2.1. FRAP Assay
The antioxidant activity of the new alkyl nitroderivatives (6b
the alkyl side chain. In comparison to NO2HT ( ), ethyl (6b) and butyl (6c) NO2HT ethers presented the
–e) varied depending on the length of
2
same or higher antioxidant activity, whereas the more lipophilic hexyl (6d) and octyl (6e) derivatives
showed lower activity, specifically that of 6e corresponded to HT (1).
2.2.2. ABTS Assay
The radical scavenging activities of the new compounds showed a similar trend to the reducing
activity. In this sense, among the nitro derivatives, the compounds with shorter side chain, i.e.,
ethyl (6b) and butyl (6c) NO2HT ethers, showed higher antioxidant activity. In contrast, the rest of the
tested nitro compounds (6d and 6e) presented lower antioxidant capacity than NO2HT (
than that of HT (1).
2) but higher
2.2.3. ORAC Assay
The oxygen radical scavenging capacities of alkyl NO2HT ethers were in agreement with the
results from ABTS analysis. The highest activity observed corresponded to ethyl NO2HT ether (6b
followed by the rest of the tested nitro derivatives, but always higher than HT (1).
)
3. Discussion
The results of a previous study on acyl NO2HT derivatives [16], prompted us to synthetize
a new series, alkyl NO2HT derivatives, with small and medium side chain length (6b–e).
According to Trujillo et al. [16], the length of the acyl side chain determined the antioxidant activity of
the compounds, so that shorter chains maintained or even enhanced the antioxidant activity compared
to NO2HT, but derivatives with longer side chain (>8 carbon atoms) showed a significantly lower

Molecules 2016, 21, 656
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antioxidant activity compared to NO2HT, even HT. Similarly, in the present study, the shorter alkyl side
chains (from two to four carbon atoms) enhanced or maintained the antioxidant activity of NO2HT,
whereas longer alkyl side chains (six and eight carbon atoms) showed lower antioxidant activity. All
the synthetized compounds (6b–e) showed higher activity than HT, except for the reducing activity of
6e determined by FRAP assay, which was slightly lower than HT. These results pointed out that the
nitro group in the catecholic ring of HT positively affects the antioxidant activity of the compound, in
accordance with the antioxidant activity of acyl NO2HT derivatives described by Trujillo et al. [16].
The higher antioxidant activity of NO2HTy compared to HT is also in agreement with the stabilization
of the phenoxy radical with electro-donating substituents at positions ortho described by Pokorny [22
]
and Chimi [23]. Furthermore, the decrease in the antioxidant activity associated with the longer
length of the acyl side chain could be due to steric hindrance. However, this disagrees with the
polar paradox that states that polar antioxidants are more active in bulk lipids than their nonpolar
counterparts, whereas nonpolar antioxidants are more effective in oil-in-water emulsions than their
polar homologs [24]. The present results indicate that increased hydrophobicity does not always lead
to increased antioxidant efficacy in non-fat environments, in accordance with previous results observed
with alkyl hydroxytyrosyl ethers [9], and other lipophilic HT derivatives such as nitrohydroxytyrosyl
esters [16], homovanillyl esters [21], as well as chlorogenate esters [25], and rosmarinate esters [26],
among others.
Moreover, a nonlinear association between biological activity and the lipophilic nature of
homologous series of molecules had already been described in different cell lines; ester derivatives
of gallic acid were cytotoxic in L1210 leukemia cells [27], hydroxytyrosyl ethers showed antiplatelet
effects in blood cells from humans [28] and rats [29]. Additionally, cytotoxic activity of hydroxytyrosyl
alkyl ether derivatives against A549 lung cancer cells and MRC5 nonmalignant lung fibroblasts has
been recently described [30]. In all of the aforementioned studies, biological activity increased up
to medium length of the acyl or alkyl side chain, whereas the most lipophilic compounds showed
lower biological activities. This nonlinear phenomenon was coined by Laguerre et al. [25] as the cut-off
effect, and it relates the lower activity of the lipophilic compounds, and therefore higher molecular
weight, than hydrophilic compounds due to their reduced mobility and self-aggregation phenomena
or internalization in the organic phase, having been observed in both biological and physicochemical
systems [31].
When the antioxidant activity of alkyl NO2HT compounds were compared to that of acyl NO2HT
derivatives [16], between compounds with the same side chain length, the alkyl series were slightly
less active than the acyl. However, the influence of the chemical bond nature (acyl vs. alkyl) on the
antioxidant activity was lower than that of the side chain length and, therefore, the lipophilic nature of
the compound. The chemical nature of the side chain substituent is emerging as an important factor
in the biological activity of these kinds of compounds. Accordingly, structure-activity relationship
studies (SAR) have demonstrated their influence on COMT activity pointing out that, although the
nitrocatechol structure was mainly responsible for the “anchoring” of the inhibitor to the enzyme
active site, variations in the side chain substituent exert a profound influence on both the peripheral
selectivity and duration of COMT activity [14]. Indeed, the alkyl nitroderivatives NO2HT ether as
well as its analogous NO2HT acetate have shown the capacity to inhibit brain COMT activity [17
Additionally, all efforts in the treatment of Parkinson s disease are directed towards the development
of novel drugs that offer neuroprotection by having various central nervous system targets [32].
,18].
'
Considering all the above mentioned, it may be gathered that alkyl lipophilic nitroderivatives
with shorter chains present some interesting features as free radical scavenging and reducing activity
to become in (promising) biological compounds with a broad pharmacological potential.

Molecules 2016, 21, 656
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4. Materials and Methods
4.1. Materials
All solvents and reagents were of analytical grade unless stated otherwise. 6-Hydroxy-
2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox), anhydrous sodium sulfate, hexane, tetrahydrofuran
(THF), diethyl ether, potassium persulfate, methanol, acetone, sodium nitrite, hydrogen chloride, iron
(III) chloride hexahydrate, sodium hydrogen phosphate, potassium dihydrogen phosphate, acetic
acid, sodium acetate trihydrate, hexadeuterated dimethyl sulfoxide (DMSO-d6) were from Aldrich
1
(Madrid, Spain). Fluorescein, methylated β-cyclodextrin (RMCD), 2,2 -azinobis(3ethylbenzothiazoline-
6-sulfonic acid) diammonium salt (98%) (ABTS), 2,21-azobis(2amidinopropane) dihydrochloride
(AAPH), and 2,4,6-tri-(2-pyridyl)-1,3,5-triazine (TPTZ) were from Sigma (Madrid, Spain). HT was
recovered with 95% purity from OOWW [33] and further purified by column chromatography.
NMR spectra were recorded on a Bruker AVANCE 500 spectrophotometer (Bruker, Madrid, Spain)
operating at 500.13 MHz (1H) and 125.75 MHz (13C). Chemical shifts are given in parts per million,
with the residual solvent signals (2.49 ppm for 1H and 39.5 ppm for 13C) as references. Samples were
dissolved (10 20 mg/mL) in DMSO-d6, and spectra were recorded at 303 K. High-resolution CI mass
´
spectra (HRMS) were obtained on a Micromass AUTOSPECQ spectrometer (Micromass, Madrid,
Spain). Theoretical values of partition coefficient (Log Ptheor) of the new synthesized compounds were
determined using the ChemBioDraw Ultra software (version14.0) (CambridgeSoft).
4.2. Synthetic Procedures
4.2.1. Synthesis of Hydroxytyrosyl Ethers (5a–e)
Compounds 5a´e were obtained as previously described in Madrona et al. [
bromide (1.4 mL, 11.8 mmol) and potassium carbonate (2.9 g, 20.8 mmol) were added to a solution of
pure HT ( , 0.8 g, 5.2 mmol) in dry acetone (25 mL), and the resulting mixture was heated to reflux
9] Briefly, benzyl
1
for 24 h. The obtained suspension was filtered and concentrated to yield a crude residue, which was
further purified by column chromatography, using a mixture (1:2) of diethyl ether/hexane as eluent, to
yield
in methyl sulfoxide (12 mL) was stirred at room temperature until completion of reaction (TLC). 25 mL
of 3 M HCl was then added and the mixture extracted with CHCl3 (3 25 mL). The organic phase was
washed with 2% NaHSO3 (25 mL) and water (25 mL), dried over Na2SO4, filtered and evaporated.
Compounds 4a were purified by flash column chromatography over silica gel. Finally, palladium
over charcoal (Pd-C) was added to a solution of the corresponding ether (4a , 1 mmol) in THF (20 mL)
3. A mixture of 3 (334 mg, 1 mmol), KOH (335 mg) and the corresponding alkyl iodide (3 mmol)
ˆ
–e
–
e
and the mixture was hydrogenated at 4 bar with magnetic stirring. After 24 h at room temperature, the
catalyst was filtered off and solvent was evaporated in vacuum, yielding the desired compound in
each case (5a–e) that was purified by column chromatography.
4.2.2. General Method of Nitration
The corresponding ether (5a
followed by sodium nitrite (138 mg, 2 mmol). After 30 min at room temperature, the mixture was
extracted with ethyl acetate (6 50 mL) and the combined organic layers were dried over anhydrous
–e, 1 mmol) was added to 0.1 M acetate buffer (pH 3.8) (200 mL)
ˆ
sodium sulfate, filtered and evaporated to give a residue that was further purified by column
chromatography (different hexane/diethyl ether mixtures as eluents) to obtain the corresponding pure
alkyl nitrohydroxytyrosyl ether (6a–e).
Data for methyl nitrohydroxytyrosyl ether (6a): 68% yield. Obtained as a white solid: mp 152–154 ˝C.
NMR (500 MHz, DMSO-d6 δ ppm 10.06 (bs, 2H, 2 phenolic OH’s), 7.45 (s, 1H, H7), 6.75 (s, 1H, H4), 3.48
(t, J = 6.6 Hz, 2H, H1), 3.21 (s, 3H, H11), 2.99 (t, 2H, H2); 13C-NMR (125 MHz, DMSO-d6
δ
ppm 151.0
), 32.5 (C2); HRMS (CI)
m/z calcd for C9H11NO5 [M]+ 213.0637, found 213.0632 (2.5 ppm). Log Ptheor 1.47.
(C5), 143.9 (C6), 139.6 (C8), 127.3 (C3), 118.3 (C4), 112.0 (C7), 71.7 (C1), 57.8 (C1
´

Molecules 2016, 21, 656
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˝
Data for ethyl nitrohydroxytyrosyl ether (6b): 73% yield. Obtained as a white solid: mp 94–96 C 1H-NMR
(500 MHz, DMSO-d6 δ ppm 10.11 (bs, 2H, 2 phenolic OH’s), 7.44 (s, 1H, H7), 6.76 (s, 1H, H4), 3.51 (t,
1
1
J = 6.7 Hz, 2H, H1), 3.39 (c, J = 7.0 Hz, 2H, H1 ), 2.99 (t, J = 6.7 Hz, 2H, H2), 1.06 (t, J = 7.0 Hz, 3H, H2 );
13C-NMR (125 MHz, DMSO-d6
δ ppm 151.0 (C5), 143.9 (C6), 139.8 (C8), 127.3 (C3), 118.3 (C4), 112.0
(C7), 69.6 (C1), 65.2 (C1 ), 32.8 (C2), 15.0 (C2 ); HRMS (CI) m/z calcd for C10H14NO5 [M + H]+ 228.0872,
1
1
found 228.0867 (2.2 ppm). Log Ptheor 1.84.
1
Data for n-butyl nitrohydroxytyrosyl ether (6c): 63% yield. Obtained as a syrup: H-NMR (500 MHz,
DMSO-d6 δ ppm 10.2 (bs, 2H, 2 phenolic OH’s), 7.44 (s, 1H, H7), 6.75 (s, 1H, H4), 3.51 (t, J = 6.8 Hz, 2H,
1
1
H1), 3.33 (t, J = 6.5 Hz, 2H, H1
´
), 2.99 (t, J = 6.8 Hz, 2H, H2), 1.42 (q, 2H, H2 ), 1.26 (m, 2H, H3 ), 0.84 (t,
1
J = 7.0 Hz, 3H, H4 ); 13C-NMR (125 MHz, DMSO-d6 δ ppm 151.0 (C5), 143.8 (C6), 139.8 (C8), 127.4 (C3),
118.4 (C4), 112.0 (C7), 69.8 (C1), 69.6 (C11), 32.8 (C2), 31.2 (C21), 18.8 (C3 ), 13.7 (C41); HRMS (CI) m/z
´
calcd for C12H18NO5 [M + H]+ 256.1185, found 256.1183 (0,8 ppm). Log Ptheor 2.75.
1
Data for n-hexyl nitrohydroxytyrosyl ether (6d): 74% yield. Obtained as a syrup: H-NMR (500 MHz,
DMSO-d6)
δ ppm 10.02 (bs, 2H, 2 phenolic OH’s), 7.44 (s, 1H, H7), 6.75 (s, 1H, H4), 3.50 (t, J = 6.7 Hz,
2H, H1), 3.32 (t, J = 6.6 Hz, 2H, H11), 2.99 (t, 2H, H2); 1.43 (q, 2H, H21), 1.26–1.20 (m, 6H, H31-H51),
0.83 (t, J = 7,0 Hz, 3H, H61); 13C-RMN (125 MHz, DMSO-d6)
δ ppm 151.0 (C5), 143.8 (C6), 139.8 (C8),
127.4 (C3), 118.4 (C4), 112.0 (C7), 69.9 (C1), 69.8 (C11), 32.7 (C2), 31.0 (C41), 29.1 (C21), 25.2 (C31), 22.0
(C51), 13.8 (C61); HRMS (CI) m/z calcd for C14H22NO5 [M + H]+ 284.1498, found 284.1494 (1,4 ppm).
Log Ptheor 3.66.
˝
Data for n-octyl nitrohydroxytyrosyl ether (6e): 66% yield. Obtained as a white solid: mp 84–86 C;
1H-NMR (500 MHz, DMSO-d6)
δ
ppm 10.02 (bs, 2H, 2 phenolic OH’s ), 7.44 (s, 1H, H7),16.75 (s, 1H,
1
H4), 3.50 (t, J = 6.7 Hz, 2H, H1), 3.32 (t, J = 6.6 Hz, 2H, H1 ), 2.99 (t, 2H, H2); 1.43 (q, 2H, H2 ), 1.26–1.20
(m, 10 H, H3 -H7 ), 0.84 (t, J = 7.0 Hz, 3H, H8 ); 13C-RMN (125 MHz, DMSO-d6)
δ ppm 151.0 (C5), 143.8
1
1
1
(C6), 139.7 (C8), 127.4 (C3), 118.3 (C4), 112.0 (C7), 69.9 (C1), 69.8 (C11), 32.7 (C2), 31.2 (C61), 29.1 (C21),
28.7 (C41), 28.6 (C51), 25.6 (C31), 22.0 (C71), 13.9 (C81); HRMS (CI) m/z calcd for C16H26NO5 [M + H]+
312.1811, found 312.1808 (1.0 ppm). Log Ptheor 4.57.
4.3. Antioxidant Activity Determinations
4.3.1. Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay was carried out according to the procedure described by Pulido et al. [34]. The
antioxidant potential of the synthesized compounds was estimated from their ability to reduce the
ferric tripyridyltriazine (TPTZ-Fe(III)) complex to its stable ferrous form (TPTZ-Fe(II)). Briefly, the
FRAP reagent contained 2.5 mL of a 10 mM TPTZ solution in 40 mM HCl, plus 2.5 mL of 20 mM
FeCl3
and warmed to 37 ˝C prior to use. Nine hundred microliters of FRAP reagent was mixed with 90 µL
of distilled water and 30 L of either a standard, methanol (as appropriate reagent blank), or a test
sample (ranging from 50 to 400 M for ethers with short (<6) alkyl chain and from 100 to 1000
for ethers with medium ( 6) alkyl chain). All compounds were dissolved in methanol. Once the
¨
6H2O and 25 mL of 0.3 M acetate buffer to a final pH of 3.6. This reagent was freshly prepared
µ
µ
µM
ě
mixture was shaken, readings at the abso˝rption maximum at 595 nm were taken every 20 s, and the
reaction was monitored up to 30 min at 37 C, using a UV visible Varian (Cary 50 BIO, Varian, Madrid,
´
Spain) spectrophotometer equipped with a thermostatic autocell-holder. The reading at 30 min was
selected in each case for the calculation of FRAP values. Methanol solutions of Trolox were used for
calibration. The FRAP values are expressed as millimolar TEAC (Trolox equivalent,). All analyses
were run in triplicate.
4.3.2. ABTS Assay
The free radical scavenging capacity was measured using the ABTS discoloration method [35
]
with some modifications. The method is based on the capacity of different components to scavenge

Molecules 2016, 21, 656
9 of 12
the ABTS radical cation (ABTS‚+) compared to a standard antioxidant (Trolox). Briefly, ABTS was
dissolved in a 2.45 mM potassium persulfate solution and stored in the dark at room temperature
for 12–16 h, to set a 7 mM concentration of ABTS radical cation (ABTS‚+) solution. The ABTS‚+
stock solution was diluted with methanol to get an absorbance of 0.70
addition of 0.1 mL of sample dissolved in methanol (ranging from 50 to 400
˘
0.02 at 730 nm. After the
M for ethers up to
µ
butyl ‚a+nd from 100 to 500
spectrophotometer. The percentage inhibition of absorbance was plotted against time, and the area
under the curve (0–360 s) was calculated. Methanol solutions of known concentrations of Trolox were
used for calibration. Results are expressed in millimolar TEAC (Trolox equivalent). Each value is the
average of three determinations.
µ
M for octyl), methanol as a blank, or Trolox standard to 3.9 mL of diluted
˝
ABTS solution, absorbance readings were taken every 20 s at 30 C over 6 min, using a UV
´visible
4.3.3. Oxygen Radical Scavenging Capacity (ORAC) Assay
The oxygen radical scavenging capacity was measured by the lipophilic ORAC assay according
to the method developed by Huang et al. [36] with some modifications. The assay is based on the
fluorescence decay of a reference substance (fluorescein) after the addition of a peroxyl radical (AAPH),
which acts as an initiator of the oxidative reaction. Nitroderivatives (6b´e) from 5 to 25
standard (6.25, 12.5, 25, 50, 75 and 100 M) were dissolved in 7% methylated cyclodextrin (RMCD)
in acetone/water (1:1, v/v) solution. Then, 25 L of either Trolox, test sample, or solvent as blank
µM and Trolox
µ
β
µ
was added to a 96-well microplate followed by the addition of 150
µ
L of fluorescein work solution
´5
(8.5 ˆ 10 mM) prepared in 75 mM phosphate buffer (pH 7.4). The microplate reader (Bio-Tek,
Winooski, VT, USA) was programmed to record every 2 min for 120 min at 485 and 528 nm excitation
and emission wavelengths, respectively, the fluorescence after the addition of 30 µL of AAPH (153 mM)
as peroxyl radical generator, which was also prepared in 75 mM phosphate buffer (pH 7.4). Trolox was
used for calibration, and values are expressed as millimolar TEAC (Trolox equivalent). All analyses
were run in quadruplicate.
4.4. Statistical Analysis
Results are expressed as means
˘
standard deviation of three measurements for the ABTS and
FRAP assays and four determinations for the ORAC assay. Results were statistically studied by
one-way analysis of variance (ANOVA) using the SPSS statistical package (version 20.0; SPSS, Inc.,
IBM Madrid, Spain). The level of significance was set at p < 0.05.
5. Conclusions
In conclusion, among the series of alkyl nitrohydroxytyrosyl ether derivatives that have been
synthesized from natural olive oil phenol HT, compounds with short alkyl side chain lengths showed
higher antioxidant activity, determined by FRAP, ABTS and ORAC assays, compared to HT, so that
the longer the length of chain, the lower the antioxidant activity, in accordance with the cut-off effect.
Acknowledgments: This work was supported by Grant P09-AGR-5098 from Junta de Andalucía (Spain). E.G.
thanks Junta de Andalucía for a predoctoral fellowship.
Author Contributions: E.G., R.P-V., I.G. and J.L.E. participated in the synthetic procedures and structural
determinations; E.G., B.S., L.B. and R.M. performed the antioxidant assays; J.P.D. was involved in revising its
content; R.M. and J.L.E. conceived and designed the experiments. E.G., B.S., L.B., R.M. and J.L.E. collaborated in
the preparation of the manuscript.
Conflicts of Interest: The authors declare no competing financial interest.
Abbreviations
The following abbreviations are used in this manuscript:
AAPH:
ABTS:
2,21-azobis(2-amidinopropane) dihydrochloride
2,21-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt

Molecules 2016, 21, 656
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COMT:
DMSO-d6:
EFSA:
FRAP:
HMBC:
HRMS:
HSQC:
HT:
Catechol orto-Methyl Transferase
hexadeuterated dimethyl sulfoxide
European Food Safety Authority
Ferric Reducing Antioxidant Power
Hetero Multiple Bond Correlation
High-Resolution Mass Spectrometry
Hetero Single Quanta Correlation
Hydroxytyrosol
NMR:
Nuclear Magnetic Resonance
Nitrohydroxytyrosol
Olive Oil Wastewaters
Oxygen Radical Scavenging Capacity
Parkinson’s Disease
NO2HT:
OOWW:
ORAC:
PD:
TEAC:
TPTZ:
Trolox Equivalent Antioxidant Capacity
2,4,6-tri-(2-pyridyl)-1,3,5-triazine
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Sample Availability: Samples of the compounds not available.
©
2016 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/).