Development of the first potential covalent inhibitors of anandamide cellular uptake

Article abstract of DOI:10.1021/jm051226l

On the basis of the chemical structures of two previously developed metabolically stable and relatively potent inhibitors of anandamide uptake, OMDM-1,2, two series of potential covalent inhibitors of anandamide cellular reuptake, which might be used for the molecular characterization of the protein(s) involved in the membrane transport of endocannabinoids, have been designed and synthesized. Most of the compounds inhibited uptake to a varied extent and in a generally enantio-sensitive manner when co-incubated with [ ¹⁴C]anandamide, but only three of them, the photoactivatable 1a (OMDM-37), 1b (OMDM-39), and 8 (Lo395), also produced a significant inhibition of uptake following the preincubation only of the cells, and this effect was significantly enhanced following UV exposure only in the case of 8. None of the new compounds inhibited [¹⁴C]anandamide hydrolysis with IC ₅₀ < 50 μM, except for 1b.

Full text of DOI:10.1021/jm051226l

2320
J. Med. Chem. 2006, 49, 2320-2332
Development of the First Potential Covalent Inhibitors of Anandamide Cellular Uptake
Aniello Schiano Moriello,† Laurence Balas,‡ Alessia Ligresti,† Maria Grazia Cascio,†,§ Thierry Durand,‡ Enrico Morera,|
Giorgio Ortar,| and Vincenzo Di Marzo*,†
Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Via dei Campi Flegrei 34, 80078
Pozzuoli (Napoli), Italy, UMR-CNRS 5074, UniVersite´ Montpellier I, aVenue Charles Flahault, 34093 Montpellier, France, Dipartimento di
Scienze Farmaceutiche, UniVersita` degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy, and Dipartimento di Studi
Farmaceutici, UniVersita` di Roma 'La Sapienza', piazzale Aldo Moro 5, 00185 Roma, Italy
ReceiVed December 7, 2005
On the basis of the chemical structures of two previously developed metabolically stable and relatively
potent inhibitors of anandamide uptake, OMDM-1,2, two series of potential covalent inhibitors of anandamide
cellular reuptake, which might be used for the molecular characterization of the protein(s) involved in the
membrane transport of endocannabinoids, have been designed and synthesized. Most of the compounds
inhibited uptake to a varied extent and in a generally enantio-sensitive manner when co-incubated with
[14C]anandamide, but only three of them, the photoactivatable 1a (OMDM-37), 1b (OMDM-39), and 8
(Lo395), also produced a significant inhibition of uptake following the preincubation only of the cells, and
this effect was significantly enhanced following UV exposure only in the case of 8. None of the new
compounds inhibited [14C]anandamide hydrolysis with IC50 < 50 µM, except for 1b.
Introduction
These compounds enhance some pharmacological actions of
AEA,19 elevate brain endocannabinoid levels,20 and exert
beneficial effects in animal models of disorders where endocan-
nabinoids seem to play a protective function, for example, during
both the symptoms and inflammatory consequences of experi-
mental allergic encephalomyelitis.19,21,22
To date, two G-protein-coupled receptors for Cannabis
psychoactive principle ∆9-tetrahydrocannabinol have been
cloned: cannabinoid CB1 and CB2 receptors.1 Endocannabinoids
are defined as endogenous agonists of these receptors2 and have
been implicated in several physiological and pathological
conditions in mammals.3,4 The levels of the two major endocan-
nabinoids, anandamide (AEA)5 and 2-arachidonoylglycerol (2-
AG)6,7 are regulated by specific biosynthetic and degradative
reactions catalyzed by enzymes that have been recently char-
acterized and cloned.3 The possible existence of a plasma
membrane protein mediating both the release from the cells of
endocannabinoids after biosynthesis on demand and the cellular
re-uptake following the activation of CB1 and CB2 cannabinoid
receptors, according to the AEA concentration gradient across
the cell membrane, has been suggested but is still contro-
versial.2,8-13 Nevertheless, numerous synthetic substances are
now available that can selectively inhibit the cellular re-uptake
of AEA14,15 without interfering with other proteins or the major
enzyme involved in the degradation of this compound, fatty acid
amide hydrolase (FAAH).16 In some cases, very subtle changes
in its chemical structure or the inversion of the stereochemistry
of a chiral center can dramatically affect the capability of a
certain compound to inhibit AEA re-uptake,9,17,18 thus supporting
the existence of a specific protein facilitating this process.
Nevertheless, a protein capable of specifically binding AEA and
other endocannabinoids with the ability to facilitate their
transport across the cell membrane has not been identified yet.
In the present study, we have designed and synthesized 21
structural analogues of OMDM-1,2 (Figure 1) aiming to develop
covalent inhibitors of the putative AEA membrane transporter.
For this purpose, we have modified either the polar aromatic
head or the alkyl tail of either compound by introducing
chemical functions potentially capable of forming covalent
bonds with amino acid residues. We have then examined the
effects of the new compounds on AEA uptake from RBL-2H3
or C6 cells, where a putative AEA transporter has been
preliminarily characterized,23,24 either under normal cell culturing
conditions or after brief exposure to UV light. We report data
suggesting that the putative mechanism(s) mediating AEA
transport across the cell membrane is indeed mediated by one
or more photoaffinity labelable proteins.
Chemistry
The 21 compounds in Figure 1 were prepared as detailed
below and as summarized in Schemes 1-5. The commercially
available N-Boc-4-iodophenylalanines (S)-9 and (R)-9 were
reduced to N-Boc-4-iodophenylalaninols (S)-10 and (R)-10 via
mixed anhydrides. The alcoholic function has been protected
as the acetate, and the resulting N-Boc-4-iodophenylalaninol
acetates (S)-11 and (R)-11 have been converted into 4-trimeth-
ylstannyl analogues 12. The carbonylative Stille reaction25 of
(S)-12 and (R)-12 with C6H5I or t-BuOCOOC6H4I under
atmospheric CO pressure in the presence of PdCl2/2PPh3
proceeded smoothly to give 4-benzoyl derivatives (S)-13 and
(R)-13. Alkaline hydrolysis of the ester group to give alcohols
(S)-14 and (R)-14 followed by the removal of the Boc protecting
group with SOCl2-MeOH afforded amine hydrochlorides (S)-
15 and (R)-15, which were condensed with oleic acid using
1-hydroxybenzotriazole (HOBt)/N-ethyl-N′-(3-dimethylamino-
We have previously shown that selective and metabolically
stable inhibitors of AEA cellular uptake can be obtained by
functionalizing the polar head of oleoylethanolamine with
aromatic groups, as in the case of the two relatively potent
enantiomers, OMDM-1 and OMDM-2, and their analogues.9,15,18
* To whom correspondence should be addressed. Phone: +39-81-
8675093. Fax: +39-81-8041770. E-mail: vdimarzo@icmib.na.cnr.it.
† Institute of Biomolecular Chemistry.
‡ Universite´ de Montpellier.
§ Universita` degli Studi di Salerno.
| Universita` di Roma ‘La Sapienza’.
10.1021/jm051226l CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/04/2006

Inhibitors of Anandamide Uptake
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2321
bethoxy analogues (S)-16 and (R)-16. The removal of the Boc
protecting group with dry HCl in AcOEt afforded amine
hydrochlorides (S)-17 and (R)-17, which were condensed with
oleic acid using HOBt/EDC as the carboxylate activator to give
amides (S)-18 and (R)-18. Alkaline hydrolysis of the ester
function and the protection of the alcohol group gave acids (S)-
19 and (R)-19. The reaction of (trimethylsilyl)diazomethane with
the acyl chlorides derived from 19 by the action of oxalyl
chloride in the presence of catalytic dimethylformamide (DMF)
yielded diazoketones (S)-20 and (R)-20, which were converted
to 1c and 2c by the hydrolysis of the acetate group. The
treatment of 1c with dry HCl in AcOEt furnished chloromethyl
ketone 1d (Scheme 2).
Commercially available N-Boc-tyrosines (S)-21 and (R)-21
were converted to corresponding R-diazomethyl ketones (S)-
22 and (R)-22 by the formation of the mixed anhydride with
isobutyl chloroformate, followed by trapping with diaz-
omethane.26 The treatment of diazomethyl ketones 22 with a
solution of dry HCl in AcOEt provided chloromethyl ketone
derivatives (S)-23 and (R)-23, which were subsequently con-
verted to 3a and 4a by coupling with oleic acid activated as
mixed anhydride by a treatment with isobutyl chloroformate.
Nucleophilic displacement with sodium iodide in dry acetone
afforded corresponding iodide derivatives 3b and 4b, which,
in turn, were transformed into 3c and 4c by treatment with 2,6-
dichlorobenzoic acid in DMF in the presence of potassium
fluoride (Scheme 3).
Commercially available 8-bromooctan-1-ol 24 was trans-
formed into its corresponding tetrahydropyranyl (THP) and
t-butyldimethylsilyl (TBDMS) ethers 25 and 26 as described
previously.27,28 Silyl ether 26 was converted to 10-[(t-butyldi-
methylsilyl)oxy]-1-decyne (27) by reaction with lithium acetyl-
ide-ethylenediamine complex according to a literature proce-
dure.28 Terminal alkyne 27 was alkylated with 25 to furnish
1-[(t-butyldimethylsilyl)oxy]-18-[(2-tetrahydropyranyl)oxy]-9-
octadecyne (28). Stereoselective reduction of 28 with P-2
nickel29 to give (Z)-alkene 29, followed by the deprotection of
the TBDMS protecting group with n-Bu4NF and the oxidation
of resulting alcohol 30 with pyridinium dichromate (DCC) in
DMF afforded a carboxylic acid that was directly converted to
corresponding methyl ester 31. The deprotection of the THP
protecting group with pyridinium p-toluenesulfonate30 gave
alcohol 32, which was converted to bromide 33 by treatment
with PPh3/CBr4.31 Hydrolysis of the ester group with LiOH
produced acid 34, which was used in the condensation reactions
Figure 1. Structures of OMDM-1,2 and the novel compounds
synthesized and tested in this study.
propyl)carbodiimide hydrochloride (EDC) as the carboxylate
activator to give 1a (OMDM-37), 2a, 1b (OMDM-39), and 2b
(Scheme 1).
Palladium-catalyzed alkoxycarbonylation of N-Boc-4-iodo-
phenylalaninols (S)-10 and (R)-10 (see above) provided 4-car-
Scheme 1a
a Reagents and conditions: (a) i-BuOCOCl, N-Methylmorpholine, THF, -15 °C, 15 min, then NaBH4, H2O, -15 °C, 45 min; (b) (CH3CO)2O, pyridine,
rt, 18 h; (c) Pd(OAc)2/2PPh3, Me3SnSnMe3, toluene, 100 °C, 15 min; (d) C6H5I or t-BuOCOOC6H4I, CO, PdCl2/2PPh3, DMF, 90 °C, 3.5 h; (e) 2N NaOH,
MeOH, rt, 4 h; (f) SOCl2, MeOH, 50 °C, 3 h; (g) oleic acid, HOBt/EDC, Et3N, DMF, rt, 12 h.

2322 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Schiano Moriello et al.
Scheme 2a
Scheme 4a
a Reagents and conditions: (a) Pd(OAc)2/2PPh3, CO, EtOH, Et3N, DMF,
80 °C, 12 h; (b) dry HCl, AcOEt, rt, 5 h; (c) oleic acid, HOBt/EDC, Et3N,
DMF, rt, 12 h; (d) 1M LiOH, THF-H2O, 50 °C, 18 h; (e) (CH3CO)2O,
pyridine, rt, 4 h; (f) (COCl)2, DMF, 0 °C, 1h, then Me3SiCHN2, Et3N, 0
°C, 12 h; (g) 1M LiOH, THF-H2O, 0 °C, 3.5 h; (h) dry HCl, AcOEt, rt,
1 h.
Scheme 3a
a Reagents and conditions: (a) 3,4-dihydro-2H-pyran, pyridinium p-
toluenesulfonate, CH2Cl2, rt, 18 h; (b) t-BuMe2SiCl, Et3N,DMAP, CH2Cl2,
rt, 18 h; (c) lithium acetylide ethylenediamine complex, DMSO, rt, 24 h;
(d) 1.2 M n-BuLi, THF, -25 °C, 15 min, then 25, -25 °C, 2 h, then rt, 12
h; (e) P-2 Nickel, EtOH, rt, 2 h; (f) n-Bu4NF, THF, rt, 2 h; (g) PDC, DMF,
rt, 48 h; (h) CH2N2, Et2O, 0 °C, 45 min; (i) pyridinium p-toluenesulfonate,
EtOH, 55 °C, 12 h; (j) CBr4, PPh3, CH2Cl2, 0 °C, 40 min; (k) 1 M LiOH,
THF-H2O, rt, 12 h; (l) (S)- or (R)-tyrosinol, DCC, DMF, rt, 12 h; (m)
NaI, acetone, rt, 12 h; (n) KCN, DMSO, rt, 12 h.
a Reagents and conditions: (a) i-BuOCOCl, Et3N, THF, -15 °C f rt,
then CH2N2, rt, 2 h; (b) dry HCl, AcOEt, rt, 48 h; (c) oleic acid, i-BuOCOCl,
N-Methylmorpholine, THF-DMF, -15 °C f rt, then 23, rt, 24 h; (d) NaI,
acetone, rt, 16 h; (e) KF, 2,6-dichlorobenzoic acid, DMF, rt, 16 h.
transporter in RBL-2H3 cells and C6 glioma cells. Protocol #1
was used to evaluate the affinity of all compounds for the
putative protein(s) involved in cellular [14C]AEA uptake and
consisted of co-incubating RBL-2H3 cells with [14C]AEA for
5 min and increasing the concentrations of the compounds. In
some cases, the compounds were added 10 min prior to [14C]-
AEA. Protocols #2 and 3 were used only for those substances
that showed some activity when protocol #1 was used and for
nonphotoactivatable and UV-activatable compounds, respec-
tively. Protocol #2 was carried out in RBL-2H3 cells preincu-
bated with the inhibitors for 1 h, then thoroughly washed and
incubated with [14C]AEA for 5 min in the absence of inhibitors.
Protocol #3 was carried out in C6 cells preincubated with the
inhibitors for 8 min with or without exposure to UV light, then
thoroughly washed and incubated with [14C]AEA for 5 min in
the absence of inhibitors. Finally, all compounds were assessed
for their capability to inhibit [14C]AEA hydrolysis using rat brain
membranes.
with (S)- and (R)-tyrosinol in the presence of N,N′-dicyclo-
hexylcarbodiimide (DCC) to furnish 5a and 6a. Bromides 5a
and 6a were converted to corresponding iodides 5b and 6b and
nitriles 5c and 6c by treatment with NaI in acetone and KCN
in DMSO, respectively (Scheme 4).
The synthesis of 8 and its noniodinated and nonphotoacti-
vatable analogue 7 was carried out in 8 steps from azelaic acid
monomethyl ester 35 via key common intermediate 39 (Scheme
5).32 The first three steps to reach Z-derivative 37 are the
reduction of carboxylic acid 35 using diborane followed by a
Swern oxidation to afford aldehyde 36 according to Okuma and
co-workers,33 then a three carbon atom elongation in the
presence of 3-(2-tetrahydropyranyloxy)propyl triphenylphos-
phonium bromide as described previously34 to give Z-derivative
37. After hydrolysis with LiOH in THF, the resulting carboxylic
acid was coupled with di-O-t-butyldiphenylsilyl-L-tyrosinol (38)
to give amide 39 using the anhydride activation procedure. After
the removal of the THP group using classical acidic conditions,
key alcohol 40 was obtained. Steglich esterification of 40 with
benzoic acid, followed by fluoride cleavage of the silyl groups
provided 7. Application of the same esterification procedure
using 2-azido-5-iodobenzoic acid35 afforded photoactivatable 8
(Lo395).
Results and Discussion
The results are summarized in Tables 1 and 2 and described
below.
OMDM-1,2 analogues with headgroup modifications can
ideally be divided into two subgroups. In the first subgroup,
the ethanolamine moiety of OMDM-1,2 was left unaltered,
whereas in the aromatic region, the OH group was replaced by
an electrophilic or a photoactivatable moiety. This yielded seven
compounds, four of which (1a, 2a, 1b, and 2b) were potentially
capable of forming covalent bonds with proteins only after
Biological Evaluation
Three types of assays were used to investigate the activity
of the novel compounds on the putative AEA membrane

Inhibitors of Anandamide Uptake
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2323
Scheme 5a
a Reagents and conditions: (a) B2H6, THF; (b) (COCl)2, DMSO, Et3N; (c) KHMDS, THF, -78 °C; (d) LiOH, THF; (e) i-BuOCOCl, N-Methylmorpholine,
Et3N, then di-O-t-Butyldiphenylsilyl-L-tyrosinol (38); (f) TsOH‚H2O, MeOH; (g) PhCO2H, DCC, DMAP, CH2Cl2; (h) n-Bu4NF, THF; (i) 2-azido-5-iodobenzoic
acid, DCC, DMAP.
Table 1. Inhibitory Effect of Compounds on [14C]Anandamide Uptake
Table 2. Inhibition of [14C]Anandamide Uptake by C6 Cells by 1a, 1b,
and 8a
by Intact RBL-2H3 Cellsa
protocol #1
protocol #2
compd, concn, N
% inhibition, dark
% inhibition, UV
compd
1a
2a*
1b
2b*
1c
2c*
1d
3a
4a*
3b
4b*
3c
4c*
5a
6a*
5b
6b*
5c
6c*
7
8 (Lo395)
(co-incubation only)
(1 h preincubation only)
1a, 5 µM, N ) 3
1a, 10 µM, N ) 3
1b, 5 µM, N ) 3
1b, 10 µM, N ) 3
8, 10 µM, N ) 6
37.5 ( 1.5
65.8 ( 6.5
28.3 ( 0.9
73.6 ( 7.5
32.8 ( 8.6
30.3 ( 3.1 (P > 0.1)
57.2 ( 5.5 (N > 0.1)
68.6 ( 24.0 (P ) 0.1)
73.2 ( 6.9 (P > 0.1)
60.0 ( 13.9 (P ) 0.038)
6.0 ( 0.9
10.0 ( 1.2b
10.1 ( 1.3
8.1 ( 0.8
>25
N. T. (protocol #3)
N. T.
N. T. (protocol #3)
N. T.
N. T.
N. T.
N. T.
>25
N. T.
a According to protocol #3 (8 min preincubation of cells with inhibitors
only, either in the dark or under UV irradiation, followed by washing of
the cells, and incubation with [14C]anandamide only; see Biological
Evaluation and Experimental Section for a detailed description of this
protocol). Data are means ( SE of N ) 3 or 6 experiments. Statistically
significant differences between dark and UV are shown and were calculated
by ANOVA followed by the Student Newman Keuls test.
>25
>25
10.0 ( 4.1
>25b
>25
N. T.
N. T.
>25
16.1 ( 2.6
7.5 ( 1.5b
10.2 ( 1.4
20.1 ( 2.3b
10.8 ( 1.5
14.0 ( 1.1b
23.1 ( 2.6
>25b
>25
>25
>25
require exposure to UV light necessary to photoactivate the two
compounds, although the low dose of 1b tended to be more
efficacious in the presence of UV light (P ) 0.1). Finally, of
the seven compounds of this subgroup, only 1b exerted some
inhibition of [14C]AEA hydrolysis by rat brain membranes (IC50
) 10.2 ( 2.1 µM, mean ( SE, N ) 3). This weak inhibitory
effect was not improved after UV irradiation (data not shown).
The second subgroup of headgroup-modified OMDM-1,2
analogues, comprises compounds 3a, 4a, 3b, 4b, 3c, and 4c,
where the aromatic region of OMDM-1,2 was left unaltered,
whereas the ethanolamine moiety was replaced by an electro-
philic halo- or acyloxymethyl ketone group. Within this
subgroup, only three compounds (3a, 3c, and 4c) exhibited
moderate potency in inhibiting [14C]AEA uptake in RBL-2H3
when co-incubated with the radiolabeled substrate (IC50 values
ranging between 7.5 and 16.0 µM, protocol #1), whereas the
other three did not inhibit the uptake up to a 25 µM concentra-
tion. However, 3a, 3c, and 4c were no longer active when they
were only preincubated with cells for up to 60 min in the
absence of the substrate and then washed before adding [14C]-
AEA (protocol #2). This indicates that none of these putative
covalent inhibitors binds stably to any of the potential proteins
involved in AEA cellular uptake under our experimental
conditions. Finally, none of the six compounds of this second
group of headgroup-modified OMDM-1,2 analogues inhibited
[14C]AEA hydrolysis by rat brain membranes (IC50 > 50 µM,
data not shown).
N. T.
>25
>25
N. T.
N. T
N. T.
N. T. (protocol #3)
>25
11.0 ( 1.6
a Assessed by using protocol #1 (co-incubation of cells with inhibitors
and [14C]anandamide) and, only for those compounds exhibiting IC50
<
20 µM with protocol #1, also by using protocol #2 (1 h preincubation of
cells with inhibitors only, followed by washing and incubation with
[14C]anandamide only; see Biological Evaluation and Experimental Section
for a detailed description of the two protocols). N. T., not tested or, when
indicated, tested with protocol #3 (Table 2). Asterisks denote those
compounds that are the R enantiomers of the compounds preceding them
in the list. Data are means ( SE of N ) 3 experiments. b Denotes a mean
significantly different from the IC50 value of the corresponding enantiomer
using ANOVA followed by the Student Newman Keuls test, and P < 0.05
as the threshold of significance.
exposure to UV, whereas the other three, 1c, 2c, and 1d, could
form covalent bonds without UV light. Of these seven com-
pounds, only the former four UV-sensitive derivatives inhibited
[14C]AEA uptake in RBL-2H3 cells with moderate potency
when co-incubated with the radiolabeled substrate (IC50 values
ranging between 6.0 and 10.1 µM, protocol #1) and little or no
enantioselectivity. Two of the four potential UV-sensitive
ligands, that is, 1a and 1b, still inhibited [14C]AEA uptake with
approximately the same potency when they were only prein-
cubated with C6 cells for 8 min in the absence of the substrate
and then washed before adding [14C]AEA (protocol #3), thus
pointing to a possible irreversible inhibition of the uptake
mechanism. However, this effect of 1a and 1b did not seem to
To the group of OMDM-1,2 analogues with alkyl tail
modifications belong eight compounds, six of which were
designed to covalently bind to the putative AEA membrane

2324 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Schiano Moriello et al.
transporter under normal cell culturing conditions and one,
compound 8, only after exposure to UV light. The eighth
compound of this group, 7, was synthesized as a noncovalent
reference compound for 8. All but one (i.e., 6c) of the six
potential covalent inhibitors exhibited affinity for putative
transporter by inhibiting [14C]AEA uptake in co-incubation
experiments (IC50 values ranging between 10.2 and >25 µM,
protocol #1), the most potent of which were 5a, 5b, and 6b
(IC50 ) 10.2, 10.8, and 14.0 µM, respectively). In this series, a
marked enantioselectivity was observed with all three couples
of enantiomers similar to that previously reported for other
OMDM-1,2 analogues.18 Interestingly, three more potent enan-
tiomers in each couple, that is, 5a, 5b, and 5c, became
significantly more potent if preincubated with cells for 10 min
and then co-incubated with [14C]AEA (IC50 ) 2.1 ( 0.3, 2.4
( 0.3, and 2.5 ( 0.4 µM, respectively, mean ( SE, N ) 3).
This suggests that allowing more time to the compounds of these
series to interact with the cells might sensibly improve their
potency. However, none of these three compounds inhibited the
uptake when they were only preincubated with cells for up to
1 h in the absence of [14C]AEA and then washed prior to the
introduction of the radiolabeled substrate in the incubation
medium (protocol #2).
lectivity of the chiral carbon atom (generally S) is required for
optimal inhibitory activity.
Possibly the most novel finding of our present study is that
although all novel compounds were designed to form covalent
bonds with the putative AEA transporter either following long
preexposure of the cells under normal light culturing conditions
or after UV irradiation three of them were indeed capable of
inhibiting AEA cellular uptake in a way that resisted thorough
washes of the cells with a BSA-containing medium; therefore,
this suggests a possible irreversible binding with the putative
protein(s) mediating this process. In fact, the observation that
out of 21 analogues with similar chemical properties the
inhibitory effects of only 3 compounds were resistant to repeated
washes validates our procedure as a possible means to assess
covalent or at least stable bonds with inhibitors of AEA uptake.
However, these three compounds, that is, 1a, 1b, and 8, were
designed to form covalent bonds with specific proteins only
after UV irradiation, and only the inhibitory effect of the latter
was significantly improved by UV irradiation, thus indicating
that perhaps the resistance to repeated washes of the inhibitory
effects is not always diagnostic of covalent inhibitors. However,
it must be emphasized that at least at one of the two concentra-
tions tested the inhibitory effect of 1b showed a strong trend
(P ) 0.1) toward being enhanced following UV irradiation. It
is therefore possible that photoactivatable labeling of the putative
protein(s) mediating AEA cellular uptake can occur in parts of
the binding site(s) that correspond to the positioning of both
the headgroup and the alkyl chain of the inhibitor. However,
because 8 did not inhibit AEA hydrolysis, either when co-
incubated or when preincubated with rat brain membranes in
the presence or absence of UV irradiation, it is very unlikely
that its stable inhibition of AEA cellular uptake is due to the
inhibition of intracellular AEA hydrolysis by FAAH.
Compound 8 and not 7 exhibited some affinity for the putative
transporter when using protocol #1 (IC50 ) 11.0 µM) and was
therefore tested using protocol #3 with and without exposure
to UV light. In this case, 10 µM 8 could inhibit [14C]AEA uptake
when only preincubated with cells and then washed prior to
introduction of [14C]AEA, and UV exposure significantly
enhanced this effect of the compound, thus suggesting a possible
photoactivation of 8 binding to the protein(s) involved in AEA
membrane transport.
Finally, none of the eight compounds of this second group
of OMDM-1,2 analogues inhibited [14C]AEA hydrolysis by rat
brain membranes under all conditions tested (IC50 >50 µM,
either with or without UV irradiation; data not shown).
In conclusion, we have reported here the development of
several novel compounds designed with the purpose of obtaining
covalent labeling of the putative AEA membrane transporter
or the other protein(s) participating in AEA cellular uptake. Most
of the new compounds exhibited an affinity for this process, as
shown by their capability to inhibit the uptake of radiolabeled
AEA when co-incubated with this compound. Furthermore, three
UV-sensitive compounds, selected among those with the highest
affinity for the putative AEA membrane transporter, stably
inhibited AEA uptake even after repeated washes of the cells,
and one of them did so more effectively following UV
irradiation. A recent elegant study showed that high-affinity
binding sites for some tetrazole-based urea derivatives are
present on the membrane of RBL-2H3 cells and are responsible
for the facilitated uptake of AEA by these cells.39 However,
the authors did not investigate the stable (possibly covalent)
binding of any of their compounds to the putative membrane
transporter. Therefore, our data simultaneously provide further
evidence for the existence of specific protein(s) mediating AEA
cellular uptake and new tools that might be useful for the
isolation and molecular characterization of these proteins.
Of the headgroup derivatives, the ones whereupon the
p-hydroxy-phenyl group of OMDM-1,2 was substituted with a
benzophenone group, either underivatized (1a, 2a) or containing
a p-hydroxy group (1b, 2b), and the ones where the hydroxym-
ethyl group of the ethanolamine portion was substituted with a
2-chloromethyl ketone group (3a) or an o,o′-dichloro benzoy-
loxymethyl ketone group (4c) were the ones with the highest
affinity for the putative transporter. However, none of the new
headgroup analogues was more potent than the parent com-
pounds OMDM-1,2. These findings confirm that if there is any
membrane protein specifically involved in AEA cellular re-
uptake its binding site has lower affinity for inhibitors with
headgroups that are too big and/or polar and at the same time
do not exhibit a high degree of aromatization.36 Furthermore,
even with those headgroups possessing several aromatic rings
(3c, 4c), or that are small but hydrophobic (3a, 4a), a certain
configuration (generally, but not always, S) is required for
optimal activity, as previously shown for other analogues of
OMDM-1,2.18
Experimental Section
Regarding the alkyl chain derivatives of OMDM-1,2, again
none of the new compounds developed here exhibited a higher
affinity than the parent compounds for the putative AEA
transporter, in agreement with previous findings with arvanil
and its alkyl chain derivatives.37,38 However, we found here that
with some of the new derivatives belonging to this subgroup a
short preincubation with the cells can significantly enhance their
capability to inhibit AEA uptake and that a certain enantiose-
Chemistry. All chemical reagents were commercially available.
Melting points were determined on a Kofler hot-stage apparatus
and are uncorrected. Optical rotations were measured at 20 °C with
a Schmidt-Haensch Polartronic D polarimeter (1 dm-cell) in 1%
CHCl3 solutions unless otherwise indicated. IR spectra were
recorded on a Perkin-Elmer 1000 FT-IR spectrophotometer as KBr
1
disks unless otherwise indicated. H and 13C NMR spectra were
obtained on a Varian Mercury 300 or a Bruker AMX300 spec-

Inhibitors of Anandamide Uptake
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2325
trometer using CDCl3 as the solvent unless otherwise indicated and
TMS as the internal standard. The ESI mass spectra were run on a
FINNIGAN LQC advantage spectrometer. FAB spectra were
recorded on a JEOL JMX DX300 spectrometer. Satisfactory
elemental analyses were obtained for the newly synthesized
compounds (C, H, N ( 0.4%).
(S)-N-Boc-4-benzoylphenylalaninol Acetate ((S)-13a). A mix-
ture of (S)-12 (209 mg, 0.5 mmol), C6H5I (0.062 mL, 0.55 mmol),
PdCl2 (5 mg, 0.025 mmol), and PPh3 (13 mg, 0.05 mmol) in DMF
(2 mL) was purged with carbon monoxide for 15 min and then
stirred at 90 °C for 8 h under a CO baloon. The reaction mixture
was then diluted with AcOEt (10 mL) and stirred at room
temperature with saturated aqueous KF (1 mL) for 30 min. The
precipitate was filtered off, and the organic phase was washed three
times with brine, dried (Na2SO4), and evaporated under vacuum.
The residue (252 mg) was chromatographed on silica gel (8 g) using
hexane/AcOEt ) 7/3 as eluent to give (S)-13a as a white solid
(168 mg, 84%). Mp 92-94 °C; [R]D -16°; IR: 3363, 2983, 1720,
Compounds 25,27 26,28 27,28 37,34 and 2-azido-5-iodobenzoic
acid35 have been described in the literature.
(S)-N-Boc-4-iodophenylalaninol ((S)-10). To a stirred solution
of (S)-N-Boc-4-iodophenylalanine ((S)-9) (1.60 g, 4.10 mmol) in
anhydrous THF (35 mL), N-methylmorpholine (4.5 mL, 4.10 mmol)
and isobutyl chloroformate (0.55 mL, 4.10 mmol) were added at
-15 °C. After stirring at -15 °C for 15 min, the solid was removed
by filtration and washed with anhydrous THF, and the filtrate was
treated dropwise with a solution of NaBH4 (235 mg, 6.20 mmol)
in H2O (1.5 mL) at -15 °C under stirring. The reaction mixture
was stirred for 30 min at -15 °C and then diluted with H2O at
room temperature. The resulting precipitate was filtered off after
30 min, washed with H2O and hexane, and dried under vacuum
(1.19 g, 89%). Mp 143-145 °C; [R]D -23°; IR: 3377, 3273, 1659,
1549, 1318, 1175 cm-1; 1H NMR (300 MHz) δ: 1.40 (9H, s), 2.78
(2H, d, J ) 6.9 Hz and 1H br s, overlapped), 3.52 (1H, dd, J )
11.1, 5.1 Hz), 3.62 (1H, dd, J ) 11.1, 3.9 Hz), 3.82 (1H, m), 4.88
(1H, m), 6.97 (2H, d, J ) 8.1 Hz), 7.61 (2H, d, J ) 8.1 Hz); 13C
NMR (75 MHz): δ 28.32, 36.92, 53.41, 63.81, 79.86, 91.73, 128.53,
129.29, 131.39, 137.52, 156.04. Anal. (C14H20INO3) C, H, N.
1
1689, 1670, 1609, 1525, 1367, 1265, 1168 cm-1; H NMR (300
MHz) δ: 1.42 (9H, s), 2.11 (3H, s), 2.86-2.99 (2H, m), 4.07 (2H,
d, J ) 4.5 Hz), 4.15 (1H, m), 4.75 (1H, d, J ) 7.8 Hz), 7.31-7.79
(9H, m); 13C NMR (75 MHz): δ 20.79, 28.28, 38.03, 50.51, 65.04,
79.64, 128.05, 128.99, 129.73, 130.21, 132.12, 135.84, 137.43,
142.11, 154.87, 170.53, 195.95. Anal. (C23H27NO5) C, H, N.
(R)-N-Boc-4-benzoylphenylalaninol Acetate ((R)-13a). Pre-
pared following the same procedure that was used for the synthesis
of (S)-enantiomer. Yield 79%; mp 90-92 °C; [R]D +16°. Anal.
(C23H27NO5) C, H, N.
(S)-N-Boc-4-benzoylphenylalaninol ((S)-14a). To a solution of
(S)-13a (150 mg, 0.38 mmol) in MeOH (1.5 mL), 2 N NaOH (0.38
mL) was added, and the mixture was stirred at room temperature
for 4 h, diluted with water, and extracted with AcOEt. The organic
solution was washed with water, dried (Na2SO4), and evaporated
under vacuum. The residue (121 mg) was chromatographed on silica
gel (3.5 g) using hexane/AcOEt ) 1/1 as eluent to give (S)-14a as
a white solid (112 mg, 84%). Mp 122-124 °C; [R]D -28°; IR:
(R)-N-Boc-4-iodophenylalaninol ((R)-10). The title compound
was prepared following the same procedure that was used for the
synthesis of the (S)-enantiomer. Yield 83%; mp 144-146 °C; [R]D
+23°. Anal. (C14H20INO3) C, H, N.
1
3356, 2970, 1686, 1655, 1527, 1278, 1170, 1007 cm-1; H NMR
(S)-N-Boc-4-iodophenylalaninol Acetate ((S)-11). To a stirred
solution of (S)-10 (1.18 g, 3.13 mmol) in anhydrous pyridine (6.9
mL) was added acetic anhydride (3.4 mL) at 0 °C. The reaction
mixture was stirred overnight at room temperature and then poured
into ice/water. The resulting solid was filtered off, washed with
H2O, and dried under vacuum to give 1.21 g (100%) of ester (S)-
11 as a white solid. Mp 118-120 °C; [R]D -2°; IR: 3363, 2980,
(300 MHz) δ: 1.41 (9H, s), 2.56 (1H, br s), 2.95 (2H, d, J ) 7.8
Hz), 3.59 (1H, dd, J ) 11.0, 5.1 Hz), 3.69 (1H, dd, J ) 11.0, 3.9
Hz), 3.92 (1H, m), 4.88 (1H, m), 7.33-7.79 (9H, m); 13C NMR
(75 MHz): δ 28.36, 37.50, 53.53, 64.10, 79.86, 128.30, 128.31,
129.31, 130.00, 130.44, 132.40, 135.91, 137.69, 143.18, 156.01,
196.53. Anal. (C21H25NO4) C, H, N.
(R)-N-Boc-4-benzoylphenylalaninol ((R)-14a). The title com-
pound was prepared following the same procedure that was used
for the synthesis of the (S)-enantiomer. Yield 93%; mp 121-123
°C; [R]D +28°. Anal. (C21H25NO4) C, H, N.
(S)-4-Benzoylphenylalaninol Hydrochloride ((S)-15a). To a
solution of (S)-14a (102 mg, 0.29 mmol) in anhydrous MeOH (1
mL), SOCl2 (0.022 mL, 0.29 mmol) was added, and the mixture
was stirred at 50 °C for 3 h. Evaporation of the solution under
vacuum furnished (S)-15a as a white solid (84 mg, 100%), which
was used without further purification in the next step.
(R)-4-Benzoylphenylalaninol Hydrochloride ((R)-15a). The
title compound was prepared following the same procedure that
was used for the synthesis of the (S)-enantiomer.
1
1739, 1682, 1530, 1444, 1368, 1299, 1170, 1059 cm-1; H NMR
(300 MHz) δ: 1.41 (9H, s), 2.08 (3H, s), 2.73 (1H, dd, J ) 13.5,
8.4 Hz), 2.80 (1H, dd, J ) 13.5, 6.0 Hz), 4.02 (2H, d, J ) 4.0 Hz),
4.06 (1H, m), 4.65 (1H, m), 6.94 (2H, d, J ) 8.4 Hz), 7.61 (2H, d,
J ) 8.4 Hz); 13C NMR (75 MHz): δ 20.84, 28.33, 37.53, 50.58,
79.84, 131.30, 131.32, 136.94, 137.65, 155.02, 170.86. Anal.
(C16H22INO4) C, H, N.
(R)-N-Boc-4-iodophenylalaninol Acetate ((R)-11). The title
compound was prepared following the same procedure that was
used for the synthesis of the (S)-enantiomer. Yield 83%; mp 117-
118 °C; [R]D +2°. Anal. (C16H22INO4) C, H, N.
(S)-N-Boc-4-trimethylstannylphenylalaninol Acetate ((S)-12).
A stirred mixture of (S)-11 (0.605 g, 1.44 mmol), Me3SnSnMe3
(0.42 mL, 2.02 mmol), Pd(OAc)2 (13 mg, 0.06 mmol), and PPh3
(30 mg, 0.12 mmol) in toluene (5.8 mL) was purged with N2 for
15 min at room temperature and then heated at 100 °C for 15 min
under N2. The reaction mixture was then filtered through a short
pad of silica gel, diluted with Et2O, washed twice with brine, dried
(Na2SO4), and evaporated under vacuum. The residue (0.73 g) was
chromatographed on silica gel (22 g) using hexane/AcOEt ) 9/1
as eluent to give 0.58 g (88%) of (S)-12 as a white solid. Mp 60-
62 °C; [R]D -5°; IR (CHCl3): 3367, 2982, 1731, 1689, 1529, 1263,
(Z)-N-{(1S)-2-Hydroxy-1-[(4-benzoylphenyl)methyl]ethyl}-9-
octadecenamide (1a). To a stirred solution of oleic acid (81 mg,
0.29 mmol) in DMF (1 mL) were added at 0 °C HOBt (41 mg,
0.30 mmol) and EDC (58 mg, 0.30 mmol). The mixture was stirred
for 15 min at 0 °C and for 1 h at room temperature, and (S)-15a
(84 mg, 0.29 mmol) and Et3N (0.040 mL, 0.29 mmol) were added,
and the mixture was stirred overnight at room temperature. The
mixture was diluted with brine and extracted with AcOEt. The
organic phase was washed with 10% citric acid solution, saturated
NaHCO3 and brine, dried (Na2SO4), and evaporated under vacuum.
The residue (145 mg) was chromatographed on silica gel (6 g) using
hexane/AcOEt ) 7/3 as eluent to give 96 mg (64%) of 1a as a
white solid. Mp 69-70 °C; [R]D -16°; IR: 3296, 2925, 1646, 1541,
1
1173 cm-1; H NMR (300 MHz) δ: 0.27 (9H, t, J ) 28.0 Hz),
1.41 (9H, s), 2.09 (3H, s), 2.77 (1H, dd, J ) 13.5, 8.4 Hz), 2.87
(1H, dd, J ) 13.5, 6.0 Hz), 4.02 (2H, d, J ) 4.8 Hz), 4.09 (1H,
m), 4.66 (1H, m), 7.16 (2H, d, J ) 7.6 Hz), 7.42 (2H, d, J ) 7.6
Hz); 13C NMR (75 MHz): δ -9.59 (t, J ) 175 Hz), 20.87, 28.35,
37.94, 50.60, 65.13, 79.29, 129.1, 136.08, 137.11, 140.19, 155.20,
170.92. Anal. (C19H31NO4Sn) C, H, N.
1
1446, 1317, 1277, 1176 cm-1; H NMR (300 MHz) δ: 0.88 (3H,
t, J ) 6.6 Hz), 1.26 (20H, m), 1.55-1.59 (2H, m), 1.99 (4H, m),
2.15 (2H, t, J ) 7.6 Hz), 2.96-2.99 (2H, m), 3.18 (1H, br s), 3.61
(1H, dd, J ) 11.0, 5.1 Hz), 3.66 (1H, dd, J ) 11.0, 3.9 Hz), 4.21
(1H, m), 6.00 (1H, d, J ) 7.9 Hz), 7.34-7.78 (9H, m); 13C NMR
(75 MHz): δ 14.12, 22.68, 25.73, 27.17, 27.23, 29.14, 29.21, 29.27,
29.31, 29.33, 29.53, 29.72, 29.77, 31.90, 36.82, 37.03, 52.43, 63.74,
(R)-N-Boc-4-trimethylstannylphenylalaninol Acetate ((R)-12).
The title compound was prepared following the same procedure
that was used for the synthesis of the (S)-enantiomer. Yield 86%;
mp 57-61 °C; [R]D +5°. Anal. (C19H31NO4Sn) C, H, N.

2326 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Schiano Moriello et al.
128.31, 129.24, 129.70, 129.97, 130.01, 130.46, 132.44, 135.95,
137.60, 143.11, 173.80, 196.48. Anal. (C34H49NO3) C, H, N.
(R)-N-{(1S)-2-Hydroxy-1-[(4-benzoylphenyl)methyl]ethyl}-9-
octadecenamide (2a). The title compound was prepared following
the same procedure that was used for the synthesis of the
(S)-enantiomer. Yield 66%; mp 70-71 °C; [R]D +16°. Anal.
(C34H49NO3) C, H, N.
29.33, 29.55, 29.73, 29.78, 31.92, 36.74, 36.79, 36.99, 52.33, 52.42,
63.24, 115.31, 128.97, 129.12, 129.73, 130.04, 130.11, 132.92,
136.63, 142.43, 161.66, 174.47, 196.01. Anal. (C34H49NO4) C, H,
N.
(Z)-N-{(1R)-2-Hydroxy-1-[[4-(4-hydroxybenzoyl)phenyl]methyl]-
ethyl}-9-octadecenamide (2b). The title compound was prepared
from (R)-15b, following the same procedure that was used for the
synthesis of 1a. Yield 62%; mp 114-116 °C; [R]D +14°. Anal.
(C34H49NO4) C, H, N.
(S)-N-Boc-4-(ethoxycarbonyl)phenylalaninol ((S)-16). A mix-
ture of (S)-10 (2.45 g, 6.49 mmol), Pd(OAc)2 (44 mg, 0.19 mmol),
PPh3 (102 mg, 0.39 mmol), EtOH (7.6 mL, 130 mmol), and Et3N
(1.82 mL, 13 mmol) in DMF (26 mL) was purged with carbon
monoxide for 5 min at room temperature and then stirred under a
CO baloon at 80 °C overnight. The mixture was diluted with brine
and extracted with AcOEt. The organic phase was washed three
times with brine, dried (Na2SO4), and evaporated under vacuum.
The residue (2.64 g) was chromatographed on silica gel (105 g)
using hexane/AcOEt ) 75/25 as eluent to give 1.13 g (54%) of
(S)-16 as a white solid. Mp 103-104 °C; [R]D -25°; IR: 3351,
2981, 1718, 1684, 1611, 1529, 1446, 1368, 1276, 1179, 1104, 1006
cm-1; 1H NMR (300 MHz) δ: 1.39 (3H, t, J ) 7.0 Hz), 1.40 (9H,
s), 2.64 (1H, br s), 2.91 (2H, d, J ) 6.6 Hz), 3.54 (1H, dd, J )
11.0, 4.6 Hz), 3.65 (1H, dd, J ) 11.0, 3.7 Hz), 3.88 (1H, br s),
4.37 (2H, q, J ) 7.0 Hz), 4.88 (1H, m), 7.30 (2H, d, J ) 8.2 Hz),
7.97 (2H, d, J ) 8.2 Hz); 13C NMR (75 MHz): δ 14.34, 28.33,
37.43, 53.47, 60.93, 63.97, 79.88, 128.80, 129.33, 129.79, 143.41,
156.02, 166.60. Anal. (C17H25NO5) C, H, N.
(S)-N,O-BisBoc-4-(4-hydroxybenzoyl)phenylalaninol Acetate
((S)-13b). A mixture of (S)-12 (228 mg, 0.5 mmol), O-Boc-4-
hydroxy-1-iodobenzene (176 mg, 0.55 mmol), PdCl2 (5 mg, 0.025
mmol), and PPh3 (13 mg, 0.05 mmol) in DMF (2 mL) was purged
with carbon monoxide for 15 min and then stirred at 90 °C for 3.5
h under a CO baloon. The reaction mixture was then diluted with
AcOEt (10 mL) and stirred at room temperature with a saturated
aqueous KF solution (1 mL) for 30 min. The precipitate was filtered
off, and the organic phase was washed three times with brine, dried
(Na2SO4), and evaporated under vacuum. The residue (336 mg)
was chromatographed on silica gel (14 g) using hexane/AcOEt )
75/25 as eluent to give (S)-13b as a white solid (133 mg, 52%).
Mp 72-75 °C; [R]D -13°; IR: 3358, 2982, 1759, 1720, 1685,
1
1607, 1525, 1370, 1272, 1148, 1047 cm-1; H NMR (300 MHz)
δ: 1.42 (9H, s), 1.58 (9H, s), 2.09 (3H, s), 2.87-2.94 (2H, m),
4.07 (2H, d, J ) 4.4 Hz), 4.15 (1H, m), 4.72 (1H, m), 7.27-7.84
(8H, m); 13C NMR (75 MHz): δ 20.83, 27.69, 28.33, 38.06, 50.57,
65.16, 79.80, 84.21, 121.12, 129.28, 130.36, 131.56, 135.00, 135.97,
142.45, 151.21, 154.19, 170.85, 195.09. Anal. (C28H35NO8) C, H,
N.
(R)-N,O-BisBoc-4-(4-hydroxybenzoyl)phenylalaninol Acetate
((R)-13b). The title compound was prepared following the same
procedure that was used for the synthesis of the (S)-enantiomer.
Yield 54%; mp 75-77 °C; [R]D +13°. Anal. (C28H35NO8) C, H,
N.
(R)-N-Boc-4-(ethoxycarbonyl)phenylalaninol ((R)-16). The title
compound was prepared following the same procedure that was
used for the synthesis of (S)-16. Yield 53%; mp 102-104 °C; [R]D
+25°. Anal. (C17H25NO5) C, H, N.
(S)-4-(Ethoxycarbonyl)phenylalaninol Hydrochloride ((S)-17).
To a solution of (S)-16 (1.021 g, 3.16 mmol) in AcOEt (6.7 mL)
was added a saturated solution of anhydrous HCl in AcOEt (13.5
mL). The solution was stirred at room temperature overnight and
then evaporated under vacuum to leave 880 mg (100%) of (S)-17
as a white powder, which was used in the next step without further
purification.
(S)-N-Boc-4-(4-hydroxybenzoyl)phenylalaninol ((S)-14b). To
a solution of (S)-13b (114 mg, 0.22 mmol) in MeOH (2 mL), 2 N
NaOH (0.5 mL) was added, and the mixture was stirred at room
temperature for 4 h, diluted with water, acidified with 2 N HCl to
pH 3-4, and extracted with AcOEt. The organic solution was
washed with water until neutral pH, dried (Na2SO4), and evaporated
under vacuum to give 80 mg (98%) of (S)-14b as a white solid.
Mp 210-212 °C; [R]D -31° (CHCl3/MeOH ) 1/1, c 1.0); IR:
1
3351, 3180, 2971, 1686, 1638, 1517, 1278, 1082 cm-1; H NMR
(R)-4-(Ethoxycarbonyl)phenylalaninol Hydrochloride ((R)-
17). The title compound was prepared following the same procedure
that was used for the synthesis of (S)-17. Yield 100%.
(300 MHz, CDCl3/CD3OD ) 1/1) δ: 1.40 (9H, s), 2.87 (1H, dd,
J ) 13.5, 6.0 Hz), 2.96 (1H, dd, J ) 13.5, 8.4 Hz), 3.54 (1H, dd,
J ) 11.2, 4.8 Hz), 3.59 (1H, dd, J ) 11.2, 4.4 Hz), 3.87 (1H, m),
5.75 (1H, m), 6.88-7.76 (8H, m); 13C NMR (75 MHz, CDCl3/
CD3OD ) 1/1): δ 28.43, 37.76, 53.80, 63.53, 79.85, 115.48,
129.21, 129.58, 130.24, 133.25, 136.69, 143.60, 156.74, 156.78,
162.27. Anal. (C21H25NO5) C, H, N.
(R)-N-Boc-4-(4-hydroxybenzoyl)phenylalaninol ((R)-14b). The
title compound was prepared following the same procedure that
was used for the synthesis of the (S)-enantiomer. Yield 99%; mp
213-215 °C; [R]D +32° (CHCl3/MeOH ) 1/1, c 1.0). Anal. (C21H25-
NO5) C, H, N.
(S)-4-(4-Hydroxybenzoyl)phenylalaninol Hydrochloride ((S)-
15b). The title compound was prepared following the same
procedure that was used for the synthesis of (S)-15a.
(R)-4-(4-Hydroxybenzoyl)phenylalaninol Hydrochloride ((R)-
15b). The title compound was prepared following the same
procedure that was used for the synthesis of (R)-15a.
(Z)-N-{(1S)-2-Hydroxy-1-[[4-(ethoxycarbonyl)phenyl]methyl]-
etil}-9-octadecenamide ((S)-18). To a stirred solution of oleic acid
(741 mg, 2.62 mmol) in DMF (10 mL) was added at 0 °C HOBt
(466 mg, 2.76 mmol) and EDC (529 mg, 2.76 mmol). The mixture
was stirred for 15 min at 0 °C and for 1 h at room temperature,
then a solution of (S)-17 (883 mg, 3.16 mmol) in DMF (10 mL)
and Et3N (0.44 mL, 3.16 mmol) was added, and the mixture was
stirred overnight at room temperature. The mixture was diluted with
brine and extracted with AcOEt. The organic phase was washed
with 10% citric acid solution, saturated NaHCO3 and brine, dried
(Na2SO4), and evaporated under vacuum. The residue (1.77 g) was
chromatographed on silica gel (55 g) using CH2Cl2/AcOEt ) 1/1
as eluent to give 944 mg (74%) of (S)-18 as an oil. [R]D -17°; IR:
3435, 2928, 2856, 1711, 1658, 1509, 1465, 1280, 1207, 1180 cm-1
;
1H NMR (300 MHz) δ: 0.88 (3H, t, J ) 6.6 Hz), 1.26 (20H, m),
1.38 (3H, t, J ) 7.2 Hz), 1.52-1.59 (2H, m), 1.97-2.01 (4H, m),
2.13 (2H, t, J ) 7.5 Hz), 2.87-3.00 (2H, m), 3.53 (1H, br s), 3.56
(1H, dd, J ) 11.1, 4.8 Hz), 3.64 (1H, dd, J ) 11.1, 3.7 Hz), 4.16-
4.22 (1H, m), 4.33 (2H, q, J ) 7.2 Hz), 5.28-5.39 (2H, m), 6.08
(1H, d, J ) 8.4 Hz), 7.30 (2H, d, J ) 8.1 Hz), 7.96 (2H, d, J ) 8.1
Hz). 13C NMR (75 MHz): δ 14.13, 14.33, 22.69, 25.74, 27.19,
27.24, 29.15, 29.19, 29.28, 29.34, 29.54, 29.74, 29.78, 31.92, 36.81,
37.02, 52.38, 60.96, 63.62, 128.86, 129.28, 129.70, 129.81, 130.02,
143.38, 166.56, 173.83. Anal. (C30H49NO4) C, H, N.
(Z)-N-{(1S)-2-Hydroxy-1-[[4-(4-hydroxybenzoyl)phenyl]meth-
yl]etil}-9-octadecenamide (1b). The title compound was prepared
from (S)-15b, following the same procedure that was used for the
synthesis of 1a. Yield 65%; mp 115-116 °C; [R]D -14°; IR: 3302,
1
3145, 2923, 1645, 1602, 1541, 1318, 1297, 1168, 1044 cm-1; H
NMR (300 MHz) δ: 0.87 (3H, t, J ) 6.6 Hz), 1.26 (20H, m), 1.57
(2H, m), 1.98 (4H, m), 2.17 (2H, t, J ) 7.2 Hz), 2.49 (2H, m),
2.86-2.99 (2H, m), 3.55 (1H, dd, J ) 11.1, 4.5 Hz), 3.63 (1H, dd,
J ) 3.9 Hz), 4.19 (1H, m), 5.26-5.38 (2H, m), 6.42 (1H, d, J )
8.4 Hz), 6.87 (2H, d, J ) 8.7 Hz), 7.29 (2H, d, J ) 8.1 Hz), 7.62
(2H, d, J ) 7.8 Hz), 7.68 (2H, d, J ) 8.7 Hz); 13C NMR (75
MHz): δ 14.12, 22.70, 25.78, 27.19, 27.23, 29.16, 29.22, 29.27,
(Z)-N-{(1R)-2-Hydroxy-1-[[4-(ethoxycarbonyl)phenyl]methyl]-
etil}-9-octadecenamide ((R)-18). The title compound was prepared
following the same procedure that was used for the synthesis of
(S)-18. Yield 70%; [R]D +16°. Anal. (C30H49NO4) C, H, N.

Inhibitors of Anandamide Uptake
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2327
(Z)-N-{(1S)-2-Hydroxy-1-[(4-carboxyphenyl)methyl]etil}-9-
octadecenamide Acetate ((S)-19). To a stirred solution of (S)-18
(544 mg, 1.18 mmol) in THF/H2O ) 5/1 (19.5 mL) was added a
1 N LiOH solution (1.8 mL) dropwise at room temperature. The
mixture was stirred overnight at 50 °C, acidified to pH 4 with 2 N
HCl, and extracted with AcOEt. The organic phase was washed
twice with brine, dried (Na2SO4), and evaporated under vacuum.
The residue of the crude hydroxy acid (541 mg, 100%) was
dissolved in anhydrous pyridine (9.3 mL), and acetic anhydride
(0.52 mL, 5.5 mmol) was added, and the solution was stirred for 4
h at room temperature. The mixture was poured into ice/water, and
the resulting suspension was stirred for 30 min at room temperature
and extracted with AcOEt. The organic phase was washed with 2
N HCl and brine until neutral, dried (Na2SO4), and evaporated under
vacuum. The residue (560 mg) was chromatographed on silica gel
(17 g) using CH2Cl2/AcOEt ) 7/3 as eluent to give 363 mg (65%)
of (S)-19 as a colorless oil. [R]D -3°; IR: 3439, 2928, 2856, 1736,
(22 mg) was chromatographed on silica gel (900 mg) using
CH2Cl2/AcOEt ) 1/1 as eluent to give 15 mg of 1d as a white
solid. Mp 82-83 °C; [R]D -5 °C; IR: 3436, 2928, 1660, 1606,
1507, 1465, 1234 cm-1; 1H NMR (300 MHz) δ: 0.88 (3H, t, J )
6.6 Hz), 1.26 (20H, m), 1.54 (2H, m), 2.00 (4H, m), 2.14 (2H, t, J
) 7.5 Hz), 2.52 (1H, br s), 2.96 (2H, d, J ) 6.6 Hz), 3.57-3.69
(2H, m), 4.22 (1H, m), 4.69 (2H, s), 5.28-5.40 (2H, m), 5.91 (1H,
d, J ) 6.6 Hz), 7.37 (2H, d, J ) 7.9 Hz), 7.89 (2H, d, J ) 7.9 Hz);
13C NMR (75 MHz): δ 14.12, 22.69, 25.70, 27.19, 27.25, 29.15,
29.19, 29.27, 29.34, 29.54, 29.73, 29.78, 31.92, 36.82, 37.14, 45.86,
52.26, 63.77, 128.90, 129.70, 129.82, 130.07, 132.76, 144.86,
173.74, 190.74. Anal. (C29H46ClNO3) C, H, N.
(1S)-N-Boc-1-amino-3-azo-1-{[(4-hydroxyphenyl)methyl]pro-
pan-2-one} ((S)-22). To a solution of (S)-21 (737 mg, 2.6 mmol)
in anhydrous THF (9 mL) were added i-butyl chloroformate (0.38
mL, 2.9 mmol) and Et3N (0.75 mL, 5.3 mmol) at -15 °C under a
nitrogen atmosphere, and the mixture was stirred for 30 min at
room temperature. An ethereal solution of diazomethane (24 mL,
ca. 12 mmol) was added, and after stirring for a further 2 h, the
solvent was evaporated under vacuum, and the residue was diluted
with saturated NaHCO3 and extracted with Et2O. The organic phase
was washed two times with brine, dried (Na2SO4), and evaporated
under vacuum. Chromatography of the residue (961 mg) on silica
gel (30 g) using hexane/AcOEt ) 6/4 as eluent afforded (S)-22
(544 mg, 68%) as a white solid. Mp 132-133 °C (lit.26 136-137
°C); [R]D +12°; IR (KBr): 3369, 3074, 2123, 1676, 1607, 1591,
1526, 1453, 1339, 1253, 1172 cm-1; 1H NMR (300 MHz) δ: 1.42
(9H, s), 2.93 (2H, d, J ) 6.6 Hz), 4.36 (1H, m), 5.22 (2H, m), 6.49
(1H, s), 6.75 (2H, d, J ) 8.4 Hz), 7.01 (2H, d, J ) 8.4 Hz); 13C
NMR (75 MHz): δ 28.32, 37.87, 54.81, 58.66, 80.45, 115.62,
126.69, 130.48, 155.20, 155.41, 193.77. Anal. (C15H19N3O4) C, H,
N.
1
1693, 1666, 1508, 1235, 1203, 1042 cm-1; H NMR (300 MHz)
δ: 0.88 (3H, t, J ) 6.6 Hz), 1.26 (20H, m), 1.55-1.59 (2H, m),
1.97-2.01 (4H, m), 2.11 (3H, s), 2.16 (2H, t, J ) 7.2 Hz), 2.86-
3.00 (2H, m), 4.05 (1H, dd, J ) 11.4, 4.2 Hz), 4.13 (1H, dd, J )
11.4, 5.6 Hz), 4.51 (1H, m), 5.28-5.39 (2H, m), 5.75 (1H, d, J )
8.7 Hz), 7.31 (2H, d, J ) 8.1 Hz), 8.04 (2H, d, J ) 8.1 Hz); 13C
NMR (75 MHz): δ 14.12, 20.84, 22.69, 25.64, 27.16, 27.22, 29.13,
29.25, 29.32, 29.53, 29.72, 29.77, 31.90, 36.77, 37.65, 49.32, 64.87,
128.16, 129.35, 129.73, 130.00, 130.50, 143.27, 170.97, 171.19,
173.17. Anal. (C30H47NO5) C, H, N.
(Z)-N-{(1R)-2-Hydroxy-1-[(4-carboxyphenyl)methyl]etil}-9-
octadecenamide Acetate ((R)-19). The title compound was pre-
pared following the same procedure that was used for the synthesis
of (S)-19. Yield 57%; [R]D +4°. Anal. (C30H49NO4) C, H, N.
(Z)-N-{(1S)-2-Hydroxy-1-[[4-(2-diazoacetyl)phenyl]methyl]-
etil}-9-octadecenamide (1c). To a solution of (S)-19 (408 mg, 0.82
mmol) in anhydrous CH2Cl2 (7.6 mL) were added DMF (26 µL,
0.33 mmol) and oxalyl chloride (0.28 mL, 3.28 mmol) at 0 °C under
stirring. The mixture was stirred for 1 h at 0 °C, evaporated under
vacuum at room temperature, redissolved in DMF (7.6 mL), and
treated with Et3N (0.34 mL, 2.46 mmol) and a 2 M ethereal solution
of Me3SiCHN2 (1.23 mL) at 0 °C. The mixture was stirred for 5 h
at 0 °C and then partitioned between brine and AcOEt. The organic
phase was washed twice with brine, dried (Na2SO4), and evaporated
under vacuum. The residue (478 mg) was chromatographed on silica
gel (16 g) with CH2Cl2/AcOEt ) 9/1 as eluent to give 56 mg (13%)
of (S)-20. Intermediate (S)-20 was dissolved in THF/H2O ) 5/1 (2
mL), and a 1 N LiOH solution (0.24 mL) was added under stirring
at 0 °C. The mixture was stirred at 0 °C for 3.5 h, diluted with
water, and extracted with AcOEt. The organic phase was washed
with brine, dried (Na2SO4), and evaporated under vacuum. The
residue (54 mg) was chromatographed on silica gel (16 g) with
CH2Cl2/AcOEt ) 1/1 as eluent to give 24 mg (68%) of 1c as a
white solid. Mp 72-74 °C; [R]D -28°; IR: 3437, 2928, 2110, 1659,
1619, 1508, 1465, 1363, 1236 cm-1; 1H NMR (300 MHz) δ: 0.88
(3H, t, J ) 6.7 Hz), 1.26 (20H, m), 1.55 (2H, m), 2.00 (4H, m),
2.13 (2H, t, J ) 6.6 Hz), 2.87-3.00 (2H, m), 3.54 (1H, dd, J )
10.9, 4.3 Hz), 3.62 (1H, dd, J ) 10.9, 3.6 Hz), 4.18 (1H, m), 5.28-
5.39 (2H, m), 5.94 (1H, s), 6.19 (1H, d, J ) 8.1 Hz), 7.31 (2H, d,
J ) 8.4 Hz), 7.67 (2H, d, J ) 8.1 Hz); 13C NMR (75 MHz): δ
14.12, 22.69, 25.74, 27.19, 27.24, 29.17, 29.20, 29.30, 29.33, 29.53,
29.75, 29.77, 31.91, 36.78, 36.97, 52.33, 54.28, 63.45, 126.96,
129.62, 129.69, 130.04, 134.96, 143.58, 173.81, 186.21. Anal.
(C29H45N3O3) C, H, N.
(1R)-N-Boc-1-amino-3-azo-1-{[(4-hydroxyphenyl)methyl]pro-
pan-2-one} ((R)-22). The title compound was prepared following
the same procedure that was used for the synthesis of (S)-22. Yield
60%; mp 132-133 °C; [R]D -14°. Anal. (C15H19N3O4) C, H, N.
(1S)-1-Amino-3-chloro-1-[(4-hydroxyphenyl)methyl]propan-
2-one Hydrochloride ((S)-23). (S)-22 (507 mg, 1.66 mmol) was
dissolved in a 0.6 M solution of HCl in AcOEt (18 mL). The
reaction mixture was stirred for 48 h at room temperature. The
resulting precipitate was filtered off, washed with anhydrous diethyl
ether, and dried under vacuum to give (S)-23 (331 mg, 80%), which
was used in the next step without further purification.
(1R)-1-Amino-3-chloro-1-[(4-hydroxyphenyl)methyl]propan-
2-one Hydrochloride ((R)-23). The title compound was prepared
from (R)-22 following the same procedure that was used for the
synthesis of (S)-enantiomer. Yield 76%.
(Z)-N-{(1S)-3-Chloro-1-[(4-hydroxyphenyl)methyl]propan-2-
one}-9-octadecenamide (3a). To a stirred solution of oleic acid
(374 mg, 1.32 mmol) in anhydrous THF (7 mL) were added i-butyl
chloroformate (0.17 mL, 1.32 mmol) and N-methylmorpholine (0.29
mL, 2.64 mmol) at -15 °C under a nitrogen atmosphere. After 15
min, a solution of (S)-23 (331 mg, 1.32 mmol) in DMF (1 mL)
was added, and the mixture was stirred for 24 h at room
temperature. The reaction mixture was diluted with water and
extracted with AcOEt. The organic phase was dried (Na2SO4) and
evaporated under vacuum. The residue (734 mg) was chromato-
graphed on silica gel (29 g) using hexane/AcOEt ) 8/2 as eluent
to provide 3a (550 mg, 87%) as a white solid. Mp 89-91 °C; [R]D
+26°; IR (KBr): 3369, 2921, 2852, 1735, 1611, 1515, 1268, 1202
cm-1; 1H NMR (300 MHz) δ: 0.87 (3H, t, J ) 6.8 Hz), 1.26 (20H,
m), 1.57 (2H, m), 2.00 (4H, m), 2.16 (2H, t, J ) 7.6 Hz), 2.90-
3.04 (2H, m), 3.96 (1H, d, J ) 16.2 Hz), 4.14 (1H, d, J ) 16.2
Hz), 4.92-4.99 (1H, m), 5.28-5.40 (2H, m), 6.08 (1H, d, J ) 7.6
Hz), 6.30 (1H, br s), 6.77 (2H, d, J ) 8.4 Hz), 7.00 (2H, d, J ) 8.4
Hz); 13C NMR (75 MHz): δ 14.13, 22.69, 25.49, 27.17, 27.23,
29.10, 29.13, 29.19, 29.33, 29.53, 29.71, 29.77, 31.91, 36.30, 36.98,
47.59, 57.26, 115.97, 126.56, 129.73, 130.05, 130.19, 155.67,
173.76, 201.33. Anal. (C28H44ClNO3) C, H, N.
(Z)-N-{(1R)-2-Hydroxy-1-[[4-(2-diazoacetyl)phenyl]methyl]-
etil}-9-octadecenamide (2c). The title compound was prepared
following the same procedure that was used for the synthesis of
1c. Overall yield 8%; mp 72-74 °C; [R]D +27°. Anal. (C29H45N3O3)
C, H, N.
(Z)-N-{(1S)-2-Hydroxy-1-[[4-(2-chloroacetyl)phenyl]methyl]-
etil}-9-octadecenamide (1d). To a solution of 1c (21 mg, 0.04
mmol) in AcOEt (1 mL) was added a saturated solution of
anhydrous HCl in AcOEt (21 µL). The mixture was stirred at room
temperature for 1 h and then evaporated under vacuum. The residue
(Z)-N-{(1R)-3-Chloro-1-[(4-hydroxyphenyl)methyl]propan-2-
one}-9-octadecenamide (4a). The title compound was prepared

2328 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Schiano Moriello et al.
from oleic acid and (R)-23, following the same procedure that was
used for the synthesis of the (S)-enantiomer. Yield 79%; mp 86-
87 °C; [R]D -27°. Anal. (C28H44ClNO3) C, H, N.
26.11, 26.32, 26.55, 29.15, 29.48, 29.67, 29.71, 30.07, 31.11, 33.19,
62.54, 63.55, 67.89, 80.39, 80.41, 99.00. Anal. (C29H56O3Si) C, H,
N.
(Z)-N-{(1S)-3-Iodo-1-[(4-hydroxyphenyl)methyl]propan-2-
one}-9-octadecenamide (3b). A mixture of 3a (300 mg, 0.63
mmol) and NaI (900 mg, 6.3 mmol) in acetone (6 mL) was stirred
for 16 h at room temperature. The mixture was diluted with water
and extracted with AcOEt. The organic phase was washed twice
with water, dried (Na2SO4), and evaporated under vacuum. The
residue (448 mg) was chromatographed on silica gel (14 g, hexane/
AcOEt ) 9/1) to provide 3b (218 mg, 61%) as a white solid. Mp
87-89 °C; [R]D +19°; IR (KBr): 3358, 2922, 2851, 1723, 1610,
1540, 1513, 1325, 1266, 1217 cm-1; 1H NMR (300 MHz) δ: 0.88
(3H, t, J ) 6.8 Hz), 1.26 (20H, m), 1.58 (2H, m), 2.00 (4H, m),
2.18 (2H, t, J ) 7.2 Hz), 3.00-3.03 (2H, m), 3.74 (1H, d, J )
10.8 Hz), 3.78 (1H, d, J ) 10.8 Hz), 5.08 (1H, m), 5.33-5.36
(2H, m), 6.08 (1H, d, J ) 7.2 Hz), 6.28 (1H, br s), 6.78 (2H, d, J
) 8.4 Hz), 7.02 (2H, d, J ) 8.4 Hz); 13C NMR (75 MHz): δ 4.40,
14.13, 22.69, 25.49, 27.18, 27.24, 29.11, 29.14, 29.21, 29.34, 29.54,
29.71, 29.78, 31.91, 36.44, 37.72, 57.01, 115.90, 129.54, 129.73,
130.05, 130.23, 155.42, 173.34, 201.71. Anal. (C28H44INO3) C, H,
N.
(Z)-1-[(t-Butyldimethylsilyl)oxy]-18-[2-(tetrahydropyranyl)-
oxy]-9-octadecene (29). To a stirred solution of Ni(OAc)2‚4H2O
(77 mg, 0.3 mmol) in 95% EtOH (5.2 mL), a 1 M solution of
NaBH4 in 95% EtOH (0.3 mL) was added at room temperature
under nitrogen. To the suspension of the in situ generated catalyst,
a solution of 28 (1.21 g, 2.5 mmol) in 95% EtOH (10 mL) was
added after 30 min. The mixture was hydrogenated at room
temperature for 2 h under atmospheric pressure, then filtered through
a Celite pad, and evaporated under vacuum to afford 1.18 g (97%)
1
of 29 as a colorless liquid. H NMR (300 MHz,) δ: 0.04 (6H, s),
0.89 (9H, s), 1.25-1.85 (30H, m), 1.98-2.02 (4H, m), 3.37 (1H,
td, J ) 9.9, 6.6 Hz), 3.49 (1H, m), 3.59 (2H, t, J ) 6.6 Hz), 3.73
(1H, td, J ) 9.9, 6.6 Hz), 3.87 (1H, m), 4.57 (1H, m), 5.34 (1H,
m); 13C NMR (75 MHz, CDCl3): δ -4.84, 18.73, 20.06, 25.88,
26.16, 26.34, 26.60, 27.56, 29.61, 29.77, 29.84, 30.10, 31.14, 33.23,
62.58, 63.59, 67.94, 99.04, 130.03. Anal. (C29H58O3Si) C, H, N.
(Z)-1-Hydroxy-18-[2-(tetrahydropyranyl)oxy]-9-octadecene (30).
Compound 29 (1.18 g, 2.4 mmol) was treated under stirring with
a 1 M solution of N-tetrabutylammonium fluoride in THF (12.2
mL) for 2 h at room temperature under nitrogen. The solution was
concentrated and partitioned between AcOEt and water. The organic
phase was washed with brine, dried (Na2SO4), and evaporated under
vacuum. The residue (1.50 g) was chromatographed on silica gel
(45 g) using CH2Cl2/AcOEt ) 95/5 as eluent to give 0.83 g (92%)
(Z)-N-{(1R)-3-Iodo-1-[(4-hydroxyphenyl)methyl]propan-2-
one}-9-octadecenamide (4b). The title compound was prepared
from 4a following the same procedure that was used for the
synthesis of the (S)-enantiomer. Yield 55%; mp 86-88 °C; [R]D
-18°. Anal. (C28H44INO3) C, H, N.
1
of 30 as a colorless liquid. H NMR (300 MHz) δ: 1.24-1.84
(Z)-N-{(1S)-3-(2,6-Dichlorobenzoyloxy)-1-[(4-hydroxyphenyl)-
methyl]propan-2-one}-9-octadecenamide (3c). A mixture of 3b
(66 mg, 0.114 mmol) and KF (13 mg, 0.23 mmol) in DMF (0.5
mL) was stirred for 1 min at room temperature. 2,6-Dichlorobenzoic
acid (44 mg, 0.23 mmol) was then added and stirring was continued
for 16 h at room temperature. The mixture was partitioned between
water and AcOEt. The organic phase was washed twice with water,
dried (Na2SO4), and evaporated under vacuum. The residue (103
mg) was chromatographed on silica gel (3 g, hexane/AcOEt ) 8/2
as eluent) to provide 3c (52 mg, 72%) as an oil. [R]D +23°; IR
(CHCl3): 3424, 3011, 2928, 2855, 1737, 1667, 1515, 1434, 1264,
1206 cm-1; 1H NMR (300 MHz) δ: 0.87 (3H, t, J ) 6.7 Hz), 1.26
(20H, m), 1.56 (2H, m), 2.00 (4H, m), 2.18 (2H, t, J ) 7.2 Hz),
2.97 (1H, dd, J ) 14.0, 6.8 Hz), 3.11 (1H, dd, J ) 14.0, 6.8 Hz),
4.92 (2H, m), 5.00 (1H, m), 5.32-5.35 (2H, m), 6.17 (1H, d, J )
7.6 Hz), 6.74 (2H, d, J ) 7.6 Hz), 7.02 (2H, d, J ) 7.6 Hz), 7.33
(3H, m); 13C NMR (75 MHz): δ 14.13, 22.70, 25.52, 27.21, 27.26,
29.13, 29.17, 29.18, 29.33, 29.55, 29.73, 29.79, 31.92, 36.42, 56.70,
68.07, 115.86, 126.72, 128.06, 129.79, 129.97, 130.04, 131.42,
132.26, 132.29, 155.59, 164.00, 173.66, 201.38. Anal. (C35H47Cl2-
NO5) C, H, N.
(30H, m), 1.97-2.01 (4H, m), 3.37 (1H, td, J ) 9.9, 6.6 Hz), 3.49
(1H, m), 3.61 (2H, t, J ) 6.6 Hz), 3.72 (1H, td, J ) 9.9, 6.6 Hz),
3.86 (1H, m), 4.57 (1H, m), 5.33 (1H, m); 13C NMR (75 MHz): δ
20.03, 25.84, 26.11, 26.57, 27.53, 29.57, 29.81, 30.07, 31.11, 33.13,
62.61, 63.23, 67.98, 99.06, 130.05. Anal. (C23H44O3) C, H, N.
(Z)-18-[2-(Tetrahydropyranyl)oxy]-9-octadecenoic Acid Meth-
yl Ester (31). A mixture of 30 (605 mg, 1.64 mmol) and pyridinium
dichromate (3.70 g, 9.84 mmol) in DMF (33 mL) was stirred at
room temperature for 48 h and then was partitioned between AcOEt
and water. The organic phase was washed three times with brine,
dried (Na2SO4), and evaporated under vacuum. The residue (657
mg) was treated with an excess of an ethereal solution of
diazomethane at 0 °C for 45 min. The ether was evaporated under
vacuum and the residue (663 mg) was chromatographed on silica
gel (15 g) with CH2Cl2 as eluent to give 553 mg (85%) of 31 as a
colorless oil. 1H NMR (300 MHz) δ: 1.22-1.81 (28H, m), 1.97-
1.99 (4H, m), 2.27 (2H, t, J ) 7.5 Hz), 3.35 (1H, td, J ) 9.9, 6.6
Hz), 3.47 (1H, m), 3.64 (3H, s), 3.70 (1H, td, J ) 9.9, 6.6 Hz),
3.84 (1H, m), 4.55 (1H, m), 5.31 (2H, m); 13C NMR (75 MHz): δ
19.67, 24.91, 25.49, 26.21, 27.12, 27.17, 29.08, 29.11, 29.22, 29.45,
29.64, 29.71, 30.75, 34.04, 51.31, 62.20, 67.55, 98.65, 129.53,
129.70, 173.95. Anal. (C24H44O4) C, H, N.
(Z)-N-{(1R)-3-(2,6-Dichlorobenzoyloxy)-1-[(4-hydroxyphenyl-
)methyl]propan-2-one}-9-octadecenamide (4c). The title com-
pound was prepared from 4b following the same procedure that
was used for the synthesis of 3c. Yield 61%; [R]D -24°. Anal.
(C35H47Cl2NO5) C, H, N.
(Z)-18-Hydroxy-9-octadecenoic Acid Methyl Ester (32). A
solution of 31 (1.05 g, 2.65 mmol) and pyridinium p-toluene-
sulfonate (67 mg, 0.27 mmol) in EtOH (22 mL) was stirred
overnight at 55 °C. The solvent was evaporated under vacuum,
and the residue was filtered through a short pad of silica gel using
CH2Cl2 as the eluent to give 797 mg (96%) of 32 as a colorless
1-[(t-Butyldimethylsilyl)oxy]-18-[2-(tetrahydropyranyl)oxy]-
9-octadecyne (28). To a stirred solution of 2728 (1.19 g, 5 mmol)
in anhydrous THF (10 mL), a solution of 1.2 M n-BuLi in hexane
(5 mL, 6 mmol) was added dropwise at -25 °C under nitrogen.
After the mixture was stirred for 10 min at -25 °C, anhydrous
hexamethylphosporamide (10 mL) was added, followed by a
dropwise addition of a solution of 2527 (1.62 g, 5 mmol) in
anhydrous THF (10 mL). The mixture was maintained at -25 °C
for an additional 2 h, then stirred overnight at room temperature,
and poured into ice/water (50 mL). Extraction with AcOEt, followed
by three washings with brine, drying (Na2SO4), and evaporation
under vacuum afforded a residue (2.51 g) that was chromatographed
on silica gel (100 g) using hexane/AcOEt ) 97/3 as eluent to give
1
oil. H NMR (300 MHz) δ: 1.30 (18H, m), 1.52-1.64 (4H, m),
1.93 (1H, br s), 1.98-2.02 (4H, m), 2.30 (2H, t, J ) 7.5 Hz), 3.62
(2H, t, J ) 6.6 Hz), 3.66 (3H, s), 5.34 (2H, m); 13C NMR (75
MHz): δ 24.91, 25.74, 27.12, 27.15, 29.04, 29.09, 29.11, 29.18,
29.39, 29.46, 29.63, 29.69, 32.75, 34.04, 51.36, 62.82, 129.56,
129.69, 174.07. Anal. (C19H36O3) C, H, N.
(Z)-18-Bromo-9-octadecenoic Acid Methyl Ester (33). To a
solution of 32 (797 mg, 2.55 mmol) in anhydrous CH2Cl2 (28 mL)
were added at 0 °C CBr4 (1.11 g, 3.34 mmol) and PPh3 (938 mg,
3.58 mmol), and the mixture was stirred at 0 °C for 40 min. The
solution was concentrated under vacuum and partitioned between
AcOEt and water. The organic phase was washed with brine, dried
(Na2SO4), and evaporated under vacuum. The residue (1.97 g) was
chromatographed on silica gel (40 g) using hexane/CH2Cl2 ) 6/4
1
1.95 g (81%) of 28 as a colorless liquid. H NMR (300 MHz) δ:
0.03 (3H, s), 0.87 (9H, s), 1.28-1.87 (30H, m), 2.11 (4H, t, J )
6.9 Hz), 3.35 (1H, td, J ) 9.9, 6.6 Hz), 3.48 (1H, m), 3.57 (2H, t,
J ) 6.6 Hz), 3.71 (1H, td, J ) 9.9, 6.6 Hz), 3.85 (1H, m), 4.55
(1H, m); 13C NMR (75 MHz): δ -4.88, 18.70, 19.10, 20.03, 25.86,
1
as eluent to give 889 mg (94%) of 33 as a colorless oil. H NMR

Inhibitors of Anandamide Uptake
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2329
(300 MHz) δ: 1.25-1.44 (18H, m), 1.59-1.64 (2H, m), 1.80-
1.90 (2H, m), 1.98-2.02 (4H, m), 3.40 (2H, t, J ) 6.9 Hz), 3.66
(3H, s), 5.34 (2H, m); 13C NMR (75 MHz): δ 24.91, 27.13, 28.13,
28.70, 29.12, 29.28, 29.40, 29.64, 32.78, 33.87, 34.04, 51.32,
129.61, 173.93. Anal. (C19H35BrO2) C, H, N.
(1H, br s), 4.11 (1H, m), 5.33 (2H, m), 6.26 (1H, d, J ) 8.4 Hz),
6.72 (2H, d, J ) 8.5 Hz), 6.98 (2H, d, J ) 8.5 Hz), 8.17 (1H, br
s); 13C NMR (75 MHz): δ 17.05, 25.25, 25.70, 27.07, 27.14, 28.57,
28.65, 29.01, 29.09, 29.17, 29.57, 29.65, 36.13, 36.70, 52.91, 63.54,
115.37, 119.75, 128.45, 129.55, 129.67, 129.94, 154.99, 162.44,
174.28. Anal. (C28H44N2O3) C, H, N.
(Z)-N-{(1R)-2-Hydroxy-1-[(4-hydroxyphenyl)methyl]ethyl}-
18-cyano-9-octadecenamide (6c). The title compound was prepared
from 6a, following the same procedure that was used for the
synthesis of the (S)-enantiomer. Yield 99%; mp 34-36 °C; [R]D
+12°. Anal. (C26H44N2O3) C, H, N.
Di-O-t-butyldiphenylsilyl-L-tyrosinol (38). To a solution of
L-tyrosinol (1.52 g, 7.45 mmol) in CH2Cl2 (15 mL), Et3N (4.36
mL, 31.29 mmol) and 4-(dimethylamino)pyridine (90.4 mg, 0.74
mmol) were added at room temperature. The reaction mixture was
stirred for 30 min at room temperature, and then upon cooling at
-4 °C, t-butyldiphenyl chloride (5.4 mL, 20.86 mmol, 2.8 eq) was
added dropwise. An immediate precipitate was formed, and the
reaction was stirred for 16 h at 25 °C. Upon cooling at 0 °C and
dilution with CH2Cl2 (45 mL), the reaction mixture was quenched
with ice/water (15 mL) and then acidified with 5% HCl (10 mL).
After stirring for 15 min and the addition of a saturated Na2CO3
solution (10 mL), the aqueous layer was separated and extracted
with CH2Cl2 (3 × 50 mL). The combined organic extracts were
dried (MgSO4), filtered, and evaporated under vacuum. The residue
(375 mg) was chromatographed on silica gel using CH2Cl2/CH3-
OH ) 95/5 as eluent to give 4.03 g (84%) of 38 as a pale-yellow
oil. IR: 3071, 3053, 2931, 2857, 1608, 1589, 1508, 1472, 1427,
1390, 1361, 1254, 1189, 1172, 1111, 1007, 998, 918, 821, 740,
699 cm-1; MS (FAB+, NBA) m/z: 645 [M + H+], 567 [M - Ph];
1H NMR (300 MHz) δ: 1.06 (9H, s), 1.10 (9H, s), 2.44 (1H, dd,
J ) 8.2, 13.5 Hz), 2.66 (1H, dd, J ) 5.4, 13.5 Hz), 2.72 (2H, br s),
3.00-3.11 (1H, m), 3.49 (1H, dd, J ) 6.4, 9.9 Hz), 3.60 (1H, dd,
J ) 4.4, 9.9 Hz), 6.67 (2H, d, J ) 8.4 Hz), 6.87 (2H, d, J ) 8.4
Hz), 7.30-7.50 (12H, m), 7.60-7.68 (4H, m), 7.68-7.75 (4H, m);
13C NMR (75 MHz): δ 19.17, 19.34, 26.47, 26.82, 39.00, 54.14,
67.59, 119.54, 127.61, 129.59, 129.74, 129.80, 130.97, 132.93,
133.32 135.41, 153.94. Anal. (C41H49NO2Si2) C, H, N.
(Z)-N-{(1S)-2-Hydroxy-1-[(4-hydroxyphenyl)methyl]ethyl}-
18-bromo-9-octadecenamide (5a). To a solution of 33 (210 mg,
0.56 mmol) in THF/H2O ) 5/1 (9.4 mL), a 1 M aqueous solution
of LiOH (0.94 mL) was added, and the mixture was stirred
overnight at room temperature. The solution was acidified to pH
4-5 with 2 N HCl and extracted with AcOEt. The organic phase
was washed with water until neutral, dried (Na2SO4), and evaporated
under vacuum. To a stirred solution of the residue of the (Z)-18-
bromo-9-octadecenoic acid (34) (202 mg, 0.56 mmol) in DMF (3
mL), a solution of L-tyrosinol (127 mg, 0.62 mmol) in DMF (2
mL) and DCC (129 mg, 0.62 mmol) were added. The mixture was
stirred overnight at room temperature and then partitioned between
AcOEt and brine. The organic phase was washed with a 2 N HCl,
water, saturated NaHCO3, and water until neutral, dried (Na2SO4),
and evaporated under vacuum. The residue (375 mg) was chro-
matographed on silica gel using CH2Cl2/AcOEt ) 6/4 as eluent to
give 118 mg (82%) of 5a as a white solid. Mp 63-65 °C; [R]D
-13° (CHCl3, c 1.0); IR (CHCl3): 3432, 3343, 2929, 2855, 1652,
1
1614, 1513, 1464, 1242, 1194, 1172 cm-1; H NMR (300 MHz,
CDCl3) δ: 1.25-1.55 (20H, m), 1.80-1.89 (2H, m), 1.99 (4H,
m), 2.14 (2H, t, J ) 7.5 Hz), 2.66-2.80 (2H, m), 3.40 (2H, t, J )
6.9 Hz), 3.52 (1H, dd, J ) 11.1, 5.1 Hz), 3.62 (1H, dd, J ) 11.1,
3.6 Hz), 3.91 (1H, br s), 4.12 (1H, br s), 5.33 (2H, m), 6.06 (1H,
d, J ) 7.3 Hz), 6.72 (2H, d, J ) 8.2 Hz), 6.98 (2H, d, J ) 8.2 Hz),
8.14 (1H, br s); 13C NMR (75 MHz, CDCl3): δ 25.70, 27.16, 28.12,
28.69, 29.12, 29.21, 29.65, 32.78, 34.01, 36.22, 36.77, 52.94, 63.79,
115.43, 128.49, 129.63, 129.65, 129.94, 154.91, 174.25. Anal.
(C27H44BrNO3) C, H, N.
(Z)-N-{(1R)-2-Hydroxy-1-[(4-hydroxyphenyl)methyl]ethyl}-
18-bromo-9-octadecenamide (6a). The title compound was pre-
pared following the same procedure that was used for the synthesis
of the (S)-enantiomer. Yield 74%; mp 63-65 °C; [R]D +13°. Anal.
(C27H44BrNO3) C, H, N.
(Z)-N-{(1S)-2-Hydroxy-1-[(4-hydroxyphenyl)methyl]ethyl}-
18-iodo-9-octadecenamide (5b). A solution of 5a (102 mg, 0.2
mmol) and NaI (150 mg, 1 mmol) in acetone (2 mL) was stirred
overnight at room temperature, diluted with water, and extracted
with AcOEt. The organic phase was dried (Na2SO4) and evaporated
under vacuum to give 110 mg (99%) of 5b as a white solid. Mp
67-68 °C; [R]D -14°; IR (CHCl3): 3433, 3345, 2928, 2855, 1654,
1514, 1465, 1234, 1199, 1172 cm-1; 1H NMR (300 MHz) δ: 1.25-
1.54 (20H, m), 1.76-1.85 (2H, m), 1.99 (4H, m), 2.13 (2H, t, J )
7.5 Hz), 2.65-2.78 (2H, m), 3.17 (2H, t, J ) 6.9 Hz), 3.51 (1H,
dd, J ) 11.1, 5.1 Hz), 3.60 (1H, dd, J ) 11.1, 3.6 Hz), 3.80 (1H,
br s), 4.11 (1H, br s), 5.33 (2H, m), 6.19 (1H, d, J ) 7.8 Hz), 6.71
(Z)-N-[(1S)-2-[[(1,1-Dimethylethyl)diphenylsilyl]oxy]-1-[[4-
[[(1,1-dimethylethyl)diphenylsilyl]oxy]phenyl]methyl]ethyl]-12-
[(tetrahydro-2H-pyran-2-yl)oxy]-9-dodecenamide (39). A 0.5 M
aqueous solution of LiOH (0.5M, 3.84 mL) was added dropwise
to a stirred solution of ester 3732 (200 mg, 0.64 mmol) in THF (6.4
mL) at 0 °C under argon. The ice bath was removed, and the
mixture was stirred for 3 h at 30 °C. Upon cooling at 0 °C, the
reaction was quenched with 1 M NaHSO4 (1.92 mL). The pH was
adjusted to 5-6. The solution was extracted with AcOEt. The
organic phase was washed with brine, dried (MgSO4), and
evaporated under vacuum to give 204 mg (100%) of the carboxylic
acid as a colorless liquid, which was used in the subsequent step
without further purification. IR: 3372-2460 (br), 3007, 2925, 2854,
3078, 1708, 1440, 1410, 1352, 1260, 1200, 1183, 1136, 1119, 1075,
1031, 984, 922, 905, 869, 814, 725 cm-1; 1H NMR (300 MHz) δ:
1.23-1.41 (8H, m), 1.42-1.90 (8H, m), 1.95-2.09 (2H, m), 2.20-
2.30 (2H, m), 2.30-2.40 (2H, m), 3.32-3.43 (1H, m), 3.43-3.55
(1H, m), 3.65-3.75 (1H, m), 3.80-3.94 (1H, m), 4.53-4.64 (1H,
m), 5.29-5.49 (2H, m), 10.74 (1H, br s); 13C NMR (75 MHz): δ
19.46, 24.68, 25.39, 27.18, 27.87, 28.95, 29.01, 29.45, 30.61, 34.44,
62.16, 67.05, 98.63, 125.48 131.81, 179.80. To a solution of the
carboxylic acid (191 mg, 0.64 mmol) in CH3CN (3.8 mL) were
added N-methylmorpholine (141 µL, 1.28 mmol) and i-butyl
chloroformate (116 µL, 0.90 mmol) at -4 °C. After stirring for 60
min at -5 °C (thick white suspension), a solution of 38 (494 mg,
0.77 mmol) in CH3CN (2.0 mL) was cannulated. The resulting
mixture was stirred for 1 h at room temperature, then diluted with
AcOEt (15 mL), and quenched with water (1 mL). The organic
layer was washed twice with brine. The combined aqueous phases
were extracted with AcOEt. The combined organic extracts were
dried (MgSO4) and evaporated under vacuum. Chromatography of
the residue on silica gel using cyclohexane/AcOEt ) 99/1 to 90/
10 as eluent provided amide 39 (491 mg, 83%) as a colorless oil.
(2H, d, J ) 8.1 Hz), 6.97 (2H, d, J ) 8.1 Hz), 8.09 (1H, br s); 13
C
NMR (75 MHz): δ 7.35, 25.69, 27.15, 28.44, 29.11, 29.20, 29.25,
29.62, 30.42, 33.48, 36.19, 36.75, 52.89, 63.60, 115.41, 128.44,
129.60, 129.63, 129.93, 154.90, 174.33. Anal. (C27H44INO3) C, H,
N.
(Z)-N-{(1R)-2-Hydroxy-1-[(4-hydroxyphenyl)methyl]ethyl}-
18-iodo-9-octadecenamide (6b). The title compound was prepared
from 6a following the same procedure that was used for the
synthesis of the (S)-enantiomer. Yield 99%; mp 67-68 °C; [R]D
+14°. Anal. (C27H44INO3) C, H, N.
(Z)-N-{(1S)-2-Hydroxy-1-[(4-hydroxyphenyl)methyl]ethyl}-
18-cyano-9-octadecenamide (5c). A solution of 5a (102 mg, 0.2
mmol) and KCN (26 mg, 0.4 mmol) in DMSO (2 mL) was stirred
overnight at room temperature, diluted with water, and extracted
with AcOEt. The organic phase was washed twice with water, dried
(Na2SO4), and evaporated under vacuum to give 91 mg (100%) of
5c as a white solid. Mp 34-36 °C; [R]D -12°; IR (CHCl3): 3433,
1
3345, 2929, 2856, 2247, 1654, 1514, 1465, 1234, 1172 cm-1; H
NMR (300 MHz) δ: 1.23-1.69 (22H, m), 1.99 (4H, m), 2.13 (2H,
t, J ) 7.6 Hz), 2.33 (2H, t, J ) 6.9 Hz), 2.66-2.79 (2H, m), 3.51
(1H, dd, J ) 11.1, 5.1 Hz), 3.60 (1H, dd, J ) 11.1, 3.6 Hz), 3.90

2330 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Schiano Moriello et al.
IR: 3301, 3071, 2929, 2856, 1641, 1608, 1541, 1508, 1472, 1427,
1390, 1361, 1253, 1200, 1112, 1070, 1031, 997, 984, 919, 869,
821, 739 cm-1; MS (FAB+, NBA) m/z: 947 [M + Na+], 923 [M
- H+], 867 [M - tBu], 841 [M + 2H+ - THP], 685 [M -
TBDPS]; 1H NMR (300 MHz) δ: 1.05 (9H, s), 1.07 (9H, s), 1.20-
1.30 (8H, m), 1.40-1.90 (8H, m), 1.95-2.06 (4H, m), 2.32 (2H,
q, J ) 6.8 Hz), 2,74 (2H, d, J ) 7.2 Hz), 3.33-3.43 (1H, m),
3.43-3.50 (1H, m), 3.50-3.57 (2H, m), 3.65-3.76 (1H, m), 3.80-
3.90 (1H, m), 4.03-4.15 (1H, m), 4.54-4.62 (1H, m), 5.30-5.49
(2H, m), 5.54 (1H, d, J ) 8.7 Hz), 6.61 (2H, d, J ) 8.4 Hz), 6.85
(2H, d, J ) 8.4 Hz), 7.26-7.43 (12H, m), 7.52-7.63 (4H, m),
7.64-7.72 (4H, m); 13C NMR (75 MHz): δ 19.31, 19.39, 19.53,
25.44, 25.61, 26.50, 26.92, 27.26, 27.92, 29.06, 29.19, 29.56, 30.67,
36.27, 36.81, 51.33, 62.20, 63.49, 67.04, 98.67, 119.54, 125.53,
127.64, 127.71, 129.78, 130.01, 130.32, 131.82, 133.02, 133.25,
135.47, 154.05, 172.15. Anal. (C58H77NO5Si2) C, H, N.
positive 468.4 (M + 1), negative 466.7 (M - 1), MS2 positive )
1
346 (M - PhCOO), 450.2 (M - H2O), 468.1 (M + 1); H NMR
(300 MHz) δ: 1.43-1.10 (8H, m), 1.45-1.60 (2H, m), 1.95-2.05
(2H, m), 2.10 (2H, t, J ) 7.5 Hz), 2.40-2.55 (2H, m), 2.62-2.85
(2H, m), 3.54 (1H, dd, J ) 11.1, 5.2 Hz), 3.63 (1H, dd, J ) 11.1,
3.3 Hz), 4.00-4.20 (1H, m), 4.29 (2H, t, J ) 6.9 Hz), 5.35-5.60
(2H, m), 5.85-6.05 (1H, m), 6.73 (2H, d, J ) 8.1 Hz), 6.99 (2H,
d, J ) 8.1 Hz), 7.40 (2H, t, J ) 7.3 Hz), 7.53 (1H, tt, J ) 1.3, 7.3
Hz), 8. 01 (2H, dd, J ) 7.3, 1.3 Hz); 13C NMR (75 MHz): δ 25.64,
26.94, 27.25, 29.00, 29.08, 29.46, 29.66, 36.18, 36.77, 53.07, 64.30,
64.59), 115.60, 124.22,128.34, 128.76, 129.56, 130.13, 130.28
132.95, 133.07, 155.25, 166.92, 174.38. Anal. (C28H37NO5) C, H,
N.
(Z)-N-[(1S)-2-Hydroxy-1-[(4-hydroxyphenyl)methyl]ethyl]-12-
[(2-azido-5-iodo)benzoyloxy]-9-dodecenamide (8). To a stirred
solution of DCC (37 mg, 177 µmol), DMAP (5 mg, 40 µmol), and
2-azido-5-iodobenzoic acid35 (35 mg, 120 µmol) in CH2Cl2 (0.9
mL) was added a solution of 40 (60 mg, 72 µmol) in CH2Cl2 (1.3
mL) at 0 °C. After stirring at room temperature for 15 h in the
dark, the mixture was diluted with CH2Cl2 and filtered through a
short pad of silica gel using cyclohexane/Et2O ) 7/3 as eluent in
the dark. The residue was chromatographed on fluorisil (1.5 g) using
cyclohexane/Et2O ) 95/5 to 50/50 as eluent to give 8 (65.4 mg,
82%) as a colorless oil. MS m/z: (ESI >0) 1133.2 [M + Na+];
IR: 3298, 3071, 2929, 2856, 2125, 2095, 1728, 1644, 1509, 1472,
1428, 1290, 1250, 1113, 1089, 920, 822 cm-1; 1H NMR (300 MHz)
δ: 1.06 (9H s), 1.07 (9H, s), 1.15-1.38 (8H, m), 1.43-1.57 (2H,
m), 1.95-2.10 (4H, m), 2.48 (2H, q, J ) 6.9 Hz), 2.75 (2H, d, J
) 7.2 Hz), 3.53 (2H, d, J ) 3.4 Hz), 4.03-4.15 (1H, m), 4.27
(2H, t, J ) 6.7 Hz), 5.32-5.45 (1H, m), 5.45-5.55 (1H, m), 5.55
(1H, d, J ) 8.9 Hz), 6.61 (2H, d, J ) 8.5 Hz), 6.85 (2H, d, J ) 8.5
Hz), 6.95 (1H, d, J ) 8.5 Hz), 7.20-7.45 (12H, m), 7.51-7.63
(4H, m), 7.63-7.72 (4H, m), 7.77 (1H, dd, J ) 2.1, 8.5 Hz), 8.11
(1H, d, J ) 2.1 Hz); 13C NMR (75 MHz): δ 19.35, 19.44, 25.65,
26.54, 26.83, 26.96, 27.34, 29.12, 29.24, 29.56, 29.67, 36.31, 36.85,
51.38, 63.51, 65.07, 87.33, 119.60, 121.75, 124.06, 127.69, 127.77,
129.83, 130.06, 130.34, 133.07, 133.20, 135.52, 139.98, 140.32,
141.71, 154.10, 163.68, 172.22. A 1 M solution of N-tetrabuty-
lammonium fluoride (TBAF) in THF (525 µL) was added in the
dark to a solution of the 2-azido 5-iodobenzoate ester (58 mg,
53 µmol) in THF at 0 °C. The mixture was stirred at 0 °C for 10
min, then at 20 °C for 1 h, and evaporated under vacuum. The
residue was filtered through a short pad of silica gel using
CH2Cl2/CH3OH ) 98/2 as eluent. Evaporation under vacuum and
chromatography on silica gel of the filtrate with CH2Cl2/CH3OH
) 99/1 to 97/3 as eluent gave 8 (25.0 mg, 75%) as a pale yellow
oil. [R]D ) +2.8 (c ) 0.05, CH2Cl2); IR: 3600-3100, 3017, 2927,
2855, 2125, 1717, 1642, 1539, 1515, 1475, 1380, 1295, 1240, 1132,
1089, 814 cm-1; MS (electrospray): positive 657.1 (M + 23),
negative 633.3 (M - 1), MS2 positive ) 502.3 (M - N2I); 629.2
(Z)-N-[(1S)-2-[[(1,1-Dimethylethyl)diphenylsilyl]oxy]-1-[[4-
[[(1,1-dimethylethyl)diphenylsilyl]oxy]phenyl]methyl]ethyl]-12-
hydroxy-9-dodecenamide (40). A solution of p-toluenesulfonic
acid (1.2 mg, 6 µmol) in MeOH (1.0 mL) was added dropwise to
a stirred solution of 39 (250 mg, 0.27 mmol) in MeOH (1.4 mL)
at 0 °C. The reaction mixture was stirred at 25 °C for 2.5 h,
concentrated under vacuum, and diluted with Et2O. The mixture
was neutralized with a few drops of a saturated NaHCO3 solution,
and the organic layer was washed with brine, dried (MgSO4), and
evaporated under vacuum. The residue was chromatographed on
silica gel using heptanes/AcOEt ) 7/3 as eluent to give 40 as a
colorless oil (205 mg, 51%). IR: 3600-3100 (br), 3068, 3050,
2930, 2855, 1645, 1509, 1428, 1255, 1113, 921, 822 cm-1; MS
(FAB+, NBA) m/z: 840 [M], 763 [M - Ph], 627 [M - TBDPS],
1
569 [M - TBDPS - tBu]; H NMR (300 MHz) δ: 1.06 (9H, s),
1.08 (9H, s), 1.18-1.39 (8H, m), 1.45-1.70 (3H, m), 1.95-2.10
(4H, m), 2.30 (2H, dd, J ) 6.8, 13.5 Hz), 2.75 (2H, d, J ) 7.2
Hz), 3.53 (2H, d, J ) 3.3 Hz), 3.61 (2H, t, J ) 6.5 Hz), 4.04-4.16
(1H, m), 5.28-5.40 (1H, m), 5.47-5.62 (2H, m), 6.62 (2H, d, J )
8.2 Hz), 6.86 (2H, d, J ) 8.3 Hz), 7.26-7.45 (12H, m), 7.53-
7.64 (4H, m), 7.64-7.73 (4H, m); 13C NMR (75 MHz): δ 19.36,
19.45, 25.59, 26.54, 26.97, 27.25, 28.97, 29.12, 29.54, 29.69, 30.84,
36.31, 36.82, 51.38, 62.31, 63.53, 119.60, 125.12, 127.69, 127,77,
129.83, 130.06 130.34, 133.07, 133.27, 135.52, 154.10, 172.24.
Anal. (C53H69NO4Si2) C, H, N.
(Z)-N-[(1S)-2-Hydroxy-1-[(4-hydroxyphenyl)methyl]ethyl]-12-
(benzoyloxy)-9-dodecenamide (7). A solution of DCC (29 mg,
142 µmol), DMAP (4 mg, 32 µmol), 40 (54 mg, 64 µmol), and
benzoic acid (12 mg, 96 µmol) in CH2Cl2 (1.3 mL) was stirred for
16 h at room temperature. The mixture was diluted with CH2Cl2,
filtered over silica gel, and concentrated in vacuo. The residue was
chromatographed on silica gel using heptanes/AcOEt ) 9/1 as
eluent to give (Z)-N-[(1S)-2-[[(1,1-dimethylethyl)diphenylsilyl]oxy]-
1-[[4-[[(1,1-dimethylethyl)diphenylsilyl]oxy]phenyl]methyl]ethyl]-
12-(benzoyloxy)-9-dodecenamide (30.1 mg, 50%) as a colorless
oil. IR: 3400-3200, 3071, 2930, 2857, 1720, 1643, 1608, 1509,
1472, 1428, 1270, 1113, 1070, 998, 920, 822 cm-1; MS (FAB+,
1
(M - N2), 657.1 (M + 23); H NMR (300 MHz) δ: 1.05-1.43
(8H, m), 1.45-1.60 (2H, m), 1.72 (1H, br s), 1.90-2.10 (2H m),
2.12 (2H, t, J ) 7.4 Hz), 2.40-2.55 (2H, m), 2.60-2.85 (2H, m),
3.00 (1H, br s), 3.55 (1H, dd, J ) 10.8, 4.8 Hz), 3.65 (1H, dd, J )
10.8, 3.0 Hz), 4.05-4.20 (1H, m), 4.27 (2H, t, J ) 6.9 Hz), 5.30-
5.44 (1H, m), 5.45-5.60 (1H, m), 5.87 (1H, d, J ) 7.6 Hz), 6.73
(d, 2H, d, J ) 8.3 Hz), 6.95 (d, 1H, d, J ) 8.5 Hz), 6.99 (d, 2H,
d, J ) 8.3 Hz), 7.77 (1H, dd, J ) 2.1, 8.5 Hz), 8.10 (1H, d, J )
2.1 Hz); 13C NMR (75 MHz): δ 25.64, 26.81, 27.28, 29.03, 29.11,
29.48, 36.19, 36.78, 53.07, 64.39, 65.17, 87.34, 115.60, 121.75,
123.96, 124.35, 128.85, 130.14, 133.31, 140.00, 140.32, 141.80,
155.12, 163.94, 174.33. Anal. (C28H35IN4O5) C, H, N.
1
NBA) m/z: 945 [M + H]; H NMR (300 MHz) δ: 1.06 (9H, s),
1.08 (9H, s), 1.19-1.40 (8H, m), 1.45-1.57 (2H, m), 1.95-2.12
(4H, m), 2.50 (2H, q, J ) 6.8 Hz), 2.76 (2H, d, J ) 7.2 Hz), 3.54
(2H, d, J ) 3.4 Hz), 4.05-4.17 (1H, m), 4.30 (2H, t, J ) 6.8 Hz),
5.35-5.53 (2H, m), 5.57 (1H, d, J ) 8.7 Hz), 6.62 (2H, d, J ) 8.4
Hz), 6.86 (2H, d, J ) 8.4 Hz), 7.26-7.45 (14 H, m), 7.48-7.64
(5H, m), 7.65-7.73 (4H, m), 8.03 (2H, d, J ) 7.2 Hz); 13C NMR
(75 MHz): δ 19.30, 19.40, 25.59, 26.50, 26.91, 27.27, 29.03, 29.16,
29.50, 36.27, 36.80, 51.35, 63.49, 64.39, 119.55, 124.32, 127.64,
127.72, 128.24, 129.50, 129.78, 130.00 130.30, 132.76, 132.88,
133.02, 135.47, 154.06, 166.52, 172.20. A 1 M solution of
N-tetrabutylammonium fluoride (TBAF) in THF (89 µL, 89 µmol)
was added to a solution of the benzoate ester (28 mg, 29.6 µmol)
in THF (500 µL) at 0 °C. The mixture was stirred for 5 min at 0
°C, then for 1 h at 20 °C, and evaporated under vacuum. The residue
was filtered on silica gel, using CH2Cl2/CH3OH ) 98/2 as eluent
to give 7 (14 mg, 98%) as a colorless oil. [R]D ) + 3.1 (c ) 0.05,
CH2Cl2); IR 3600-3080 (br), 1717, 1704 cm-1; MS (electrospray):
Assay of AEA Cellular Reuptake. The effect of compounds
on the uptake of [14C]AEA by rat basophilic leukemia (RBL-2H3)
cells was studied by using 2.4 µM (10,000 cpm) of [14C]AEA. Three
protocols were used. In the first case (protocol #1), a previously
described procedure was used.23 This consisted of incubating the
cells with [14C]AEA for 5 min at 37 °C in the presence or absence
of varying concentrations of the inhibitors. In some cases, a 10
min preincubation of the cells with the inhibitors preceded the
addition of the radiolabeled substrate. The second protocol (protocol

Inhibitors of Anandamide Uptake
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2331
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#2) consisted of preincubating RBL-2H3 cells at room temperature
(25 °C) for 60 min under normal laboratory lighting conditions
with nonphotoactivatable compounds. The preincubation was then
followed by 3 washes of the cells with a cell culture medium
containing 0.2% BSA, 2 washes with BSA-free culture medium,
and finally, by a 5 min incubation with [14C]AEA in the absence
of inhibitors. The third protocol (protocol #3) consisted of prein-
cubating rat C6 glioma cells at room temperature (25 °C) for either
8 min in the presence of UV light (315-400 nm, 125W) or for 8
min in the dark with photoactivatable compounds. The preincuba-
tion was then followed by 3 washes of the cells with a cell culture
medium containing 0.2% BSA, 2 washes with BSA-free culture
medium, and finally, by a 5 min incubation with [14C]AEA in the
absence of inhibitors. One or two fixed concentrations of the
inhibitors, roughly corresponding to their IC50 values obtained using
protocol #1, were used for protocol #2 or #3. In the case of protocol
#3, cell viability after UV exposure was checked after each
experiment, and the incubation time of 8 min was selected as the
longest time interval that did not cause cell damage as assessed
with trypan blue. C6 cells were used instead of RBL-2H3 cells
because they were more resistant to UV exposure. In all cases,
residual [14C]AEA in the incubation medium after extraction with
CHCl3/CH3OH 2:1 (by volume), determined by scintillation count-
ing of the lyophilized organic phase, was used as a measure of the
amount of AEA that was taken up by the cells.40 Data are expressed
as the concentration exerting 50% inhibition of AEA uptake (IC50)
calculated by GraphPad. Nonspecific binding of [14C]AEA to cells
and plastic dishes was determined in the presence of 100 µM AEA
and was never higher than 40% of total uptake with both RBL-
2H3 and C6 cells.
Assay of Fatty Acid Amide Hydrolase (FAAH). The effect of
compounds on the enzymatic hydrolysis of AEA was studied as
described previously,23 using membranes prepared from rat brain,
incubated with the test compounds and [14C]AEA (2.4 µM) in 50
mM Tris-HCl at pH 9 for 30 min at 37 °C. The [14C]ethanolamine
produced from [14C]AEA hydrolysis was measured by scintillation
counting of the aqueous phase after extraction of the incubation
mixture with 2 volumes of CHCl3/CH3OH 2:1 (by volume). Some
experiments were likewise performed by preincubating membranes
from C6 cells with 1b and 8 for 10 min at 37 °C, in the dark or
with concomitant exposure to UV light, followed by the addition
of [14C]AEA in the presence of the inhibitors. With active
compounds, data are expressed as the concentration exerting 50%
inhibition of AEA hydrolysis (IC50) calculated by GraphPad.
Supporting Information Available: Elemental analysis data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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