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Astarita et al.
visceral malaise, anxiety-like behaviors, or stress hormone acid chloride (Nu-Chek Prep, Elysian, MN) with a 10-fold excess of
ethanolamine (Sigma-Aldrich Co.) in dichloromethane. The reaction
was conducted at 0–4°C under stirring for 15 min, and the products
were washed with water, dehydrated over sodium sulfate, filtered,
and dried under reduced pressure. The crude product was purified by
flash-chromatography on silica gel (EtOAc) to yield white solids (96%
for 1 and 95% for 2). (Z)-(R)-9-Octadecenamide,N-(2-hydroxyethyl,1-
methyl) (3, KDS-5104), (Z)-(S)-9-octadecenamide,N-(2-hydroxyethyl,
1-methyl (4), and (Z)-9-octadecenamide,N-(2-hydroxyethyl),N-
methyl (5) were synthesized by the stoichiometric reaction of oleoyl
chloride with the respective amine: (R)-(Ϫ)-2-aminopropanol, (S)-
(ϩ)-2-aminopropanol, or N-methyl-N-ethanolamine (Sigma-Aldrich
Co.) in the presence of triethylamine (Sigma-Aldrich Co.) (Lin et al.,
1998). The reaction was conducted in dichloromethane at 0–4°C
under stirring for 12 h. The solvent was removed under vacuum, and
the mixture was dissolved in tetrahydrofuran/ethanol (1:1) and
treated with an aqueous solution of 3 N KOH (2 Eq). The solution
was kept at reflux for 30 min, after which the solvent was removed.
The residue was dissolved in ethyl acetate and washed sequentially
with water, sodium hydroxide (2 N), hydrochloric acid (2 N), and
brine. The organic phase was dried over sodium sulfate, filtered, and
concentrated under reduced pressure. Crude products were purified
by flash-chromatography on silica gel (EtOAc) to yield 96% of white
solid products (3 and 4) and 97% of an oily product (5). (Z)-3-Hy-
droxypropionamide,N-(8-heptadecenyl) (6) was synthesized in two
steps. The first step consisted in the synthesis of (Z)-8-heptadece-
nylamine; oleoyl chloride was reacted with an aqueous solution of
sodium azide in dichloromethane in phase-transfer conditions using
tetrabutylammonium bromide as surfactant. The resulting acyl
azide was converted to isocyanate through Curtius rearrangement in
refluxing toluene (Pfister and Wymann, 1983). Subsequent treat-
ment with 1 N NaOH in tetrahydrofuran at reflux afforded (Z)-8-
heptadecenylamine. Reaction of this product with trimethylalumi-
nium and -propiolactone in dichloromethane (Lin et al., 1998)
yielded (Z)-3-hydroxypropionamide,N-(8-heptadecenyl) (6). The reac-
tion mixture was refluxed for 6 h, quenched with 50 ml of 1 N HCl,
and extracted with CH2Cl2. The organic layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
product was purified by flash-chromatography on silica gel (EtOAc)
to yield a solid (33%), with the following chemical-physical proper-
ties. The purity of all compounds was Ͼ98% by liquid chromatogra-
phy/mass spectrometry (LC/MS). The syntheses of compounds 4 and
6 have never been reported before.
release (Rodr´ıguez de Fonseca et al., 2001; Proulx et al.,
2005).
The nuclear receptor peroxisome proliferator-activated re-
ceptor-␣ (PPAR-␣) is a key regulator of lipid metabolism and
energy balance in mammals (Desvergne and Wahli, 1999).
Although OEA may bind to multiple receptors (Wang et al.,
2005; Overton et al., 2006), three distinct lines of evidence
indicate that PPAR-␣ mediates the satiety-inducing effects of
this compound. First, OEA binds with high affinity to the
purified ligand-binding domain (LBD) of mouse and human
PPAR-␣ (KD 37 and 40 nM, respectively) and activates with
high-potency PPAR-␣-driven transactivation in a heterolo-
gous expression system (EC50 ϭ 120 nM) (Fu et al., 2003).
Second, two structurally different synthetic PPAR-␣ ago-
nists, the compounds GW7647 and Wy-14643, exert anorex-
iant effects that are due to a selective increase in feeding
latency and thus are behaviorally identical to those produced
by OEA (Fu et al., 2003). Third, mutant mice in which the
PPAR-␣ gene has been deleted by homologous recombination
(PPAR-␣Ϫ/Ϫ mice) do not respond to OEA or synthetic
PPAR-␣ agonists, although they retain normal responses to
two mechanistically different anorexiants, cholecystokinin-
octapeptide and fenfluramine (Fu et al., 2003). Together, the
findings outlined above suggest that endogenous OEA, pro-
duced in the proximal small intestine during feeding, regu-
lates between-meal satiety by activating PPAR-␣.
The biological deactivation of OEA is not fully understood
but is likely to involve the enzymatic hydrolysis of this lipid
amide to oleic acid and ethanolamine (Lo Verme et al.,
2005b). Two structurally distinct OEA-hydrolyzing enzymes
have been characterized: fatty-acid amide hydrolase (FAAH),
an intracellular membrane-bound serine hydrolase (De´sar-
naud et al., 1995; Hillard et al., 1995; Ueda et al., 1995;
Cravatt et al., 1996), and N-acylethanolamine-hydrolyzing
acid amidase, a lysosomal cysteine hydrolase (Tsuboi et al.,
2005; Ueda et al., 2005). In the present study, we set out to
develop analogs of OEA that resist enzymatic hydrolysis
while retaining agonist activity at PPAR-␣. Such agents may
not only be useful to characterize the pharmacological prop-
erties of OEA in live animals but might also provide a start-
ing point for the development of novel antiobesity, antihyper-
lipidemic, and anti-inflammatory drugs (Willson et al., 2000).
Chemical Analyses. Melting points were determined on a Buchi
SMP-510 capillary melting point apparatus (Flawil, Switzerland)
and were uncorrected. 1H NMR spectra were recorded on a Bruker
Avance 200 MHz spectrometer (Bruker, Newark, DE); chemical
shifts are reported in parts per million relative to the central peak of
the solvent. Coupling constants (J values) are given in Hertz. EI-MS
spectra (70 eV) were taken on a Fisons Trio 1000 spectrometer
(Fisons Instruments, Manchester, UK). Infrared spectra were ob-
tained on a Nicolet Avatar 360 Fourier-Transfor-IR spectrometer
(ThermoNicolet, Madison, WI); absorbance is reported in centime-
terϪ1. Optical rotatory powers were obtained on a PerkinElmer 241
Materials and Methods
Animals. Adult male Wistar rats (250–300 g, 7 weeks old) and
C57/BL6 mice (20–25 g, 8 weeks old) were housed in standard
Plexiglas cages at room temperature. A 12-h light/dark cycle was set
with the lights on at 4:45 AM. Water and standard chow pellets polarimeter (PerkinElmer Life and Analytical Sciences, Boston,
(Prolab RMH 2500; PMI Nutrition International, Brentwood, MO) MA). Elemental analyses for C, H, and N were performed on a Carlo
were available ad libitum. All procedures met the National Institutes Erba analyzer and were within Ϯ0.4% of theoretical values. Com-
of Health Guidelines for the Care and Use of Laboratory Animals and pound 1 presented the following chemical-physical properties: MS
were approved by the Institutional Animal Care and Use Committee (EI) m/z 325 (Mϩ); 1H NMR (CDCl3) ␦: 5.84 (br s, 1H), 5.31 (m, 2H),
of the University of California, Irvine.
Drugs and Solvents. URB597 was synthesized as described pre- 1.59 (m, 2H), 1.31 (br d, 20H), 0.88 (t, 3H). Anal.(C20H39NO2) C, H,
viously (Mor et al., 2004). Solvents were obtained from Burdick and
N. Compound 2: M.p.: 82–84°C; MS (EI) m/z 325 (Mϩ); IR (film
Jackson (Muskegon, MI), and all other chemicals were obtained from cmϪ1): 3398, 3299, 2919, 2849, 1640; 1H NMR (CDCl3) ␦: 5.86 (br s,
Sigma-Aldrich Co. (St. Louis, MO).
1H), 5.38 (m, 2H) 3.73 (m, 2H), 3.42 (m, 2H), 2,20 (t, 2H), 1.95 (m,
Chemical Syntheses. The compounds prepared and tested in the 4H), 1.82 (br s, 1H), 1.63 (m, 2H), 1.27 (br d, 20H), 0.88 (t, 3H).
present study are illustrated in Table 1. (Z)-9-Octadecenamide, N-(2- Anal.(C20H39NO2) C, H, N. Compound 3: M.p.: 39–40°C; [a]25
3.57 (m, 2H), 3.27 (m, 2H), 2.51 (br s, 1H), 2.17 (t, 2H), 2.01 (m, 4H),
ϭ
D
hydroxyethyl) (OEA, 1) and (E)-9-octadecenamide,N-(2-hydroxyl- ϩ8.5; MS (EI) m/z 339 (Mϩ); IR (film cmϪ1): 3390, 3300, 2922, 2851,
ethyl) (2) were synthesized by the reaction of the respective fatty-
1643; 1H NMR (CDCl3) ␦: 5.87 (br d, 1H), 5.33 (m, 2H), 4.40 (br s,