Synthesis of fatty acid amides of catechol metabolites that exhibit antiobesity properties

Article abstract of DOI:10.1002/cmdc.201000161

A series of fatty acid amides of 3,4-methylenedioxymethamphetamine (MDMA) catechol metabolites were synthesized in order to evaluate their biological activities. Upon administration, all synthesized compounds resulted in negative modulation of food intake in rats. The most active compounds have affinity for the CB₁ receptor and/or PPAR-α; part of their biological activity may be caused by these double interactions.

Full text of DOI:10.1002/cmdc.201000161

DOI: 10.1002/cmdc.201000161
Synthesis of Fatty Acid Amides of Catechol Metabolites
that Exhibit Antiobesity Properties
Bruno Almeida,[a, b, f] Jesffls Joglar,*[c] María Jesffls Luque Rojas,[d] Juan Manuel Decara,[d]
Francisco Javier Bermffldez-Silva,[d] Manuel Macias-Gonzµlez,[e, f] Montserrat Fitó,[f, g]
Miguel Romero-Cuevas,[d] Magí FarrØ,[a, h] María Isabel Covas,[f, g]
Fernando Rodríguez de Fonseca,[d, f] and Rafael de la Torre*[a, b, f]
In memory of Prof. Dr. Josꢀ Manuel Concellꢁn (Universidad de Oviedo)
A series of fatty acid amides of 3,4-methylenedioxymethampheta-
mine (MDMA) catechol metabolites were synthesized in order to
evaluate their biological activities. Upon administration, all syn-
thesized compounds resulted in negative modulation of food
intake in rats. The most active compounds have affinity for the
CB1 receptor and/or PPAR-a; part of their biological activity may
be caused by these double interactions.
Introduction
The discovery of endocannabinoids, including the fatty acid
amide anandamide (arachidonylethanolamide, AEA), paved the
way for lipidomic discoveries within the growing family of en-
dogenous fatty acid amides.[1] Fatty acid amides can be formed
from metabolites of xenobiotics such as acetaminophen, or
from endogenous substrates like dopamine (Figure 1). For ex-
amide oleylethanolamide inhibits food intake, it does not inter-
act with cannabinoid type 1 (CB1) or 2 (CB2) receptors, but
rather through the peroxisome proliferator-activated nuclear
receptor-a (PPAR-a).[6]
[a] B. Almeida, Dr. M. Farrꢀ, Dr. R. de la Torre
Human Pharmacology and Clinical Neurosciences Research Group
Neuropsychopharmacology Research Program
IMIM-Hospital del Mar Research Institute
Dr. Aiguader 88, Barcelona 08003 (Spain)
Fax: (+34)933160467
E-mail: rtorre@imim.es
[b] B. Almeida, Dr. R. de la Torre
Universitat Pompeu Fabra, CEXS-UPF
Dr. Aiguader 88, Barcelona 08003 (Spain)
[c] Dr. J. Joglar
Departamento de Quꢂmica Biolꢁgica y Modelizaciꢁn Molecular
Instituto de Quꢂmica Avanzada de CataluÇa (IQAC-CSIC)
Jordi Girona 18-26, 08034 Barcelona (Spain)
E-mail: joglar@iqac.csic.es
[d] M. J. Luque Rojas, J. M. Decara, F. J. Bermffldez-Silva, M. Romero-Cuevas,
Dr. F. Rodrꢂguez de Fonseca
Fundaciꢁn IMABIS, Hospital Carlos Haya de Mµlaga
Avda. Carlos Haya 82, Pabellꢁn de Gobierno, 29010 Mµlaga (Spain)
Figure 1. Fatty acid amides of endogenous or exogenous substrates with
[e] Dr. M. Macias-Gonzµlez
cannabinoid activity.
Hospital Clꢂnico Virgen de la Victoria
Campus Universitario Teatinos, 29010 Mµlaga (Spain)
[f] B. Almeida, Dr. M. Macias-Gonzµlez, Dr. M. Fitꢁ, Dr. M. I. Covas,
Dr. F. Rodrꢂguez de Fonseca, Dr. R. de la Torre
ample, it has been proposed that the analgesic effects of acet-
aminophen are derived from the compound known as AM404,
which results from the conjugation of p-aminophenol with
arachidonic acid and its subsequent interaction with the endo-
cannabinoid system.[2–4]
CIBEROBN, Instituto de Salud Carlos III, Madrid (Spain)
[g] Dr. M. Fitꢁ, Dr. M. I. Covas
Oxidative Stress and Nutrition Research Group
Inflammation and Cardiovascular Research Program
IMIM-Hospital del Mar, Dr. Aiguader 88, Barcelona 08003 (Spain)
It is well known that cannabinoid agonists modulate food
intake, among other physiological effects, and that the inhibi-
tion of cannabinoid type 1 receptors causes an appetite de-
crease, leading to a corresponding decrease in weight.[5] It was
also discovered that, although the endogenous fatty acid
[h] Dr. M. Farrꢀ
Universitat Autònoma de Barcelona, UDIMAS-UAB
Dr. Aiguader 88, Barcelona 08003 (Spain)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000161.
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J. Joglar, R. de la Torre, et al.
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Herein we present the synthesis and biological activity of
fatty acid amides of several metabolites of 3,4-methylenedioxy-
methamphetamine (MDMA). MDMA is a widely abused, recrea-
tional drug that produces effects commonly associated with
stimulants and hallucinogens.[7,8] It is believed that long term
use can cause neurodegeneration in the serotonergic neuro-
transmission system, both in animals and humans.[9]
cannabinoids and endovanilloids that play a role in the regula-
tion of pain and inflammation.[15–18]
Some existing data supports the interaction between MDMA
and the cannabinoid system. In clinical practice, MDMA and
cannabis consumption are strongly associated. It has been
postulated that the connection does not occur randomly, or
because of drug availability, but through modulation of phar-
macological effects. It has already been established in animal
models that some in vivo effects of MDMA, such as long term
rewarding process, hyperthermia, and mood alteration, among
others, are associated with endocannabinoid system interac-
tions.[19–22] Recent results in humans have shown that cannabi-
noids regulate cardiovascular activity and body temperature
when combined with MDMA.[23] By analogy, based on what is
known of the fatty acid derivatives of dopamine and the possi-
bility that xenobiotic metabolites may form fatty acid deriva-
tives with relevant biological activities, it is postulated that
fatty acid acyl derivatives of several catecholic metabolites of
MDMA (HHA and HHMA) and their O-methyl derivatives (HMA
and HMMA) could display biological activities of interest. If
their in vivo formation is ever demonstrated, it may significant-
ly improve our understanding of MDMA pharmacology. Addi-
tionally, these amides may display a potential therapeutic pro-
file and could become useful pharmacological tools.
MDMA has two main metabolic pathways: 1) O-demethyle-
nation, followed by methylation and/or glucuronide/sulfate
conjugation; and 2) N-dealkylation. The latter of these MDMA
pathways gives rise to 3,4-methylenedioxyamphetamine
(MDA). The parent compound and MDA are further O-deme-
thylated to 3,4-dihydroxymethamphetamine (HHMA, 3) and
3,4-dihydroxyamphetamine (HHA, 1), respectively. Both HHMA
and HHA are subsequently O-methylated to 4-hydroxy-3-me-
thoxymethamphetamine (HMMA, 4) and 4-hydroxy-3-methoxy-
amphetamine (HMA, 2), respectively. These metabolites are
mainly present in plasma and urine as their glucuronide or sul-
fate conjugates.[10,11] As catechols, HHA and HHMA are highly
redox active and may form adducts with glutathione, after au-
tooxidation to their corresponding quinones. (Scheme 1).[12]
In this context, the oleic and arachidonic fatty acid amides
of MDMA metabolites HHA, HMA, and HHMA were synthesized
as racemic mixtures in order to evaluate their biological activi-
ties (Figure 2).
Scheme 1. MDMA metabolic distribution.
It must be noted that body disposition of MDMA is subject-
ed to selective enantiomeric metabolism.[13] Although the
methods reported herein would allow for the preparation of
enantiomerically pure compounds, it was considered unneces-
sary for this preliminary study of the pharmacological activity
of the synthesized compounds. However, work in our research
group is ongoing toward production of the enantiomeric
MDMA metabolites, which are crucial for ongoing and future
studies of the differential metabolism of MDMA enantiomers
and their role in the neurotoxicity and other pharmacological
effects of MDMA. Catechol metabolites of MDMA are ana-
logues of dopamine, and can be renamed as a-methyldopa-
mine for HHA, or as N-methyl-a-methyldopamine for HHMA.
Figure 2. Proposed structures of fatty acid amides. R1 =R2 =H, N-(1-(3,4-dihy-
droxyphenyl)propan-2-yl)oleamide (5); N-(1-(3,4-dihydroxyphenyl)propan-2-
yl)arachidonamide (8); R1 =H, R2 =Me, N-(1-(4-hydroxy-3-methoxyphenyl)-
propan-2-yl)oleamide (6); N-(1-(4-hydroxy-3-methoxyphenyl)propan-2-yl)ara-
chidonamide (9); R1 =Me, R2 =H, N-(1-(3,4-dihydroxyphenyl)propan-2-yl)-N-
methyloleamide (7); N-(1-(3,4-dihydroxyphenyl)propan-2-yl)-N-methylarachi-
donamide (10); N-phenethyloleamide (11).
Fatty acid amides of dopamine (N-oleyldopamine, OLDA and Results and Discussion
N-arachidonyldopamine, NADA) have been found in the brain
Chemistry
and other tissues, and their biosynthesis has recently been re-
ported.[14] OLDA and NADA act with varying potency on the
(CB1) receptor and on the transient receptor potential vanilloid
subtype 1 (TRPV1). As such, they are considered putative endo-
MDMA metabolites were synthesized according to previously
described methodology (Scheme 2).[24] Next, fatty acid amides
were synthesized by a one-pot method: the fatty acid carbox-
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Catechol Metabolites with Antiobesity Properties
Scheme 2. General procedure for the synthesis of fatty acid amides of
MDMA metabolites: a) CH3CN, Et3N, 48C, 1 h; b) DMF, Et3N, 258C, 24 h.
ylic group was activated with benzyl chloroformate, and this
intermediate was subsequently treated with MDMA metabo-
lites 1–3. (Scheme 2). Compounds 7 and 10 were obtained as a
mixture of cis and trans isomers and were identified by NMR
(see Supporting Information).
Biology
Feeding experiments
The acute food intake effect of all compounds was tested in
24 h food-deprived rats. The amide of phenylethylamine with
oleic acid (11) was synthesized as a theoretical negative con-
trol due to the lack of a catechol group. All compounds in-
duced a significant decrease in food intake (Figure 3). Com-
pounds 5 and 8 were most active and displayed an acute food
intake modulation profile and potency similar to that of oleyl
ethanolamide (OEA, a known PPAR-a selective endogenous ag-
onist), although OEA activity lasts longer.[6,25] The food intake
modulation profile is also compatible with other PPAR-a li-
gands.[26]
HHA derivatives are more potent than HMA and HHMA de-
rivatives, thus we concluded that meta-O-methylation of the
catechol ring and N-methylation of the amide group are dele-
terious with respect to food intake activity. Compound 11
showed only a minor increase in food intake at the 1 mgkgÀ1
dose. This is indicative of the importance of the catechol
group as a pharmacophore. To confirm that the negative mod-
ulation of food intake was not caused by an anesthetic effect
of the compounds, we tested 5 in an open field test using the
same doses and established that there was no effect on animal
locomotion (see Supporting Information).
Figure 3. Relative food intake (g food per kg animal weight) by male Wistar
A study of the affinity of this compound series for the CB1
receptor, as well as efficacy toward PPAR-a, was performed to
further evaluate the biological activity of the synthesized com-
pounds. The CB1 receptor binding test evaluated the capacity
of these compounds to displace [3H]SR141716 in rat cerebel-
lum homogenate. Compounds 5–8, which exhibited greatest
activity in the food ingestion test, were evaluated. Only com-
pounds 5, 6, and 8 had affinity for the CB1 receptor (Figure 4,
Table 1) (see Supporting Information). This indicates that N-
methylation is deleterious for the CB1 receptor compound af-
finity, corresponding to the decrease in activity observed for
rats deprived of food for 24 h. Food was weighed at 30, 60, 120, and
~
&
^
240 min after injection of vehicle ( ), test compounds 5–10 at 0.5 ( ), 1 ( ),
and 5 ( ) mgkg , or compound 11 at 0.3 ( ), 1 ( ), and 3 ( ) mgkgÀ1. Re-
sults are shown as the mean ÆSEM of a group of eight animals; *p<0.05,
**p<0.01, ***p<0.001 (ANOVA).
À1
~
~
~
&
compound 7 in the food intake test. These results demonstrate
that at least some of the biological activity of the compounds
may be attributed to interaction with CB1. Calculated pKi
values of our compounds are similar to that of endogenous
anandamide, using the same labeled CB1 ligand ([3H]SR141716)
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J. Joglar, R. de la Torre, et al.
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cate that the catechol moiety is not essential for PPAR activity.
The tested compounds displayed lower PPAR-a efficacy than
OEA and GW7647 (a synthetic PPAR-a ligand), although their
activities were similar to those observed in other examples of
PPAR-a ligands and food intake modulators, as described by
Cano et al.[26]
It is worth noting that compound 8, which displayed one of
the highest levels of food intake inhibition, did not exhibit any
activity toward PPAR-a. In this particular case, the food intake
activity may be due entirely to CB1 receptor interaction. On the
other hand, modifications to the fatty acid moiety may alter
the pharmacodynamics of these compounds.
We tested compounds 5–8 as potential fatty acid amide hy-
drolase (FAAH) inhibitors using Wistar rat cerebral cortex and
tritiated anandamide. Degradation of the labeled anandamide
by the FAAH present in the cortex homogenate was monitored
by beta scintillation. None of the tested compounds showed
any FAAH inhibitory activity (see Supporting Information).
Because some fatty acid amides are ligands for not only can-
nabinoid receptors but also for the TRPV1 and the PPAR-a re-
ceptors, we questioned the extent to which activities observed
for our compounds were modulated by TRPV1 receptors. In
order to study the hypothetical participation of TRPV1 recep-
tors in the observed food intake modulation, a TRPV1 antago-
nist, capsazepine,[28] at 2.5 mgkgÀ1, was co-administered with
the most active compounds (5, 6, and 8) at 5 mgkgÀ1 doses.
Capsazepine and fatty acid amides, either separately or in com-
bination, were tested on groups of animals. Capsazepine was
unable to significantly modify the effects on food intake elicit-
ed by the tested compounds (see Supporting Information),
which likely indicates that TRPV1 receptors do not contribute
to the observed biological effects.
~
&
^
Figure 4. CB1 binding assay for compounds 5 ( ), 6 ( ), and 8 ( ).
Conclusions
Table 1. Pharmacological properties of fatty acid amides.
The newly synthesized compounds presented herein have po-
tential as food intake inhibitors, as all compounds effected a
decrease in food intake to some extent. A number of these dis-
played affinity for the CB1 receptor, similar to the endocannabi-
noid anandamide, and some exhibited activity against PPAR-a.
N-methylation and catechol O-methylation were shown to be
deleterious modifications for food intake modulation. In con-
trast, the catechol group is essential for retaining this activity.
Observed pharmacodynamics of the biologically active com-
pounds could be explained by CB1, PPAR-a, or a dual CB1/
PPAR-a receptor interaction. Nevertheless, further studies are
necessary to elucidate the precise mechanism of action of
these compounds.
Compound
CB1 Ki [m]
CB1 pKi
PPAR-a EC50 [nm][a]
SR141716
anandamide
WIN55212-2
3.64”10À10
9.44
6.77
7.95
6.44
5.84
6.69
–
–
–
–
–
>10000
–
1.7”10À7[b]
1.11”10À8[b]
5
6
8
3.65”10À7
698Æ102
1022Æ206
>10000
1879Æ384
218Æ150
148Æ29
65Æ1
1.44”10À6
2.02”10À7
11
oleic acid
OEA
–
–
–
–
GW7647
[a] Values calculated with GraphPad Prism 4; results for PPAR-a are the
mean ÆSEM of three experiments. [b] Values from reference [27].
and similar experimental conditions, but lower than those of
synthetic ligands WIN55212-2 and SR141716.[27]
Experimental Section
We also tested the efficacy of compounds 5, 6, 8, and 11 for
PPAR-a by the transient transfection and luciferase reporter
assay. We used OEA and AEA as positive and negative controls,
respectively. Compounds 5, 6, and 11 exhibited an EC50 in the
micromolar range, while compound 8 showed no activity
(Table 1). The activity displayed by compound 11 could indi-
Chemistry
MDMA metabolites HHA, HMA and HHMA were synthesized as de-
scribed.[24] All reagents and solvents were commercially available
and were used without further purification unless specifically indi-
cated. A Nicolet Avatar 360 IR spectrophotometer was employed,
as well as a Sigma 3K30 ultracentrifuge. H and 13C NMR analyses
1
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Catechol Metabolites with Antiobesity Properties
were carried out with Varian Anova 500 and Varian Mercury 400
spectrometers. Progress of all reactions was monitored by TLC on
aluminum sheets pre-coated with silica gel 60 (HF-254, Merck), film
thickness 0.25 mm. Elemental analyses (C, H, N) were performed on
a Thermo Finnigan Elemental Analyzer Flash 1112 Series and were
within Æ0.3% of theoretical values. MS data were recorded using a
LCT Premier (Waters, Milford, MA, USA) ESI-TOF instrument
equipped with a 4 GHz time-to-digital converter (TDC).
1.19–1.42 (m, 20H), 1.36–1.42 (trans, m, 2H), 1.80–1.87 (cis, m, 2H)
1.94–2.00 (m, 4H), 2.06–2.09 (cis, m, 2H), 2.11–2.19 (trans, m, 2H),
2.46–2.55 (m, 2H), 2.66 (cis, s, 3H), 2.74 (trans, s, 3H), 3.95–4.06 (cis,
m, 1H), 4.64–4.75 (trans, s, 1H), 5.28–5.36 (m, 2H), 6.36–6.41 (m,
1H), 6.52–6.54 (m, 1H), 6.56–6.70 ppm (m, 1H); 13C NMR (101 MHz,
CDCl3): d=14.36, 14.41, 15.57, 19.50, 21.29, 22.92, 25.34, 25.52,
27.12, 27.45, 29.36, 29.40, 29.47, 29.55, 29.76, 29.96, 30.01, 32.14,
33.24, 34.32, 39.68, 40.00, 55.42, 60.69, 114.94, 115.62, 115.98,
120.66, 120.80, 130.01, 130.16, 143.43, 143.66, 143.65, 144.39,
144.76, 171.53, 174.76, 175.21 ppm; IR (KBr): n˜ =3354, 3227, 2926,
1597, 1514, 1454, 1283, 815 cmÀ1; MS [M+H]+ calcd for C28H47NO3:
446.3634, found: 446.3537; Anal. calcd for C28H47NO3: C 75.46, H
10.63, N 3.14, found: C 75.25, H 10.48, N 3.07.
General synthesis of fatty acid amides
In a round-bottomed flask at 48C under nitrogen atmosphere,
oleic or arachidonic acid (4.5 mmol), CH3CN (30 mL), Et3N (863 mL,
6.2 mmol), and benzyl chloroformate (771 mL, 5.4 mmol) were
added and stirred for 1 h. CH3CN was removed under reduced
pressure, and the dried residue was redissolved in DMF (30 mL).
Et3N (640 mL, 4.6 mmol) and phenylethylamine or the correspond-
ing MDMA metabolite (4.9 mmol) were added to the DMF solution
under a nitrogen atmosphere at 48C. After this addition, the cold
bath was removed, and the mixture stirred for 24 h at 258C. The
reaction was monitored by TLC until no starting materials re-
mained. The DMF was removed under reduced pressure, and the
product was purified by flash chromatography using 20% ethyl
acetate/hexane. Yields are given for isolated products and were
not optimized.
N-(1-(3,4-dihydroxyphenyl)propan-2-yl)arachidonamide (8). Com-
pound 8 was obtained as a pale-yellow oil (82 mg, 39% yield). Rf
(Hex/EtOAc, 4:1)=0.14; 1H NMR (500 MHz, CDCl3): d=0.90 (t, J=
6.93 Hz, 3H), 1.13 (d, J=6.62 Hz, 3H), 1.24–1.42 (m, 6H), 1.59–1.74
(m, 2H), 2.03–2.11 (m, 4H), 2.17 (t, 7.73 Hz, 2H), 2.59 (A of an ABX
syst., J=7.26, 13.69 Hz, 1H), 2.70 (B of an ABX syst., J=6.48,
13.68 Hz, 1H), 2.77–2.87 (m, 6H), 4.19–4.29 (X of an ABX syst., m,
1H), 5.30–5.46 (m, 8H), 5.53 (d, J=8.38 Hz, 1H), 6.54 (dd, J=1.90,
8.02 Hz, 1H), 6.78 (s, 1H), 6.78 ppm (d, J=10.89, 1H); 13C NMR
(101 MHz, CDCl3): d=14.30, 20.34, 22.80, 25.81, 25.85, 26.75, 27.44,
29.54, 31.74, 36.49, 42.26, 47.03, 115.04, 115.93, 121.39, 127.73,
128.05, 128.31, 128.52, 128.85, 129.04, 129.17, 129.94, 130.76,
143.47, 144.48, 173.82 ppm; IR (KBr): n˜ =3381, 2926, 1651, 1516,
N-(1-(3,4-dihydroxyphenyl)propan-2-yl)oleamide
(5).
Com-
1448, 1281 960, 813 cmÀ1 MS [M+H]+ calcd for C29H43NO3:
;
pound 5 was obtained as a pale-yellow oil (306 mg, 43% yield). Rf
(Hex/EtOAc, 4:1)=0.09; 1H NMR (500 MHz, CDCl3): d=0.86 (t, J=
6.95 Hz, 3H), 1.11 (d, J=6.58 Hz, 3H), 1.19–1.34 (m, 20H), 1.50–1.60
(m, 2H), 1.96–2.03 (m, 4H), 2.12 (t, J=7.78 Hz, 2H), 2.57 (A of an
ABX syst., J=7.08, 13.65 Hz, 1H), 2.66 (B of an ABX syst., J=6.55,
13.70 Hz, 1H), 4.16–4.28 (X of an ABX syst., m, 1H), 5.28–5.39 (m,
2H), 5.53 (d, J=8.28 Hz, 1H), 6.51 (dd, J=1.40, 8.00, 1H), 6.75 (s,
1H), 6.76 ppm (d, J=8.0 Hz, 1H); 13C NMR (101 MHz, CDCl3): d=
14.34, 20.37, 22.92, 26.03, 27.41, 27.46, 29.34, 29.44, 29.56, 29.77,
29.95, 30.00, 32.14, 37.20, 42.26, 46.91, 114.97, 115.90, 121.34,
129.90, 129.96, 130.22, 143.51, 144.51, 174.11 ppm; IR (KBr): n˜ =
3363, 3266, 2920, 1638, 1555, 1465, 1032, 814 cmÀ1; MS [M+H]+
calcd for C27H45NO3: 432.3478, found: 432.3490; Anal. calcd for
C27H45NO3: C 75.13, H 10.51, N 3.24, found: C 74.92, H 10.33, N
3.20.
454.3321, found: 454.3221; Anal. calcd for C29H43NO3: C 76.78, H
9.55, N 3.09, found: C 76.59, H 9.78, N 3.21.
N-(1-(4-hydroxy-3-methoxyphenyl)propan-2-yl)arachidonamide
(9). Compound 9 was obtained as a pale-yellow oil (114 mg, 40%
1
yield). Rf (Hex/EtOAc, 4:1)=0.40; H NMR (500 MHz, CDCl3): d=0.87
(t, J=6.60 Hz, 3H), 1.09 (d, J=6.63 Hz, 3H), 1.21–1.40 (m, 6H),
1.61–1.76 (m, 2H), 1.99–2.15 (m, 6H), 2.59 (A of an ABX syst., J=
7.48, 13.59 Hz, 1H) 2.73–2.86 (m, 7H, B of an ABX syst. + 6H), 3.85
(s, 3H), 4.15–4.27 (X of an ABX syst., m, 1H), 5.27–5.43 (m, 9H),
6.62 (dd, J=1.50, 7.95 Hz, 1H), 6.68 (d, J=1.40 Hz, 1H), 6.81 ppm
(d, J=7.98 Hz, 1H); 13C NMR (101 MHz, CDCl3): d=14.32, 20.18,
22.81, 25.80, 25.85, 26.86, 27.44, 29.55, 31.74, 36.52, 42.44, 46.33,
56.11, 111.89, 114.36, 122.33, 127.72, 128.06, 128.37, 128.44, 128.81,
128.95, 129.31, 130.01, 130.74, 144.47, 146.73, 172.45 ppm; IR (KBr):
n˜ =3389, 3264, 2924, 1638, 1553, 1273, 1033, 815 cmÀ1; MS
[M+H]+ calcd for C30H45NO3: 468.3478, found: 468.3459; Anal.
calcd for C30H45NO3: C 77.04, H 9.70, N 2.99, found: C 76.89, H 9.78,
N 3.11.
N-(1-(4-hydroxy-3-methoxyphenyl)propan-2-yl)oleamide
(6).
Compound 6 was obtained as a pale-yellow oil (258 mg, 49%)
1
yield. Rf (Hex/EtOAc, 4:1)=0.26; H NMR (500 MHz, CDCl3): d=0.87
(t, J=6.58 Hz, 3H), 1.09 (d, J=6.61 Hz, 3H), 1.20–1.37 (m, 20H),
1.51–1.62 (m, 2H), 1.95–2.05 (m, 4H), 2.10 (t, J=7.48, 7.48 Hz, 2H),
2.60 (A of an ABX syst., J=7.34, 13.61 Hz, 1H), 2.76 (B of an ABX
syst., J=5.70, 13.59 Hz, 1H), 3.86 (s, 3H), 4.18–4.29 (X of an ABX
syst., m, 1H), 5.24–5.41 (m, 3H), 6.63 (dd, J=1.28, 7.95 Hz, 2H),
6.69 (d, J=1.39 Hz, 1H), 6.82 ppm (d, J=7.97 Hz, 1H); 13C NMR
(101 MHz, CDCl3): d=14.36, 20.24, 22.92, 26.02, 27.40, 27.45, 29.36,
29.45, 29.51, 29.55, 29.75, 29.95, 29.99, 32.13, 37.25, 42.44, 46.25,
56.12, 111.87, 114.32, 122.34, 129.97, 130.04, 130.21, 144.45, 146.71,
172.72 ppm; IR (KBr): n˜ =3418, 3293, 2922, 1639, 1556, 1465, 1030,
N-(1-(3,4-dihydroxyphenyl)propan-2-yl)-N-methylarachidona-
mide (10). Compound 10 was obtained as a pale-yellow oil
(137 mg, 42% yield) and was a mixture of cis and trans isomers. Rf
(Hex/EtOAc, 4:1)=0.43; 1H NMR (500 MHz, [D6]DMSO): d=0.87 (t,
J=6.60 Hz, 3H), 0.95 (trans, d, J=7.00 Hz, 3H), 1.11 (cis, d, J=
6.5 Hz, 3H) 1.20–1.34 (m, 6H), 1.43–1.50 (trans, m, 2H), 1.83–1.94
(cis, m, 2H), 1.98–2.03 (m, 4H), 2.08–2.37 (m, 4H), 2.65 (cis, s, 3H),
2.73 (trans, s, 3H), 2.74–2.82 (m, 6H), 3.95–4.01 (cis, m, 1H), 4.65–
4.72 (trans, m, 1H), 5.25–5.38 (m, 8H), 6.36–6.40 (m, 1H), 6.52–6.54
(m, 1H), 6.57–6.61 ppm (m, 1H); 13C NMR (101 MHz, CDCl3): d=
17.57, 19.53, 22.81, 25.06, 25.20, 25.85, 26.83, 26.87, 27.13, 27.45,
29.35, 29.56, 31.74, 32.56, 33.65, 39.68, 39.97, 49.95, 55.37, 60.70,
114.97, 115.57, 115.99, 120.69, 120.83, 127.74, 127.77, 128.07,
128.13, 128.34, 128.39, 128.42, 128.52, 128.81, 129.81, 129.00,
129.32, 129.35, 130.05, 130.08, 130.73, 143.42, 143.67, 144.38,
144.67, 144.74, 174.42, 174.92 ppm; IR (KBr): n˜ =3534, 3272, 2928,
1603, 1518, 1447, 1282, 1117, 712 cmÀ1; MS [M+H]+ calcd for
844 cmÀ1 MS [M+H]+ calcd for C28H47NO3: 446.3634, found:
;
446.3545; Anal. calcd for C28H47NO3: C 75.46, H 10.63, N 3.14,
found: C 75.28, H 10.65, N 2.89.
N-(1-(3,4-dihydroxyphenyl)propan-2-yl)-N-methyloleamide
(7).
Compound 7 was obtained as a pale-yellow oil (142 mg, 45%
yield), and was a mixture of cis and trans isomers. Rf (Hex/EtOAc,
4:1)=0.29; 1H NMR (500 MHz, [D6]DMSO): d=0.85 (t, J=6.87 Hz,
3H), 0.95 (trans, d, J=6.80 Hz, 3H), 1.12 (cis, d, J=6.50 Hz, 3H),
ChemMedChem 2010, 5, 1781 – 1787
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J. Joglar, R. de la Torre, et al.
MED
C30H45NO3: 468.3478; found 468.3469; Anal. calcd for C30H45NO3: C
77.04, H 9.70, N 2.99, found: C 76.88, H 9.75, N 2.72.
homogenized with 50 mm Tris buffer (pH 7.4) using an Ultra-
Turraxꢁ. The homogenate was centrifuged at 25000 g for 15 min
at 48C. The supernatant was discarded and the pellet was washed
again with 5 mL of buffer, then centrifuged. The supernatant was
again discarded, and the protein concentration of the pellet was
measured using the Bradford test.
N-phenethyloleamide (11). Compound 11 was obtained as a
1
white solid (105 mg, 95% yield). Rf (Hex/EtOAc, 4:1)=0.31; H NMR
(500 MHz, CDCl3): d=0.87 (t, J=6.93 Hz, 3H), 1.21–1.37 (m, 20H),
1.53–1.63 (m, 2H), 1.96–2.05 (m, 4H), 2.11 (t, J=7.60 Hz, 2H), 2.81
(t, J=6.94 Hz, 2H), 3.49–3.53 (m, 2H), 5.30–5.37 (m, 2H), 5.53 (brs,
1H), 7.18–7.33 ppm (m, 5H).); 13C NMR (101 MHz, CDCl3): d=14.38,
22.93, 25.99, 27.41, 27.46, 29.38, 29.48, 29.51, 29.56, 29.77, 29.95,
30.01, 32.14, 35.93, 37.05, 40.75, 126.72, 128.84, 128.99, 129.97,
130.22, 139.15, 173.47 ppm; IR (KBr): n˜ =3309, 2920, 1640, 1559,
1446. 1085, 698 cmÀ1; MS [M+H]+ calcd for C26H43NO: 386.3423;
found 386.3347; Anal. calcd for C26H43NO: C 80.98, H 11.24, N 3.63,
found: C 80.75, H 11.31, N 3.58.
Binding assay
The binding assay was performed using the labeled CB1 antagonist
[3H]SR141716; 450 mL buffer A (50 mm Tris, pH 7.4, with 0.5%
bovine serum albumin (BSA)), 100–200 mg of rat brain membranes
(cerebellum), the diluted compound, and tracer [3H]SR141716 were
added to each tube. The mixture was incubated while shaking at
378C for 60 min, and the reaction was stopped with 1 mL buffer A.
The resulting mixture was centrifuged at 5000 rpm for 5 min. The
supernatant was discarded, then the pellet was washed with 1 mL
buffer A and centrifuged, and the supernatant was again discard-
ed. Scintillation liquid was added, and the samples were read
using a beta scintillator (Liquid scintillation analyzer, Tri-Carb 2100
TR, PACKARD, Packard Biosciences). All compounds were diluted in
buffer B (50 mm Tris, pH 7.4, with 0.5% bovine serum albumin
(BSA) and 0.3% DMSO) in 10À5, 10À6, 10À7, 10À8, 10À9, 10À10 and
10À11 mol concentrations. All experiments were performed in tripli-
cate.
In vivo experiments
All in vivo experiments were carried out using Wistar male rats
within a 200–450 g weight range. Animals were housed in individ-
ual cages in a room with controlled temperature (238C) and rela-
tive humidity (50%), as well as a 12 h/12 h light and dark cycle.
Water and food were available ad libitum except in specific experi-
mental proceedings. The animals were handled twice in the days
prior to the experimental sessions. Compounds were dissolved in a
mixture of DMSO/Tween-60/saline (5:5:90 v/v/v) and administered
intraperitoneally. The experiments performed in this study are in
compliance with the Spanish Real Decreto 1201/2005, October 21,
2005 (BOE number 252) concerning the protection of experimental
animals, as well as with the European Communities Council Direc-
tive of November 24, 1986 (86/609/EEC).
PPAR-a affinity assay
Biology: The compounds reported in this study were first evaluat-
ed using an in vivo reporter gene assay in order to assess which
compounds could induce interaction between PPAR-a and co-acti-
vator SRC-1 in MCF-7 cells.
Feeding experiments
Drugs: Oleylethanolamide (OEA), GW7647, anandamide (AEA) and
oleic acid were purchased from Tocris Biosciences (Cookson Ltd.
Bristol, UK). For in vitro cell culture experiments, all compounds
were dissolved and diluted in DMSO (Sigma–Aldrich, Spain).
Feeding experiments were carried out using animals that had been
deprived of food for 24 h but permitted free access to water. Thirty
minutes post-injection, a weighed amount of food was placed in
the cage. The food was weighed again at 30, 60, 120, and 240 min
from test initiation. All feeding experiments were performed with
groups of eight animals (n=8). In capsazepine experiments, capsa-
zepine was injected five minutes prior to the compound being
tested.
DNA constructs: Full-length cDNA for human PPAR-a, human
RXRa, and human SRC-1 were subcloned into the T7/SV40 promot-
er-driven pSG5 expression vector (Qiagen). These constructs were
used for viral promoter-driven overexpression of their respective
proteins in mammalian cells.
Reporter gene constructs: Four copies of the human CPTI gene
DR1-type RE (core sequence GTA GGG AAA AGG TCA) were individ-
ually fused with the thymidine kinase (tk) minimal promoter con-
trolling the firefly luciferase reporter gene.
Open field test
This test measures the natural conflict of the animal between the
tendency to explore and the avoidance reaction to protect itself.
All rats were handled for a period of 15 min over two days to mini-
mize injection stress. They were acclimated to the testing room for
30 min before commencing behavioral testing. The animal was in-
jected with the compound and placed on the center of a 40 cm ”
40 cm table with 30 cm walls. The total displacement and the time
spent by the animal on the corners or in the center of the table
were registered using a video tracking system (Smartꢁ Panlab, Bar-
celona, Spain).
Transient transfection and luciferase reporter assays[29–31]
MCF-7 human breast cancer cells were seeded into 6-well plates
(200000 cells per well) and grown overnight in phenol red-free
DMEM, supplemented with 5% charcoal-stripped fetal bovine
serum. Liposomes containing plasmid DNA were formed by incu-
bating 1 mg of an expression vector for wild-type PPAR-a, RXRa, or
SRC-1 and 1 mg of reporter plasmid with 10 mg of N-[1-(2,3-dioleoy-
loxy)propyl]-N,N,N-trimethylammonium
methylsulfate
(DOTAP,
In vitro experiments
Roche) in a total volume of 100 mL for 15 min at room tempera-
ture. After dilution with 900 mL phenol red-free DMEM, the lipo-
somes were added to the cells. Phenol red-free DMEM, supple-
mented with 500 mL 15% charcoal-stripped fetal bovine serum was
added 4 h after transfection. At this time, cells were treated for
Rat cerebellum and cortex membranes homogenization
The rats were anesthetized with Dolethalꢁ (pentobarbital) and sac-
rificed by decapitation. The cortex and cerebellum were extracted
and stored separately in dry ice. The cerebellum and cortex were
16 h with solvent (DMSO) and various concentrations (10À9, 10À8
,
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1781 – 1787

Catechol Metabolites with Antiobesity Properties
10À7, 10À6, 10À5 and 10À4 m) of OEA, GW7647, AEA, and oleic acid,
as well as the various compounds under evaluation as previously
indicated. The cells were lysed 16 h after onset of stimulation
using reporter gene lysis buffer (Roche). Both the transient trans-
fection assay and the constant light signal luciferase reporter gene
assay were performed as recommended by the suppliers (Roche
and Roche Diagnostics, respectively). Stimulation of normalized lu-
ciferase activity was calculated in comparison with solvent-induced
cells that did not overexpress protein. Data were used to calculate
the EC50 value for each compound.
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This work was supported by grants from Instituto de Salud
Carlos III – Fondo de Investigaciꢁn Sanitaria FIS (FIS CP04/0039,
PI 07/1226 to J.M.D.; PI 07/0880 to F.J.B.S.), Red de Trastornos
Adictivos (FIS-TARD06/001/0026 and RD06/001/0000), Centro de
Investigaciꢁn Biomꢀdica en Red-Fisiopatologꢂa de la Obesidad y
la Nutriciꢁn (CIBEROBN, which is an initiative of the Instituto de
Salud Carlos III, Madrid, Spain), Generalitat de Catalunya (AGAUR
2009 SGR 718), Alban Program, the European Union Program of
High Level Scholarships for Latin America, scholarship number
E06D101051BR (B.A.), European Union FP7 grant HEALTH-F2-
2008-223713, REPROBESITY (F.R.F., F.J.B.S., and M.R.C.), Consejerꢂa
de Economꢂa, Innovaciꢁn y Ciencia, Junta de Andalucꢂa (PI-0232/
2008 to F.J.B.S.), UE-FEDER grant CVI-1038, and Fundaciꢁ La
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Keywords: CB1 · endocannabinoids · obesity · PPAR-a ·
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Published online on August 18, 2010
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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