Unsaturated fatty alcohol derivatives of olive oil phenolic compounds with potential low-density lipoprotein (LDL) antioxidant and antiobesity properties

Article abstract of DOI:10.1021/jf203814r

A new route for the synthesis of fatty alcohol derivatives of hydroxytyrosol and other olive oil phenolic compounds was developed to allow the preparation of unsaturated derivatives. The biological activity of synthesized compounds was evaluated. Most of the compounds presented a significant antioxidant activity on low-density lipoprotein (LDL) particles. The activity of the tested products was significantly influenced by the number and position of unsaturations as well as modifications on the polar head of the synthesized compounds. Some of them presented modulation of food intake in rats and, due to their molecular similarity with CB₁ endogenous ligands, the endocannabinoid system and PPAR-α were also evaluated as potential targets. The pharmacodynamics could not be totally explained by CB₁ and PPAR-α receptor interactions because only two of the four compounds with biological activity showed a CB₁ activity and all of them presented low PPAR-α affinity, not justifying its whole in vivo activity. The hydroxytyrosol linoleylether (7) increased LDL resistance to oxidation with a capacity similar to that of hydroxytyrosol and was the most active in vivo compound with a hypophagic effect comparable to that of oleoylethanolamine. We consider that this compound could be a good lead compound for future drug development in obesity treatments.

Full text of DOI:10.1021/jf203814r

Article
pubs.acs.org/JAFC
Unsaturated Fatty Alcohol Derivatives of Olive Oil Phenolic
Compounds with Potential Low-Density Lipoprotein (LDL)
Antioxidant and Antiobesity Properties⊗
Bruno Almeida Cotrim,†,‡,▽ Jesus Joglar,*,§ M. Jesus L. Rojas,†,⬢ Juan Manuel Decara del Olmo,⬢
́
́
Manuel Macias-Gonzalez,▽,⊥ Miguel Romero Cuevas,⬢ Montserrat Fito,▽,□ Daniel Munoz-Aguayo,□
́
́
̃
María Isabel Covas Planells,▽,□ Magí Farre,†,▲ Fernando Rodríguez de Fonseca,⬢,▽
́
and Rafael de la Torre*,†,‡,▽
†Human Pharmacology and Clinical Neurosciences Research Group, Neurosciences Research Program,
IMIM-Hospital del Mar Research Institute, Dr. Aiguader 88, Barcelona 08003, Spain
‡Universitat Pompeu Fabra, CEXS-UPF, Dr. Aiguader 88, Barcelona 08003, Spain
§Departamento de Química Biolog
́
ica y Modelizacion
Jordi Girona 18-26, Barcelona 08034, Spain
⬢Fundacion
́ ́ ́
IMABIS, Hospital Carlos Haya de Malaga, Avda. Carlos Haya 82, Pabellon de Gobierno, Malaga 29010, Spain
́
Molecular, Instituto de Química Avanzada de Cataluna (IQAC−CSIC),
̃
́
▽CIBEROBN (CB06/03), Edificio D 1a planta, Hospital Clínico Universitario Santiago de Compostela, Choupana s/n,
Santiago de Compostela 15706, Spain
⊥Servicio de Endocrinologia y Nutricion
Campus Universitario Teatinos, Malaga 29010, Spain
́ ́
, Hospital Clínico Virgen de la Victoria (Fundacion IMABIS),
́
□Cardiovascular Risk and Nutrition Research Group, Inflammation and Cardiovascular Research Program,
IMIM-Hospital del Mar Research Institute, Dr. Aiguader 88, Barcelona 08003, Spain
▲Universitat Auton
́
oma de Barcelona, UDIMAS-UAB, Dr. Aiguader 80, Barcelona 08003, Spain
S
* Supporting Information
ABSTRACT: A new route for the synthesis of fatty alcohol derivatives of hydroxytyrosol and other olive oil phenolic
compounds was developed to allow the preparation of unsaturated derivatives. The biological activity of synthesized compounds
was evaluated. Most of the compounds presented a significant antioxidant activity on low-density lipoprotein (LDL) particles.
The activity of the tested products was significantly influenced by the number and position of unsaturations as well as
modifications on the polar head of the synthesized compounds. Some of them presented modulation of food intake in rats and,
due to their molecular similarity with CB1 endogenous ligands, the endocannabinoid system and PPAR-α were also evaluated as
potential targets. The pharmacodynamics could not be totally explained by CB1 and PPAR-α receptor interactions because only
two of the four compounds with biological activity showed a CB1 activity and all of them presented low PPAR-α affinity, not
justifying its whole in vivo activity. The hydroxytyrosol linoleylether (7) increased LDL resistance to oxidation with a capacity
similar to that of hydroxytyrosol and was the most active in vivo compound with a hypophagic effect comparable to that of
oleoylethanolamine. We consider that this compound could be a good lead compound for future drug development in obesity
treatments.
KEYWORDS: CB1, PPAR-α, obesity, ether, fatty alcohol
INTRODUCTION
■
Low-density lipoprotein (LDL) oxidation is involved in the
earliest events of atherosclerosis,1 although its role in the latter
stages of plaque evolution is nowadays questioned.2 Hydroxy-
tyrosol (2-(3,4-dihydroxyphenyl)ethanol), a phenolic compound
found in olive oil, is a potent in vitro inhibitor of LDL oxida-
tion, being capable of disrupting peroxidative chain reactions.3
Hydroxytyrosol is also a natural metabolite of dopamine, with
which it shares a structural similarity (Figure 1).4,5
Figure 1. Molecular structures of hydroxytyrosol and dopamine.
A series of saturated alkyl chain hydroxytyrosol ethers8 as-
sessed with the rancimat test already displayed good antioxidant
The poor bioavailability of hydroxytyrosol in humans6 and
the interest of the food industry in new lipophilic antioxidants
make the development of long-chain hydroxytyrosol ethers and
esters attractive.7,8
Received: September 20, 2011
Revised: December 12, 2011
Accepted: January 2, 2012
Published: January 2, 2012
© 2012 American Chemical Society
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stabilizer properties for food products, having activity equal to
or even higher than that of hydroxytyrosol.9 They have also
shown a cell protective activity against oxidation and better
bioavailability than hydroxytyrosol.10−12 It is worth noting that
the synthetic route for the hydroxytyrosol ether derivatives
previously described in the literature does not allow the syn-
thesis of unsaturated derivatives because a hydrogenation is
needed in the last step for catechol deprotection. Thus, only
saturated ether derivatives were synthesized.8
Obesity is certainly one of the most important challenges for
public health in the 21st century, because excessive weight is
associated with an increasing risk of mortality, among other
unhealthy effects.13,14 Loss of weight can have a great impact in
different scopes of public health. Bearing in mind the structural
similarity of hydroxytyrosol with dopamine and the structure of
the endocannabinoids N-oleoyldopamine and N-arachidonoyl-
dopamine (Figure 2), we hypothesize that ether derivatives of
MATERIALS AND METHODS
■
Chemistry. All reagents and solvents used were commercially avail-
able and were employed without further purification unless specifically
indicated. The infrared spectrophotometer was a Nicolet Avatar
1
360. H and 13C NMR analyses were carried out with Varian Anova
500 and Varian Mercury 400 spectrometers. The ultracentrifuge used
was a Sigma 3K30. The progress of all reactions was monitored by
TLC on aluminum sheets precoated with silica gel 60 (HF-254,
Merck), film thickness of 0.25 mm. Elemental analyses (C, H, N)
were performed on a Thermo Finnigan Elemental Analyzer Flash 1112
series and were within 0.3% of theoretical values.
Oleyl Iodide (15a). In a round-bottom flask equipped with a
magnetic stirrer, iodine (11.8 mmol), PPh3 (12.6 mmol), imidazole
(26.5 mmol), and THF (20 mL) were added at 4 °C. Oleyl alcohol
(14a) (11.2 mmol) in THF (5 mL) was slowly added, and the reaction
was maintained at room temperature for 48 h. The THF was removed
under reduced pressure, and the product was purified by flash chro-
matography using hexane. The product was obtained as a colorless
oil in 83% yield: IR (KBr), ν = 3003, 2924, 2853, 1464, 722 cm−1;
1H NMR (400 MHz, CDCl3), δ 0.88 (t, J = 6.84 Hz, 3H), 1.19−
1.46 (m, 22H), 1.76−1.88 (m, 2H), 1.97−2.05 (m, 4H), 3.19 (t, J =
7.05 Hz, 2H), 5.29−5.41 (m, 2H); 13C NMR (101 MHz, CDCl3), δ
7.35, 14.13, 22.69, 27.15, 27.21, 28.51, 29.17, 29.31, 29.32, 29.52,
29.69, 29.76, 30.49, 31.90, 33.54, 129.74, 129.99. Anal. Calcd for
C18H35I: C, 57.14; H, 9.32. Found: C, 57.01; H, 9.30.
Linoleyl Iodide (15b). This compound was synthesized as described
above for oleyl iodide but starting with linoleyl alcohol (14b). The
product was obtained as a colorless oil in 71% yield: IR (KBr), ν =
Figure 2. Structures of the endocannabinoids N-oleoyldopamine and
N-arachidonoyldopamine.
1
3008, 2926, 2854, 1464, 1179, 730, 631 cm−1; H NMR (400 MHz,
unsaturated fatty alcohols and hydroxytyrosol could also inter-
act with the cannabinoid system and display some modulatory
activity on food intake, as the role of this receptor on appetite is
well-known.15 Moreover, we hypothesize an affinity of this new
class of compounds for PPAR-α, a nuclear receptor involved in
lipid metabolism, which is often activated by CB1 ligands.16,17
Our team has previously described a group of fatty acid amides
with this dual CB1/PPAR-α interaction. We consider that the
ether derivatives of hydroxytyrosol could have a significant im-
portance in obesity management because appetite and lipid
metabolism could be simultaneously regulated.18
CDCl3), δ 0.88 (t, J = 6.53 Hz, 3H), 1.20−1.47 (m, 16H), 1.74−1.88
(m, 2H) 1.98−2.12 (m, 4H), 2.77 (t, J = 6.36 Hz, 2H), 3.19 (t, J =
7.05 Hz, 2H), 5.27−5.44 (m, 4H); 13C NMR (101 MHz, CDCl3), δ
7.36, 14.09, 22.58, 25.62, 27.18, 28.50, 29.17, 29.30, 29.34, 29.59,
30.48, 31.53, 33.53, 127.88, 128.02, 130.05, 130.21. Anal. Calcd for
C18H33I: C, 57.44; H, 8.84. Found: C, 57.56; H, 8.86.
3,4-(Dimethylmethylenedioxy)phenylacetate Methyl Ester (10).
In a round-bottom flask equipped with a Dean−Stark apparatus and
a magnetic stirrer, 3,4-dihydroxyphenylacetic acid (9) (11.9 mmol),
p-toluenesulfonic acid (1.2 mmol), 2,2-dimethoxypropane (69.2 mmol),
and toluene (30 mL) were added. The reaction was heated under
reflux for 24 h. The organic phase was washed with distilled water (two
times), then brine, and evaporated until dryness. The product was
purified by flash chromatography using ethyl acetate/hexane as eluent.
The product was obtained as a colorless oil in 94% yield: IR (KBr), ν =
A new route for the synthesis of hydroxytyrosol ether deriv-
atives (Figure 3) with natural unsaturated derived fatty alcohols
1
2990, 2952, 1740, 1498, 1255, 981, 838 cm−1; H NMR (400 MHz,
CDCl3), δ 1.66 (s, 6H), 3.52 (s, 2H), 3.69 (s, 3H), 6.64−6.71 (m,
3H); 13C NMR (101 MHz, CDCl3), δ 25.85, 40.80, 52.01, 108.04,
109.44, 117.94, 121.70, 146.51, 147.55, 172.25. Anal. Calcd for
C11H12O4: C, 63.45; H, 5.81. Found: C, 63.56; H, 5.80.
2-(3,4-(Dimethylmethylenedioxy)phenyl)ethanol (11). In a round-
bottom flask equipped with a magnetic stirrer, compound 10 (5.7 mmol),
NaBH4 (13.22 mmol) and THF (100 mL) were added at 4 °C. Iodine
(5.6 mmol) in THF was slowly added, and the reaction was heated to
reflux for 24 h. The THF was removed under reduced pressure and the
residue dissolved in ethyl acetate. The organic phase was washed with
distilled water (two times), then brine, and evaporated until dryness.
The product was purified by flash chromatography using ethyl acetate/
hexane as eluent. The product was obtained as a yellow oil in 72%
yield: IR (KBr), ν = 3370, 2989, 2937, 2872, 1739, 1498, 1445, 1255,
Figure 3. Proposed structures for fatty alcohol ethers. R1 = oleyl,
(Z)-1-(2-phenylethoxy)octadec-9-ene (1), (Z)-1-(2-(4-hydroxyphenyl)-
ethoxy)octadec-9-ene (2), (Z)-1-(2-(3,4-dihydroxyphenyl)ethoxy)octadec-
9-ene (3), (Z)-1-(2-(3,4-methylenedioxyphenyl)ethoxy)octadec-9-ene
(4); R1 = linoleyl (9Z,12Z)-1-(2-phenylethoxy)octadeca-9,12-diene (5),
(9Z,12Z)-1-(2-(4-hydroxyphenyl)ethoxy)octadeca-9,12-diene (6),
(9Z,12Z)-1-(2-(3,4-dihydroxyphenyl)ethoxy)octadeca-9,12-diene
(7), (9Z,12Z)-1-(2-(3,4-methylendioxyphenyl)ethoxy)octadeca-
9,12-diene (8).
1
1046, 981, 839, 807 cm−1; H NMR (500 MHz, CDCl3), δ 1.66 (s,
6H), 2.77 (t, J = 6.47 Hz, 2H), 3.81 (t, J = 6.07 Hz, 2H), 6.62−
6.74 (m, 3H); 13C NMR (101 MHz, CDCl3), δ 25.83, 38.85, 63.75,
108.08, 109.06, 117.71, 121.23, 131.36, 145.99, 147.57. Anal. Calcd for
C11H14O3: C, 68.02; H, 7.27. Found: C, 67.85; H, 7.29.
2-(4-(Methoxymethyleneoxy)phenyl)ethanol (13). In a round-
bottom flask equipped with a magnetic stirrer, tyrosol (12) (0.26 mmol),
MOMCl (3.51 mmol), tetrabutylamonium bromide (0.15 mmol),
CH2Cl2 (6 mL), and aqueous NaOH (30% w/v, 6 mL) were added at
room temperature, and the reaction was stirred for 24 h. The organic
such as oleic and linoleic alcohol is proposed. It is postulated that
newly synthesized compounds may have LDL antioxidant activity
with a potency similar to that of hydroxytyrosol. In addition, this
class of compounds, which shares a structural similarity with
known endocannabinoids and PPAR-α ligands, can modulate
food intake through interaction with those receptors.
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phase was separated and washed with distilled water (two times), then
brine, and evaporated until dryness. The product was purified by flash
chromatography using ethyl acetate/hexane as eluent. The product
was obtained as a white solid in 65% yield: IR (KBr), ν = 3404, 2937,
2827, 1612, 1513, 1233, 1199, 1152, 1110, 1079, 1008, 922, 825 cm−1;
1H NMR (400 MHz, CDCl3), δ 1.79 (s, 1H), 2.80 (t, J = 6.57 Hz,
2H), 3.47 (s, 3H), 3.80 (t, J = 6.60 Hz, 2H), 5.15 (s, 2H), 6.99 (d, J =
8.48 Hz, 2H), 7.14 (d, J = 8.40 Hz, 2H); 13C NMR (101 MHz,
CDCl3), δ 38.24, 55.85, 63.66, 94.39, 116.33, 129.92, 131.77, 155.75.
Anal. Calcd for C10H14O3: C, 65.91; H, 7.74. Found: C, 66.02; H, 7.75.
General Method for the Synthesis of Ethers (1, 4, 5, 8, and 17a−d).
In a round-bottom flask equipped with a magnetic stirrer, phenyleth-
anol derivatives (11, 13, 16a, or 16b) (0.60 mmol), iodoalkenes 15a
or 15b (1.32 mmol), tetrabutylammonium bromide (0.16 mmol),
aqueous KOH (30% w/v, 10 mL), and toluene (10 mL) were added,
and the reaction was heated under reflux for 48 h. The organic phase
was separated, washed with distilled water (two times), then brine, and
evaporated until dryness. The product was purified by flash chroma-
tography using ethyl acetate/hexane as eluent.
(Z)-1-(2-(4-(Methoxymethyleneoxy)phenyl)ethoxy)octadec-9-ene
(17a). This compound was synthesized following the general proce-
dure using 15a and 13 as starting materials. The product was obtained
as a colorless oil in 30.0% yield: IR (KBr), ν = 2925, 2853, 1613, 1511,
1
1465, 1233, 1153, 1114, 1080, 1010, 924 cm−1; H NMR (400 MHz,
CDCl3), δ 0.88 (t, J = 6.45 Hz, 3H), 1.20−1.41 (m, 22H), 1.48−1.68
(m, 2H), 1.93−2.11 (m, 4H), 2.83 (t, J = 7.27 Hz, 2H), 3.42 (t, J =
6.70 Hz, 2H), 3.47 (s, 3H), 3.58 (t, J = 7.34 Hz, 2H), 5.15 (s, 2H),
5.27−5.43 (m, 2H), 6.96 (d, J = 8.56 Hz, 2H), 7.14 (d, J = 8.46 Hz,
2H); 13C NMR (101 MHz, CDCl3), δ 14.11, 22.67, 26.15, 27.19,
29.24, 29.31, 29.45, 29.48, 29.51, 29.71, 29.74, 31.89, 35.49, 55.88,
71.06, 71.95, 94.50, 116.14, 129.82, 129.90, 132.44, 155.60. Anal.
Calcd for C28H48O3: C, 77.72; H, 11.18. Found: C, 77.65; H, 11.20.
(9Z,12Z)-1-(2-(4-(Methoxymethyleneoxy)phenyl)ethoxy)-
octadeca-9,12-diene (17b). This compound was synthesized fol-
lowing the general procedure using compounds 13 and 15b as starting
materials. The product was obtained as a colorless oil in 20.0% yield:
IR (KBr), ν = 3008, 2927, 2854, 1511, 1233, 1153, 1113, 1080, 1010 cm−1;
1H NMR (400 MHz, CDCl3), δ 0.88 (t, J = 6.53 Hz, 3H), 1.18−1.44
(m, 20H), 1.50−1.62 (m, 2H), 1.98−2.11 (m, 4H), 2.77 (t, J =
6.19 Hz, 2H), 2.83 (t, J = 7.26 Hz, 2H), 3.42 (t, J = 6.67 Hz, 2H), 3.47
(s, 3H), 3.58 (t, J = 7.32 Hz, 2H), 5.15 (s, 2H), 5.27−5.46 (m, 4H),
6.96 (d, J = 8.48 Hz, 2H), 7.14 (d, J = 8.23 Hz, 2H); 13C NMR (101 MHz,
CDCl3), δ 14.08, 22.57, 25.61, 26.16, 27.19, 27.22, 29.25, 29.34, 29.45,
29.49, 29.65, 29.72, 31.52, 35.50, 55.91, 71.06, 71.96, 94.51, 116.15,
127.90, 127.93, 129.83, 130.13, 130.18, 132.44, 155.60. Anal. Calcd for
C28H46O3: C, 78.09; H, 10.77. Found: C, 77.97; H, 10.78.
(Z)-1-(2-Phenylethoxy)octadec-9-ene (1). This compound was syn-
thesized following the general procedure using phenylethanol (16a)
and 15a as starting materials. The product was obtained as a colorless
oil in 20.0% yield: IR (KBr), ν = 2924, 2854, 2358, 1699, 1113, 747,
1
639 cm−1; H NMR (400 MHz, CDCl3), δ 0.89 (t, J = 6.09 Hz, 3H),
1.21−1.40 (m, 22H), 1.51−1.64 (m, 2H), 1.95−2.13 (m, 4H), 2.89 (t,
J = 7.28 Hz, 2H), 3.43 (t, J = 6.68 Hz, 2H), 3.63 (t, J = 7.34 Hz, 2H),
5.29−5.42 (m, 2H) 7.15−7.36 (m, 5H); 13C NMR (101 MHz,
CDCl3), δ 14.12, 22.68, 26.15, 27.19, 29.24, 29.32, 29.45, 29.49, 29.52,
29.71, 29.76, 31.90, 36.37, 71.07, 71.79, 126.09, 128.27, 128.87,
129.82, 129.90, 139.03. Anal. Calcd for C26H44O: C, 83.80; H, 11.90.
Found: C, 83.92; H, 11.91.
(Z)-1-(2-(3,4-Methylenedioxyphenyl)ethoxy)octadec-9-ene (4).
This compound was synthesized following the general procedure
using 2-(3,4-methylendioxyphenyl)ethanol (16b) and 15a as starting
materials. The product was obtained as a colorless oil in 35.0% yield:
IR (KBr), ν = 2924, 2854, 1506, 1490, 1246, 1113, 1042, 940, 639 cm−1;
1H NMR (400 MHz, CDCl3), δ 0.88 (t, J = 5.79 Hz, 3H), 1.19−
1.40 (m, 22H), 1.49−1.65 (m, 2H), 1.95−2.07 (m, 4H), 2.80 (t, J =
7.12 Hz, 2H), 3.42 (t, J = 6.63 Hz, 2H), 3.57 (t, J = 7.16 Hz, 2H),
5.27−5.42 (m, 2H), 5.92 (s, 2H), 6.66 (d, J = 7.99 Hz, 1H), 6.70−6.77
(m, 2H); 13C NMR (101 MHz, CDCl3), δ 14.12, 22.68, 26.16, 29.19,
29.25, 29.32, 29.45, 29.49, 29.52, 29.70, 29.76, 31.90, 36.04, 71.08,
71.93, 100.74, 108.10, 109.35, 121.66, 129.83, 129.91, 132.88, 145.81,
147.45. Anal. Calcd. for C27H44O3: C, 77.83; H, 10.64. Found: C,
78.01; H, 10.65.
(Z)-1-(2-(3,4-(Dimethylmethylendioxy)phenyl)ethoxy)octadec-9-
ene (17c). This compound was synthesized following the general
procedure using 15a and 11 as starting materials. The product was
obtained as a colorless oil in 30.0% yield: IR (KBr), ν = 2927, 2854,
−1
1736, 1653, 1498, 1445, 1375, 1253, 1234, 1113 cm ; 1H NMR (400 MHz,
CDCl3), δ 0.88 (t, J = 6.21 Hz, 3H), 1.20−1.39 (m, 22H), 1.47−1.62
(m, 2H), 1.66 (s, 6H), 1.96−2.07 (m, 4H), 2.79 (t, J = 7.28 Hz, 2H),
3.42 (t, J = 6.72 Hz, 2H), 3.57 (t, J = 7.32 Hz, 2H), 5.30−540 (m,
2H), 6.57−6.68 (m, 3H); 13C NMR (101 MHz, CDCl3), δ 14.12,
22.66, 25.84, 26.16, 27.21, 28.98, 29.25, 29.46, 29.49 29.73 31.79,
36.05, 71.06, 72.06, 107.90, 109.14, 117.52, 121.02, 129.84, 129.91,
132.09, 145.69, 147.30. Anal. Calcd for C29H48O3: C, 78.33; H, 10.88.
Found: C, 78.10; H, 10.90.
(9Z,12Z)-1-(2-(3,4-(Dimethylmethylenedioxy)phenyl)ethoxy)-
octadeca-9,12-diene (17d). This compound was synthesized follow-
ing the general procedure using compounds 15b and 11 as starting
materials. The product was obtained as a colorless oil in 53.0% yield:
IR (KBr), ν = 3009, 2927, 2855, 1499, 1253, 1234, 1113, 980 cm−1;
1H NMR (400 MHz, CDCl3), δ 0.89 (t, J = 6.51 Hz, 3H), 1.22−1.42
(m, 20H), 1.50−1.62 (m, 2H), 1.66 (s, 6H), 2.00−2.11 (m, 4H),
2.72−2.85 (m, 4H), 3.42 (t, J = 6.51 Hz, 2H), 3.57 (t, J = 7.31 Hz,
2H) 5.26−5.48 (m, 4H), 6.58−6.67 (m, 3H); 13C NMR (101 MHz,
CDCl3), δ 14.07, 22.57, 25.61, 25.82, 26.16, 26.18, 27.18, 27.21, 29.25,
29.34, 29.45, 29.49, 29.65, 29.71, 29.77, 31.51, 36.04, 70.95, 71.04,
72.05, 107.89, 109.13, 117.49, 121.00, 127.93, 130.11, 130.16, 132.08,
145.69, 147.29. Anal. Calcd for C29H48O3: C, 78.33; H, 10.88. Found:
C, 78.52; H, 10.85.
(Z)-1-(2-(4-Hydroxyphenyl)ethoxy)octadec-9-ene (2). In a round-
bottom flask equipped with a magnetic stirrer, compound 17a (0.17 mmol),
isopropanol (5 mL), and aqueous HCl 6 N (2 mL) were added at
room temperature, and the reaction was stirred for 24 h. The organic
phase was separated and washed with distilled water (two times), then
brine, and evaporated until dryness. The product was purified by flash
chromatography using ethyl acetate/hexane as eluent. The product
was obtained as a colorless oil in a 97.8% yield: IR (KBr), ν = 3373,
2924, 2854, 1614, 1516, 1464, 1236, 1190, 829 cm−1; 1H NMR
(400 MHz, CDCl3), δ 0.88 (t, J = 6.13 Hz, 3H), 1.20−1.42 (m, 22H),
1.50−1.65 (m, 2H), 1.94−2.09 (m, 4H), 2.82 (t, J = 7.24 Hz, 2H),
3.45 (t, J = 6.75 Hz, 2H), 3.60 (t, J = 7.32 Hz, 2H), 5.29−5.38 (m,
2H), 5.40 (bs, 1H), 6.73 (d, J = 8.45 Hz, 2H), 7.07 (d, J = 8.32 Hz,
2H); 13C NMR (101 MHz, CDCl3), δ 14.12, 22.67, 26.10, 27.19,
29.23, 29.31, 29.42, 29.47, 29.51, 29.60, 29.74, 29.75, 31.89, 35.33,
(9Z,12Z)-1-(2-Phenylethoxy)octadeca-9,12-diene (5). This com-
pound was synthesized following the general procedure using 16a and
15b as starting materials. The product was obtained as a colorless oil in
27.0% yield: IR (KBr), ν = 3009, 2927, 2855, 2361, 1738, 1455, 1115,
1
698 cm−1; H NMR (400 MHz, CDCl3), δ 0.89 (t, J = 6.80 Hz, 3H),
1.15−1.44 (m, 20H), 1.49−1.67 (m, 2H), 1.99−2.10 (m, 4H), 2.78 (t,
J = 6.10 Hz, 2H), 2.89 (t, J = 7.30 Hz, 2H), 3.34−3.52 (m, 2H), 3.62
(t, J = 7.30 Hz, 2H), 5.27−5.46 (m, 4H), 7.14−7.41 (m, 5H); 13C
NMR (101 MHz, CDCl3), δ 14.06, 22.57, 25.61, 26.17, 27.19, 27.22,
29.25, 29.34, 29.44, 29.49, 29.66, 29.71, 31.52, 36.37, 71.07, 71.80,
126.10, 127.90, 127.94, 128.28, 128.87, 130.13, 130.18, 139.03. Anal.
Calcd for C26H42O: C, 84.26; H, 11.42. Found: C, 84.08; H, 11.41.
(9Z,12Z)-1-(2-(3,4-Methylenedioxyphenyl)ethoxy)octadeca-9,12-
diene (8). This compound was synthesized following the general pro-
cedure using compounds 16b and 15b as starting materials. The prod-
uct was obtained as a colorless oil in 23.0% yield: IR (KBr), ν = 2927,
2855, 1741, 1489, 1246, 1113, 1043 cm−1; 1H NMR (500 MHz, CDCl3),
δ 0.89 (t, J = 6.42 Hz, 3H), 1.22−1.42 (m, 20H), 1.49−1.64 (m, 2H),
198−2.12 (m, 2H), 2.72−2.85 (m, 4H), 3.42 (t, J = 6.65 Hz, 2H), 3.57
(t, J = 7.21 Hz, 2H), 5.26−5.46 (m, 4H), 5.92 (s, 2H), 6.62−6.78 (m,
3H); 13C NMR (101 MHz, CDCl3), δ 14.08, 22.57, 25.61, 26.15,
27.19, 28.58, 29.25, 29.34, 29.45, 29.49, 29.66, 31.52, 36.04, 38.34,
71.08, 71.93, 100.74, 108.10, 109.35, 121.66, 127.90, 127.94, 130.13,
130.18, 132.88, 145.81, 147.57. Anal. Calcd for C27H42O3: C, 78.21; H,
10.21. Found: C, 78.35; H, 10.22.
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71.12, 72.06, 115.16, 129.83, 129.93, 130.80, 154.05. Anal. Calcd for
C26H44O2: C, 80.35; H, 11.41. Found: C, 80.19; H, 11.43.
Sephadex column (Pharmacia, Uppsala, Sweden), with 2.7 mL of
phosphate-buffered saline (PBS) 0.01 mol, pH 7.4, under gravity feed
at 4 °C. Apolipoprotein B100 content was determined by immuno-
turbidimetry (ABX Diagnostics, Montpellier, France).
(Z)-1-(2-(3,4-Dihydroxyphenyl)ethoxy)octadec-9-ene (3). In a
round-bottom flask equipped with a magnetic stirrer, compound 17c
(0.26 mmol) and aqueous HCl 6 N (5 mL) were added, and the
reaction was heated under reflux for 24 h. The organic phase was
separated and washed with distilled water (two times), then brine, and
evaporated until dryness. The product was purified by flash chromatog-
raphy using ethyl acetate/hexane as eluent. The product was obtained
as a colorless oil in 50.7% yield: IR (KBr), ν = 3394, 2922, 2854, 1606,
1520, 1465, 1446, 1279, 1193, 1113, 1092 cm−1; 1H NMR (500 MHz,
CDCl3), δ 0.88 (t, J = 6.91 Hz, 3H), 1.18−1.38 (m, 20H), 1.51−
1.62 (m, 2H), 1.97−2.04 (m, 4H), 2.76 (t, J = 7.14 Hz, 2H), 3.46 (t,
J = 6.83 Hz, 2H), 3.62 (t, J = 7.16 Hz, 2H), 5.30−5.41 (m, 2H), 5.63
(bs, 1H), 5.86 (bs, 1H), 6.59−6.63 (m, 1H), 6.68 (d, J = 1.66 Hz, 1H),
6.72 (d, J = 8.03 Hz, 1H); 13C NMR (101 MHz, CDCl3), δ 14.12,
22.68, 26.07, 27.19, 29.23, 29.31, 29.42, 29.48, 29.51, 29.74, 29.75,
31.89, 35.41, 71.18, 71.93, 115.20, 115.85, 121.00, 129.82, 129.93,
131.57, 142.03, 143.58. Anal. Calcd for C26H44O3: C, 77.18; H, 10.96.
Found: C, 77.15; H, 10.94.
(9Z,12Z)-1-(2-(4-Hydroxyphenyl)ethoxy)octadeca-9,12-diene (6).
This compound was synthesized as already described for compound 2
starting with 17b. The product was obtained as a colorless oil in 80.0%
yield: IR (KBr), ν = 3361, 2927, 2855, 1516, 1111, 829 cm−1; 1H NMR
(400 MHz, CDCl3), δ 0.89 (t, J = 6.30 Hz, 3H), 1.20−1.42 (m, 20H),
1.51−1.63 (m, 2H), 1.99−2.09 (m, 4H), 2.73−2.87 (m, 4H), 3.44 (t,
J = 6.75 Hz, 2H), 3.59 (t, J = 7.33 Hz, 2H), 5.26−5.46 (m, Hz, 4H),
6.74 (d, J = 8.46 Hz, 2H), 7.07 (d, J = 8.43 Hz, 2H); 13C NMR
(101 MHz, CDCl3), δ 14.08, 22.56, 25.60, 26.10, 27.18, 27.21, 29.23,
29.33, 29.42, 29.47, 29.62, 29.64, 31.51, 35.35, 71.09, 72.06, 115.15,
127.89, 127.93, 129.93, 130.12, 130.18, 130.83,154.05. Anal. Calcd. for
C26H42O2: C, 80.77; H, 10.95. Found: C, 80.90; H, 10.94.
Diene Conjugates Monitoring. Using a 96-well ELISA plate, di-
alyzed LDL (final concentration = 0.06 g of Apo-B/L) in PBS at a final
volume of 150 μL was incubated with 10 μL of methanol in the
presence or absence (control) of the dissolved analytes. Afterward,
10 μL of a 100 μM cupric sulfate (final concentration = 0.67 μmol)
solution was added to a 96-well half-area flat-bottom UV-transparent
microplate (Corning). To minimize evaporation during prolonged
incubation time, 10 μL of mineral oil (Sigma-Aldrich) was layered over
the reaction mixture and the plate was covered with adhesive optical
transparent film. Absorbance at 234 nm was continuously monitored
at 15 min intervals for 24 h at 36 °C in an Infinite M200 lector (Tecan
Iberica, Mannedorf, Switzerland).
̈
Controls and samples with the concentration of all tested com-
pounds at 0.5, 1, and 3 μM were evaluated in the same run in dupli-
cate, and each experiment was repeated three times. For data presen-
tation, the x-axis value, corresponding to the intercept of the
propagation phase tangent with the extrapolated line for the slow
propagation reaction, was calculated (lag time).
Binding and PPAR-α Affinity Assay. Rat Cerebellum and
Cortex Membranes Homogenization. The rats were anesthetized
with Dolethal (pentobarbital) and killed with a guillotine. The cortex
and cerebellum were extracted and stored separately in dry ice.
The cerebellum and cortex were homogenized with a buffer of Tris
50 mmol, pH 7.4, using an Ultraturrax. The homogenate was centri-
fuged at 25000g for 15 min at 4 °C. The supernatant was discarded.
The pellet was again washed with 5 mL of buffer and centrifuged.
The supernatant was again discarded. The protein concentration of the
pellet was measured using the Bradford test.
Binding Assay. The binding assay was performed using the labeled
CB1 antagonist [3H]-SR141716. In each tube were added 450 μL of
buffer A (50 mmol of Tris, pH 7.4, with 0.5% bovine serum albumin
(BSA)), 100−200 μg of rat brain membranes (cerebellum), the diluted
product, and the tracer [3H]-SR141716. The mixture was incubated
with shaking at 37 °C for 60 min, and the reaction was stopped with
1 mL of buffer A. The resultant mixture was centrifuged at 5000 rpm
for 5 min. The supernatant was discarded and the pellet washed with
1 mL of buffer A and centrifuged, and again the supernatant was dis-
carded. Scintillation liquid was added, and the samples were read
at a beta scintillator (liquid scintillation analyzer, Tri-Carb 2100 TR,
Packard, a Packard Bioscience Company). All products were diluted in
buffer B (50 mmol of Tris, pH 7.4, with 0.5% BSA and 0.3% DMSO)
at the concentrations of 10−5, 10−6, 10−7, 10−8, 10−9, 10−10, and 10−11 M.
All data points were performed in triplicate.
PPAR-α Affinity Assay. (a) Biology. The compounds reported in this
study were first evaluated for in vivo Reporter Gene Assay to assess
which compounds could induce interaction between PPAR-α and the
coactivator (SRC-1) into MCF-7 cells.
(b) Drugs. Oleoylethanolamide (OEA), GW7647, and WY14643
were purchased from Tocris Biosciences (Cookson Ltd., Bristol, U.K.).
For in vitro cell culture experiments, all compounds were dissolved
and diluted in DMSO (Sigma-Aldrich Spain).
(9Z,12Z)-1-(2-(3,4-Dihydroxyphenyl)ethoxy)octadeca-9,12-diene
(7). This compound was synthesized as already described for com-
pound 3 starting with 17d. The product was obtained as a colorless oil
in 27.0% yield: IR (KBr), ν = 3386, 3009, 2935, 2856, 1606, 1520,
1446, 1375, 1279, 1113, 811, 723 cm−1; 1H NMR (500 MHz, CDCl3),
δ 0.89 (t, J = 6.96 Hz, 3H), 1.22−1.42 (m, 20H), 1.50−1.62 (m, 2H),
1.96−2.12 (m, 4H), 2.72−2.82 (m, 4H), 3.42 (t, J = 6.71 Hz, 2H),
3.58 (t, J = 7.21 Hz, 2H), 4.99 (bs, 1H), 5.13 (bs, 1H), 5.27−5.45 (m,
4H), 6.61−6.70 (m, 1H), 6.71−6.81 (m, 2H); 13C NMR (101 MHz,
CDCl3), δ 14.07, 22.56, 25.60, 26.04, 27.17, 27.20, 29.23, 29.33, 29.40,
29.47, 29.63, 31.50, 35.37, 71.17, 71.93, 115.19, 115.84, 120.94,
127.89, 127.95, 130.12, 130.19, 131.44, 142.09, 143.63. Anal. Calcd for
C26H42O3: C, 77.56; H, 10.51. Found: C, 77.50; H, 10.52.
Biology. Feeding Experiments. Feeding experiments were car-
ried out using Wistar male rats with a 200−450 g weight range.
Animals were housed in individual cages in a room with controlled
temperature (23 °C) and relative humidity (50%) with a 12/12 h light
and dark cycle. Water and food were available ad libitum except in
specific experimental proceedings. The animals were handled twice in
the days prior to the experimental sessions. All products were dis-
solved in a mixture of DMSO 5%/Tween 60 5%/saline 90% and ad-
ministered intraperitoneally. The experiments performed in this study
are in compliance with Spanish regulations concerning the protection
of experimental animals (Real Decreto 1201/2005, October 21, 2005,
BOE no. 252), as well as with the European Communities Council
Directive of 24 November 1986 (86/609/EEC).
The feeding experiments were carried out using animals that had
been deprived of food for 24 h but with free access to water. Thirty
minutes after the injection, the previously weighed food was placed
in the cage. The food was weighed 30, 60, 120, and 240 min after
initiation of the test. All feeding experiments were performed with
groups of eight animals (n = 8).
In Vitro Experiments: Dienes Conjugation Experiment. LDL
Isolation. Blood from healthy volunteers was collected after an over-
night fast in tubes containing 1 g/L EDTA. Plasma was separated
by centrifugation at 1000g at 4 °C for 15 min. LDL isolation was
performed by sequential flotation ultracentrifugation. Native LDL
was dialyzed by molecular size exclusion chromatography in a G25
(c) DNA Constructs. Full-length cDNAs for human PPAR-α, human
RXRα, and human SRC-1 were subcloned into the T7/SV40 promoter-
driven pSG5 expression vector (Qiagen). Those constructs were used
for viral promoter-driven overexpression of the respective proteins in
mammalian cells.
(d) Reporter Gene Constructs. Four copies of the human CPTI gene
DR1-type RE (core sequence GTAGGGAAAAGGTCA) were indivi-
dually fused with the thymidine kinase (tk) minimal promoter driving
the firefly luciferase reporter gene.
Transient Transfection and Luciferase Reporter Assays19−21
.
MCF-7 human breast cancer cells were seeded into 6-well plates
(200 000 cells/well) and grown overnight in phenol red-free DMEM
supplemented with 5% charcoal-stripped fetal bovine serum. Plasmid
DNA containing liposomes were formed by incubating 1 μg of an
expression vector for wild type PPAR-α, RXRα, SRC-1, and 1 μg of
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reporter plasmid with 10 μg of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-
trimethylammoniummethylsulfate (DOTAP, Roche) for 15 min at
room temperature in a total volume of 100 μL. After dilution with
900 μL of phenol red-free DMEM, the liposomes were added to the
cells. Phenol red-free DMEM supplemented with 500 μL of 15%
charcoal-stripped fetal bovine serum was added 4 h after transfection.
At this time, cells were treated for 16 h with solvent (DMSO) at
different concentrations (10−9, 10−8, 10−7, 10−6, 10−5, and 10−4 mol) of
OEA, GW7647, AEA, oleic acid, and the different compounds under
evaluation as indicated. The cells were lysed for 16 h after the onset of
stimulation using the reporter gene lysis buffer (Roche). Both the
constant light signal luciferase reporter gene assay and the constant
light signal luciferase reporter gene assay were performed as recom-
mended by the suppliers (Roche and Roche Diagnostics, respectively).
Stimulation of normalized luciferase activity was calculated in com-
parison with solvent-induced cells that did not overexpress any protein.
Data were used to calculate the EC50 (nmol) for each compound.
RESULTS AND DISCUSSION
■
Chemistry. The free phenolic hydroxyl groups of the re-
quired phenylalcohols were protected (Figure 4). The synthesis
Figure 4. Protection of phenolic hydroxyl groups of tyrosol and hydro-
xytyrosol: (a) 2,2-dimethoxypropane, TsOH, Dean−Stark, 100 °C,
toluene; (b) NaBH4, I2, 66 °C, THF; (c) CH2Cl2, H2O, (nBu)4NBr,
25 °C.
of protected hydroxytyrosol was performed as described by
Gambacorta et al.22 Thus, intermediate 10, synthesized by the
reaction of 3,4-dihydroxyphenylacetic acid (9) with 2,2-dimeth-
oxypropane, was reduced with NaBH4/I2 to give compound 11.
The protection of the phenol group of tyrosol (12) was carried
out by reaction with MOMCl in the biphasic system CH2Cl2/
NaOH 30% aq using (nBu)4NBr as a phase transfer catalyst,23
yielding compound 13.
Figure 5. General synthesis of fatty alcohol ether derivatives: (a) I2,
PPh3, imidazole, 25 °C, THF; (b) toluene, KOH 30% aq, (nBu)4NBr,
100 °C; (c) HCl 6 N aq, 100 °C; (d) HCl 6 N aq, iPrOH, 80 °C.
oxidation reaction. Variables were log-transformed for statistical
analyses (Figure 6).
Iodoalkenes 15a and 15b (Figure 5) were obtained by the
reaction of oleic and linoleic alcohol (14a,b) with I2/PPh3/
imidazole at room temperature.24 The fatty alcohol ethers
(17a−d, 1, 4, 5, and 8) were prepared by conjugation of the
iodine derivatives 15a and 15b with the corresponding alcohols,
that is, 2-phenylethanol (16a), 2-(3,4-methylenedioxyphenyl)-
ethanol (16b), 2-(3,4-(dimethylmethylendioxy)phenyl)ethanol
(11), and 2-(4-(methoxymethylenoxy)phenyl)ethanol
(13).25,26 Deprotection of compounds 17a−d with aq HCl
yielded the final compounds 2, 3, 6, and 7.
Biology. LDL Antioxidant Activity. We tested the antioxi-
dant LDL activity of the synthesized compounds 1−8 using hy-
droxytyrosol (a well-known natural antioxidant27), tyrosol, and
homovanillyl alcohol as reference compounds. We monitored
the oxidation kinetics of LDL by CuSO4 using a spectrophoto-
metric technique previously standardized by our group.27 The
length of the lag phase was determined as the intercept of
the propagation phase tangent with the extrapolated line for
the slow reaction. Lag-time data were calculated as the ratio
between the observed value for each compound versus those
corresponding to the native LDL (with no added compound)
Figure 6. Log (lag time ratio) of LDL oxidation in the presence of the
tested compounds at the 0.5, 1, and 3 μM concentrations. HT,
hydroxytyrosol; MHT, homovanillyl alcohol; T, tyrosol. ∗, P trend
value <0.05; +, P value versus native LDL oxidation <0.05.
Compound 7 was the only compound that displayed an activ-
ity similar to that of hydroxytyrosol at all three concentrations
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Figure 7. Relative food intake (g of food per kg of animal weight) of 24 h food-deprived male Wistar rats. The food was weighed at 30, 60, 120, and
240 min after the injection. The results are shown as the mean SEM of a group of eight animals. (∗) p < 0.05; (∗∗) p < 0.01; (∗∗∗) p < 0.001
(ANOVA analysis).
tested. The rest of the compounds had no significant activity
at 0.5 and 1 μM. Nevertheless, at 3 μM compounds 2−4, 6,
and 8 also had significant antioxidant activity compared with
native LDL.
two compounds that carried a methylenedioxy group (4 and 8)
presented a hyperphagic effect at a 1 mg/kg dose. Compound 4
had a longer lasting activity than compound 8, as the effect of
compound 8 was seen only in the first 30 min. Using the same
methodology, tyrosol and hydroxytyrosol did not present any
food modulation activity (see the Supporting Information).
CB1 and PPAR-α Affinity Experiments. A study on the affin-
ity of this series of compounds for the CB1 receptor and PPAR-
α was performed to further characterize their biological activity.
The CB1 receptor binding test evaluated the capacity
of the synthesized compounds to displace [3H]-SR141716 in
rat cerebellum homogenate. Compounds 1, 4, 7, and 8, the four
active compounds in the food ingestion test, were evaluated.
The linoleic alcohol derivatives 7 and 8 (Figure 8) showed CB1
receptor affinity, although with a smaller pKi when compared
with endogenous anandamide and synthetic CB1 ligands, such
as WIN55212-2 and SR141716 (Table 1).30 Oleic alcohol
derivative compounds 1 and 4 did not display activity in this
test.
We also evaluated the affinity of compounds 1, 4, and 7
against PPAR-α by the transient transfection and luciferase
reporter assay (Table 1). We used OEA and WY14643 as posi-
tive controls. All tested compounds showed an EC50 at the
micromolar range (Table 1). They displayed lower PPAR-α
affinity than OEA (endogenous PPAR-α ligand) and WY14643
(synthetic PPAR-α ligand), although comparable to that ob-
served in other examples of CB1/PPAR-α ligands previously
described by our group.18
Pereira-Caro et al., in their work with saturated alkyl chain
hydroxytyrosol ethers,9 observed that these derivatives had anti-
oxidant activity similar to that of free hydroxytyrosol in the
rancimat test and that the longer the alkyl chain, the lower
the activity. Our results show that the number and position of
unsaturations in the alkyl chain could also influence the LDL
antioxidant activity, with the linoleic derivative (7) having a
higher activity than the oleic derivative (3). We also observed
that those compounds that lack the free catechol group had less
activity than the catechol derivatives.
We hypothesized that the antioxidant activity of the tested
compounds is derived from their catechol/phenol groups, in a
similar way to the naturally occurring phenol antioxidants, res-
veratrol and hydroxytyrosol.
Feeding Experiments. The effect of acute food intake of all
the products was tested in 24 h food-deprived rats. Compounds
1, 4, 7, and 8 had significant activity on food intake modula-
tion (Figure 7; the rest of the compounds are shown in the
Supporting Information. Compounds 1 and 7 presented a hy-
pophagic effect. Compound 7 was the most active; it presented
an acute food intake modulation profile and potency similar to
that of oleoyl ethanolamine (OEA, a known anorectic endoge-
nous compound that acts through PPAR-α selective activation;
data shown in the Supporting Information) and to recently
developed PPAR-α/CB1 amide ligands.18,28,29 In contrast, the
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Figure 8. CB1 binding assay for compounds 7 and 8.
Table 1. Pharmacological Properties of Fatty Alcohol Ethers
pounds. The linoleic hydroxytyrosol linoleyl ether (7) was the
most active compound in vivo with a hypophagic effect com-
parable to that of oleoylethanolamine. In addition, it was also
the compound that conferred the highest protection of LDL to
oxidation in vitro in the same low micromolar range seen for
hydroxytyrosol. For all of the above considerations, compound
7 can be regarded as a promising lead for future drug develop-
ment aiming at concomitantly combating obesity and coronary
artery disease, one of its most dangerous related diseases.
a
compound
CB1 Ki (M)
1.15 × 10−9
CB1 pKi
8.94
PPAR-α EC50 (nM)
SR141716
b
b
anandamide
1.7 × 10−7
6.55
>10.000
WIN55212-2
684 47
148 29
1165 166
791 119
1934 224
OEA
1
4
7
8
no activity
no activity
2.25 × 10−5
7.49 × 10−6
no activity
no activity
4.65
5.12
ASSOCIATED CONTENT
* Supporting Information
■
a
Values calculated in the presence of the different compounds by
S
GraphPad Prism 4. Results for PPAR-α are the mean SEM of three
1H and 13C NMR spectra of compounds 15a, 15b, 10, 11, 13,
1, 4, 5, 8, 17a, 17b, 17c, 17d, 2, 3, 6, and 7. Graphics of
“relative food intake” for tyrosol, hydroxytyrosol, oleoylethanol-
amine, and compounds 2, 3, 5, and 6, CB1 binding assay for
compounds SR141716, 1, and 4. This material is available free
of charge via the Internet at http://pubs.acs.org.
b
experiments. Values from the literature.27
In conclusion, in the dienes conjugation experiment we could
observe that most of the synthesized compounds had LDL
antioxidant activity at the 3 μM concentration. Hydroxytyrosol
linoeyl ether (7) also had significant antioxidant activity
at 0.5 and 1 μM. Both the alkenyl chain and the “head” are
important for food intake activity. In addition, depending on
structural modifications, the modulation activity could change both
in potency and from negative to positive, and vice versa. Possibly,
the pharmacodynamic profile of the synthesized compounds
operates through mechanisms other than CB1 and PPAR-α interac-
tions. This is due to the fact that among the four compounds with
modulatory food activity, only two displayed CB1 activity, and their
affinity toward PPAR-α was lower than that of OEA. These CB1
and PPAR-α affinities do not justify their whole in vivo activity.
In summary, the pharmacological profile of these new com-
pounds suggests that they can be further developed as new
medications for complicated obesity. They may provide a new
way of not only reducing feeding but also ameliorating the
negative impact derived of oxidized LDL. However, further
research is needed to identify the ADME/TOX profile of these
compounds. This a relevant issue because the two only specific
therapies against obesity, Sibutramine and Rimonabant,31 have
recently been withdrawn from the market. Although we used
the ip route for bypassing the oral route, we do not have infor-
mation on the absorption of these compounds. However,
the natural related compound OEA has been found to be
orally active and to cross major barriers, including blood−brain
barrier.32,33 Another important issue is the potential toxic
effects of these compounds. Again, initial studies have revealed
that not only OEA but also other closely related compounds
such amide or sulfonamide derivatives of oleic acid have
no effect on liver transaminase levels and cytochrome P450
isoforms, indicating a safe profile.34,35 However, this needs to
be conclusively determined for this present series of com-
DEDICATION
■
⊗In memory of Prof. Dr. Octavio Augusto Ceva Antunes
(Universidade Federal do Rio de Janeiro).
FUNDING
■
This work was supported by grants from DIUE de la
Generalitat de Catalunya (2009 SGR 718), the Instituto de
́
Salud Carlos III del Ministerio de Ciencia e Innovacion [grants
FIS FEDER PI0/81913, PI07/1226, SAF2010-20521, UE-
ERDF (CB06/03/1008)]; Red de Trastornos Adictivos [grant
RD06/0001, and the Centro de Investigacion
Red-Fisiopatologia de la Obesidad y la Nutricion
́
Biomed
(CIBEROBN,
́
ica en
́
which is an initiative of the Instituto de Salud Carlos III,
Madrid, Spain), the Spanish Ministry of Science and Innovation
[research project SAF2010-20521]. Grant EU-ERDF PAIDI CTS-
433 and grant PI45403 from the Andalusian Ministry of Economy,
Science and Innovation. B.A.C. is a recipient of a fellowship from the
Alban Program, the European Union Program of High Level
Scholarships for Latin America (E06D101051BR, B.A.). F.R.F. M.J.L.,
J.M.D. and M.R.C. are supported by the 7th Framework Program of
the European Union [grant HEALTH-F2-2008-223713, REPROBE-
SITY]. M.M.-G. is supported by the Research Stabilization Program
(CES 10/004) and M.F. by SNS contract Miguel Servet (CP06/
00100) of the Instituto de Salud Carlos III (CES 10/004).
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