Synthesis, in vitro and in vivo evaluation, and radiolabeling of aryl anandamide analogues as candidate radioligands for in vivo imaging of fatty acid amide hydrolase in the brain

Article abstract of DOI:10.1021/jm900324e

Fatty acid amide hydrolyase (FAAH) is one of the main enzymes responsible for terminating the signaling of endocannabinoids in the brain. Imaging FAAH in vivo using PET or SPECT is important to deeper understanding of its role in neuropsychiatric disorders. However, at present, no radioligand is available for mapping the enzyme in vivo. Here, we synthesized 18 aryl analogues of anandamide, FAAH's endogenous substrate, and in vitro evaluated their potential as metabolic trapping tracers. Interaction studies with recombinant FAAH revealed good to very good interaction of the methoxy substituted aryl anandamide analogues 17, 18, 19, and 20 with FAAH and they were identified as competing substrates. Compounds 17 and 18 did not display significant binding to CB₁ and CB₂ cannabinoid receptors and stand out as potential candidate metabolic trapping tracers. They were successfully labeled with ¹¹C in good yields and high radiochemical purity and displayed brain uptake in C57BL/6J mice. Radioligands [¹¹C]-17 and [ ¹¹C]-18 merit further investigation in vivo.

Full text of DOI:10.1021/jm900324e

J. Med. Chem. 2009, 52, 4613–4622 4613
DOI: 10.1021/jm900324e
Synthesis, In Vitro and In Vivo Evaluation, and Radiolabeling of Aryl Anandamide Analogues as
Candidate Radioligands for In Vivo Imaging of Fatty Acid Amide Hydrolase in the Brain
Leonie wyffels,† Giulio G. Muccioli,‡, Sylvie De Bruyne,† Lieselotte Moerman,† Johan Sambre,§ Didier M. Lambert,‡ and
Filip De Vos*,†
†Department of Radiopharmacy, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium, ‡Uniteꢀ de Chimie Pharmaceutique et de
Radiopharmacie, Universiteꢀ Catholique de Louvain, Avenue E. Mounier 73, UCL-CMFA 73-40, B-1200 Brussels, Belgium, and §University
Hospital Ghent, Cyclotron Department, De Pintelaan 185, B-9000 Ghent, Belgium. Present address: Bioanalysis and Pharmacology of Bioactive
Lipids Laboratory: Uniteꢀ d’Analyse Chimique et Physico-chimique des Meꢀdicaments et Pharmacognosie, Universiteꢀ Catholique de Louvain,
Avenue E. Mounier 72, UCL-CHAM 7230, B-1200 Brussels, Belgium.
Received March 16, 2009
Fatty acid amide hydrolyase (FAAH) is one of the main enzymes responsible for terminating the
signaling of endocannabinoids in the brain. Imaging FAAH in vivo using PET or SPECT is important to
deeper understanding of its role in neuropsychiatric disorders. However, at present, no radioligand is
available for mapping the enzyme in vivo. Here, we synthesized 18 aryl analogues of anandamide,
FAAH’s endogenous substrate, and in vitro evaluated their potential as metabolic trapping tracers.
Interaction studies with recombinant FAAH revealed good to very good interaction of the methoxy
substituted aryl anandamide analogues 17, 18, 19, and 20 with FAAH and they were identified as
competing substrates. Compounds 17 and 18 did not display significant binding to CB1 and CB2
cannabinoid receptors and stand out as potential candidate metabolic trapping tracers. They were
successfully labeled with 11C in good yields and high radiochemical purity and displayed brain uptake in
C57BL/6J mice. Radioligands [11C]-17 and [11C]-18 merit further investigation in vivo.
Introduction
local due to rapid sequential cellular reuptake and enzymatic
hydrolysis of AEA. The metabolization of AEA to arachido-
nic acid and ethanolamine is catalyzed by an amidase, known
as fatty acid amide hydrolase (FAAH).6 The enzymes respon-
siblefor biosynthesis anddegradation areconsidered tobekey
steps in the regulation of endocannabinoid levels.
The discovery in 1992 of N-arachidonoylethanolamine,
christened anandamide (AEAa),1 confirmed the existence of
an endogenous cannabinoid system in mammals. This endo-
cannabinoid system comprises, among others, cannabinoid
receptors CB1 and CB2, endogenous ligands (the endocanna-
binoids, withAEA and 2-arachidonoylglycerol being the most
studied ones), and the proteins for their synthesis and inacti-
vation. AEA was isolated from porcine brain, shortly after
cDNA cloning of the CB1 cannabinoid receptor.2 AEA is the
amide of the polyunsaturated fatty acid arachidonic acid with
ethanolamine and has an agonist action on CB1 cannabinoid
receptors.3 It functions as a retrograde synaptic messenger,
implying that it is released from postsynaptic neurons and
travels backward across synapses to activate presynaptic CB1
cannabinoid receptors, modulating neurotransmitter action
and release.4,5 This neuromodulatory function remains very
FAAH was the first endocannabinoid enzyme to be
cloned.7 It is a dimeric integral membrane enzyme widely
distributed in the brain as well as in the periphery, with a
predominant localization in microsomal and mitochondrial
fractions.8-11 FAAH constitutes the only characterized mam-
malian member of a large class of enzymes termed the amidase
signature family12 and is responsible for the metabolism of a
wide variety of fatty acid amides. It may act as a hydrolytic
enzyme not only for AEA but also for the anti-inflammatory
compound N-palmitoylethanolamine (PEA),13 the sleep in-
ducing lipid cis-9-octadecenoamide (oleamide)14 and even
esters such as 2-AG (Figure 1).15 Extensive SAR studies
revealed the structural requirements for substrate interaction.
Those requirements are relatively flexible, with a preference
for long-chain fatty acid amides, consistent with its proposed
role as the primary catabolic route for these signaling lipids in
vivo. Up to 4, the higher the number of cis-1,4-diene double
bonds in the fatty acid chain, the stronger the interaction of
the substrate with FAAH.11,16-18 This observation may be
attributed to the requirement of a hairpin conformation for
substrate recognition at the FAAH active site: a low energy
conformation involving a folding in the middle of the fatty
acid chain so that its distal part is in close proximity with the
headgroup. This hairpin conformation is best accommodated
*To whom correspondence should be addressed. Phone:
(þ32)92648066. Fax: (þ32)92648071. E-mail: filipx.devos@ugent.be
a Abbreviations: AEA, anandamide; AMT, anandamide membrane
transporter; 2-AG, 2-arachidonoylglycerol; BBB, blood-brain barrier;
CB1, cannabinoid receptor subtype-1; CB2, cannabinoid receptor sub-
type-2; CDI, carbonyldiimidazole; CHO, Chinese hamster ovary; DMF,
dimethylformamide; ECD, ethylene cysteine dimer; EDTA, ethylene
diamine tetraacetic acid; FAAH, fatty acid amide hydrolase; FDG, 2-
fluorodeoxyglucose; HPLC, high performance liquid chromatography;
MAFP, methyl fluorophosphonate; PEA, N-palmitoylethanolamine;
PET, positron emission tomography; PMSF, phenylmethanesulfonyl
fluoride; rFAAH, recombinant rat FAAH; SAR, structure-activity
relationship; SPECT, single photon emission computed tomography;
TBAH, tetrabutylammonium hydroxide.
r
2009 American Chemical Society
Published on Web 07/15/2009
pubs.acs.org/jmc

4614 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
wyffels et al.
Scheme 1. General Reaction Scheme for Synthesis of Aryl
Anandamide Analogues (R Groups are Shown in Table 1)a
Figure 1. Chemical structures of representative FAAH substrates.
by the presence of four cis nonconjugated double bonds as in
AEA but is less thermodynamically favored in the cis-diene
linolenyl and cis-ene oleyl analogues, thus decreasing the
ability of these substrates to recognize the FAAH catalytic
site.19 FAAH can accept as substrate both primary and
secondary amides with numerous substitutions, including
aromatic rings and oxygenated and branched alkyl substitu-
ents.16,19,20 Lin et al. showed that increased electronegativity
at the headgroup is accompanied with decreased biochemical
stability.21 The presence of electronegative substituents ren-
ders the carbonyl group more electrophilic, which favors
enzymatic hydrolysis.
a Reagents and conditions: (a) CDI, CH2Cl2, room temperature, 1 h,
then various amines, room temperature, 2 h, 13-88%.
fluorodeoxyglucose ([18F]-FDG) to assess rates of cerebral
glucoseutilization for detection of brain tumors, epileptic foci,
and Alzheimer’s disease48,49 and the use of [99mTc]-ethylene
cysteine dimer ([99mTc]-ECD) for the study of regional cere-
bral perfusion.50
Former attempts to synthesize useful FAAH metabolic
trapping tracers failed due to in vivo instability.51 Thus
because no PET or SPECT tracer for in vivo visualization
of FAAH exist so far, the aim of this study was the synthesis
and in vitro evaluation of aryl anandamide analogues while
focusing on the potential for discovery of radioligands that
might be useful for in vivo molecular imaging of FAAH. Here
we report that two out of 18 synthesized compounds may be
promising PET tracers for in vivo visualization of FAAH in
the brain.
The importance of FAAH in controlling endogenous ana-
ndamide levels in the brain and consequently the importance
of FAAH as a regulatory enzyme for key physiological
functions is suggested by studies with transgenic mice lacking
FAAH. They possess high endogenous levels of anandamide
and related fatty acid amides in the brain that correlate
with CB1-dependent behavioral responses including hypomo-
tility, analgesia, catalepsy, and hypothermia.22 Accumulating
data suggest that the symptoms of several neurological and
neuropsychiatric disorders could be caused by changes in
the endocannabinoid biosynthesis and degradation, includ-
ing addiction,23-28 schizophrenia,29-31 anxiety,32-34 depres-
sion,35,36 multiple sclerosis,37,38 epilepsy,39 Parkinson’s dis-
ease,40-42 and Huntington’s disease.42,43 However, the exact
mechanisms correlating the endocannabinoid system, and
more specifically FAAH, with these disorders are not fully
understood. Besides the extensive search for FAAH inhibi-
tors, few studies have been made to assess the possible
diagnostic and therapeutic application of FAAH in neurolo-
gical and neuropsychiatric diseases.
To more deeply understand the relationship between the
FAAH-endocannabinoid system and neuronal and neurop-
sychiatric disorders and to find cause-effect relationships
between changes in the expression/activity of FAAH and
pathological conditions, visualization of the enzyme in vivo
would be of great help. Noninvasive in vivo detection of
enzymes can be achieved by using in vivo molecular imaging
and the technique of metabolic trapping.44 Briefly, the prin-
ciple of this method is that a radiolabeled substrate analogue
passes the blood-brain barrier (BBB) and is taken up in the
cell and hydrolyzed by FAAH to produce a labeled metabo-
lite. Because this metabolite has limited ability to diffuse
across the BBB, it is trapped in neuronal cells at the site of
the enzyme. In that manner, we can map the enzyme and
quantify the amount and functionality of FAAH. The prin-
ciple of metabolic trapping has already been successfully
applied for evaluation of acetylcholinesterase activity in the
brain for Alzheimer’s disease diagnosis using [18F]-, [11C]-,
and [123I]-labeled acetylcholine analogues.45-47 Other well-
known examples are the use of the glucose analogue [18F]-2-
Results and Discussion
Chemistry. A total of 18 AEA-analogues was synthesized,
following the reaction scheme depicted in Scheme 1. Iodine
and methoxy substituted aryl AEA analogues were chosen
with the prospect on possible labeling with 123I, 124I, or 11C.
The structures of all 18 compounds are shown in Table 1. All
compounds, as well as the known FAAH substrates N-(2-
hydroxyethyl)linoleoylamide (23) and AEA (24) and the O-
desmethyl precursors 25 and 26 were synthesized by the
direct condensation between in situ preactivated arachidonic
acid or linoleic acid, employing the coupling reagent carbo-
nyldiimidazole (CDI) and the corresponding amine. This
reaction, which generates imidazole in situ, does not require
an additional base and is even compatible with hydrochlor-
ide salts of the amine.52 In practice, the imidazolides of
arachidonic acid and linoleic acid were preformed for 1 h
and then the appropriate amine was added to form the
corresponding acylamide. After workup, purification was
achieved by silica flash column chromatography. With the
exception of 1, 4, 7, and 14, all amines were commercially
available. Synthesis of amines 1, 4, 7 (Scheme 2), and 14
(Scheme 3) involved a reduction of the corresponding nitrile
with the BH3 THF complex and a workup as described by
3
54
Ikeuchi et al.53 or by Sole et al. Both workup methods
ꢀ
resulted in the desired amine, which could be used in the next
step without any further purification. With the exception of 2
and 3, all compounds were obtained in good to excellent
yields.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4615
Table 1. Inhibition of [3H]-AEA Metabolism for Aryl Analogues of Anandamidea
a
Values are calculated from a minimum of three experiments.
b
c Compounds 23 and 24 are used as references.
Scheme 2. Synthesis of Iodophenethylamines (1, 4, and 7)a
Scheme 3. Synthesis of 2-Iodobenzylamine (14)a
a Reagents and conditions: (a) BH3, THF, 0 °C to reflux, 2 h, 1 M
HCl-CH3OH, 0 °C.
FAAH Interaction. A prerequisite for a metabolic trapping
ligand is that the compound is a substrate of the enzyme
under investigation. Therefore, we first investigated whether
the synthesized aryl anandamide analogues were recognized
by the FAAH and then determined whether they are indeed
hydrolyzed by the enzyme.
a Reagents and conditions: (a) BH3, THF, 0 °C to reflux, 2 h, then 6 M
HCl, to basic pH with NaOH 1M, 0 °C (for 1 and 7) or 1 M HCl-
CH3OH, 0 °C (for 4).
The FAAH assay used in this study allows us to investigate
the interaction between recombinant rat FAAH (rFAAH)

4616 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
wyffels et al.
Figure 2. Inhibition of the hydrolysis by recombinant rat FAAH of 2 μM [3H]-AEA by compounds 23 and 24 (A) and compounds 17, 18, 19,
and 20 (B). Shown are means of three experiments, with no preincubation of the compounds with rFAAH prior to addition of [3H]-AEA. pIC50
values are shown as a dotted line on the figure.
and the synthesized anandamide analogues by their ability to
prevent the enzyme from hydrolyzing [3H]-AEA. It is im-
portant to emphasize that by using this approach no infor-
mation is given on the efficacy of the compounds as
substrates but rather allows for determination of their
affinity for the enzyme.55 All compounds were tested for
their ability to inhibit FAAH catalyzed [3H]-AEA hydrolysis
using 10 concentrations of test compound in the range of 3
mM to 1 nM. Potencies to inhibit the hydrolysis of [3H]-AEA
by FAAH are expressed as pIC50 (i.e., -log IC50). Com-
pound 23 is known to be a substrate for FAAH and was used
as a reference compound in this assay, as it inhibits [3H]-AEA
metabolism by acting as a competing substrate. In our hands,
23 inhibits [3H]-AEA hydrolysis with a pIC50 value of 5.12, a
value comparable with the values reported for the known
endogenous FAAH substrate palmitoylethanolamine (pIC50
value of 5.30).51,56 As an additional control, one of the most
thoroughly studied FAAH substrates, anandamide (24), was
also synthesized and tested in this assay and displayed a
pIC50 value of 4.93 (Figure 2).
The results obtained with the AEA analogues are pre-
sented in Table 1. In the series of iodinated aryl anandamide
analogues, the para and meta iodobenzyl derivatives (10, 11,
12 and 13) inhibited the hydrolysis of [3H]-AEA with clearly
higher potency than that of the iodophenethyl derivatives (2,
3, 5, 6, 8 and 9). Within the iodobenzyl series, the para and
meta substituted compounds seem to have a better potency
than the ortho substituted derivatives 15 and 16, with 10
displaying the highest pIC50 value of 4.84. Steric hindrance
caused by the bulky iodine in ortho position might contri-
bute to the weaker interaction of 15 and 16 with the enzyme.
In the iodophenethyl series, 2, 3, 5, 6, 8, and 9 displayed
inhibition of [3H]-AEA hydrolysis, only at the higher con-
centrations tested (>0.1 mM). Probably, the iodine is a too
big substituent on the phenethyl for adequate interaction
with FAAH. With the exception of 21 and 22, the methox-
yphenethyl series (17, 18, 19, 20) displayed higher pIC50
values than both iodobenzyl and iodophenethyl derivatives.
Possibly the methoxy substituted phenolic ring contributes
to the compounds’ ability to interact with the enzyme and
electron-rich aromatics such as the methoxylated ones might
give rise to a more stable enzyme-substrate complex than do
the electron-poor iodinated aromatics. Varying the position
of the electron donating methoxy substituent from para to
meta or ortho position did change the activity toward the
enzyme. Para substitution (17 and 18) clearly allowed the
best interaction with FAAH, followed by meta substitution
(19 and 20). Introduction of a methoxy in the ortho position
of the phenyl (21 and 22) resulted in pIC50 values leaning
toward those of 11, 12, 13, and 15. Looking at the difference
in FAAH interaction between the arachidonoyl-based versus
linoleoyl-based compounds, no unambiguous conclusion
could be drawn although the arachidonoyl-based counter-
part mostly demonstrated higher potencies, consistent with
endocannabinoid acyl chain SAR studies.18,20
In a second set of experiments, we wanted to distinguish
inhibitors from competing substrates by preincubating the
compounds with rFAAH prior to addition of [3H]-AEA: for
a competing substrate, the preincubation is expected to
reduce the observed potency of the compound.57 The meth-
oxyphenethyl series (17-22) were tested as those compounds
gave the best results in the former experiment as well as 3 and
11 to check the influence of the two methylenes on recogni-
tion by the catalytic site and subsequent hydrolysis. We
selected the concentration of test compound corresponding
to the IC50 in the previously described experiment or, in the
case of 3 and 11, used the concentration corresponding to the
steepest part of the curve (0.3 mM for 3 and 10 μM for 11)
and preincubated with rFAAH for 0 min, 1 h, 2 h, or 3 h prior
to addition of [3H]-AEA. Compounds 23 and 24 were used as
reference compounds because they are known FAAH sub-
strates and thus should demonstrate a decrease in potency
with longer preincubation time. Methyl arachidonyl fluor-
ophosphonate (MAFP, 0.3 nM), a potent irreversible FAAH
inhibitor, was used as negative control.
The results are presented in Figure 3. As expected for 23
and 24, a clear increase in [3H]-AEA hydrolysis or decrease in
potency of the inhibition was observed after preincubation
because of metabolization of both compounds by rFAAH
during the preincubation (P < 0.001 compared to no pre-
incubation condition). Within the methoxyphenethyl series,
17, 18, and 20 displayed a large decrease in potency with
longer preincubation, suggesting metabolization of the com-
pounds by rFAAH (P < 0.01 compared to no preincubation
condition). The influence of preincubation was most clear
for 17 and 18. On the other hand, 21 and 22 displayed a slight
increase in FAAH inhibition with longer preincubation.
These results indicate that probably 21 and 22 interact with
FAAH not as competing substrates but rather as slow and
not very potent inhibitors of the enzyme. Preincubation of 3
caused a little increase in inhibition of FAAH with longer
preincubation time. No consistent effect of preincubation

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4617
Scheme 4. Radiosynthesis of [11C]-17 and [11C]-18a
a Conditions, reagents and decay-corrected yield: (a) precursor (25 or
26), DMF, 1.5 M Bu4NOH ([11C]-17), 1 M NaH ([11C]-18), [11C]CH3I,
55 °C (for 17), 50 °C (for 18), 10 min, 39.6 ( 0.7% ([11C]-17, n = 5) and
27.5 ( 2.6% ([11C]-18, n = 5).
Figure 3. Inhibition of the hydrolysis by recombinant rat FAAH of
2 μM [3H]-AEA by compounds 3, 11, 17, 18, 19, 20, 21, and 22
following preincubation with rFAAH for 0 min, 1 h, 2 h, or 3 h prior
to addition of [3H]-AEA. Compounds 23 and 24 were used as
references, MAFP was used as a negative control. Data are means
of three experiments. Significance (P < 0.01) was assessed using
One-Way Anova followed by the Dunnett post-test.
incubation at 37 °C. HPLC analysis revealed no formation of
fatty acid, indicating inhibition of conversion of 24, 17, and
18 by FAAH with MAFP preincubation.
Overall, these assays confirm the identity of both 17 and 18
as FAAH substrates and thus potential metabolic trapping
tracers for visualization of brain FAAH.
could be demonstrated for 11. Opposite to the methoxyphe-
nethyl derivatives, the difference in pIC50 values was not
reflected in the preincubation studie for 3 and 11. No clear-
cut difference between the benzyl and phenethyl could be
demonstrated.
From the above experiments, it appears that all six meth-
oxyphenethyl derivatives clearly show a substrate-like beha-
vior when incubated with rFAAH, while the iodobenzyl and
definitely the iodophenethyl derivatives demonstrate only
weak or no interaction with the enzyme. The preincubation
experiments demonstrated that 21 and 22 interact with
FAAH probably as weak inhibitors, while 17, 18, 19, and
20 interact with FAAH as competing substrates.
Because 17 and 18 displayed the best interaction with
FAAH, both compounds were put forward as possible useful
metabolic trapping tracers for visualization of FAAH. It has
been suggested, using indirect methods, that 18 is not a
FAAH substrate.58 Therefore, to further confirm that 17
and 18 are indeed substrates for FAAH, a HPLC method
was used to demonstrate generation of linoleic acid or
arachidonic acid as a result of enzymatic metabolization
after incubation of the compounds with FAAH. An experi-
ment with the endogenous FAAH substrate 24 was per-
formed to validate the method. Because 24 is hydrolyzed to
the fatty acid arachidonic acid and ethanolamine, HPLC
analysis with UV detection of test solution containing 24 and
rFAAH reveals the generation of arachidonic acid. Incuba-
tion of 17 and 18 with rFAAH resulted in the formation of
linoleic acid and arachidonic acid, respectively. To rule out
spontaneous formation of fatty acid during incubation, the
stability of 24, 17, and 18 in assay buffer containing no
FAAH was studied by incubation at 37 °C. Test solution
(100 μL) was injected into the HPLC system after the first
minute and at 30 min intervals for 3 h. The results showed
that no formation of fatty acid was detected during this time.
Methyl arachidonoyl fluorophosphonate (MAFP) is a
potent irreversible inhibitor of FAAH. Preincubation of
FAAH with MAFP blocks the enzymatic activity, resulting
in no formation of fatty acid in case the compounds are
genuine FAAH substrates. Thus rFAAH was preincubated
with 10 nM of MAFP in assay buffer for 10 min at 37 °C,
followed by addition of 24, 17, or 18 and another 30 min
Receptor Binding Studies. A condition for 17 and 18 to be
useful as tracer for FAAH is that they do not display binding
to the cannabinoid receptors CB1 and CB2. Indeed, binding
of radiolabeled 17 or 18 to the cannabinoid receptors would
give a background signal in future imaging studies. There-
fore, both compounds were screened at 10 μM for competi-
tive binding to the hCB1 and hCB2 cannabinoid receptors
using membranes of Chinese hamster ovary (CHO) cells
selectively expressing either the hCB1 or hCB2 cannabinoid
receptors. The experiments were done in the presence of
phenylmethanesulfonyl fluoride (PMSF, 200 μM), a broad
acting serine hydrolases inhibitor, in order to prevent meta-
bolism of compounds by FAAH. Compound 17 (10 μM) as
well as 18 (10 μM) did not display significant binding to the
hCB1 cannabinoid receptor, with only 13% displacement of
bound [3H]-SR141716A for 17 and 26% displacement of
bound [3H]-SR141716A for 18. Compounds 17 and 18 did
not show significant binding to the hCB2 cannabinoid
receptor either, as they displaced only 8% and 11%, respec-
tively, of the radioligand ([3H]-CP55,940) bound to the
receptor. The obtained results are in line with cannabinoid
receptors SAR studies stating that the cannabinoid receptor
can accommodate both hydrophobic and hydrophilic
head groups, however, the size of the cavity in which the
headgroup binds is small.20 The cannabinoid receptors site
does not tolerate large head groups. Substitution of the 2-
hydroxyethyl group of AEA with an aromatic hydroxyl
group greatly decreased affinity for CB1 cannabinoid recep-
tors,16,59 a conclusion that is supported by our findings. The
observation that 18 displaces more of bound [3H]-
SR141716A from the hCB1 cannabinoid receptor compared
to 17 is as expected by SAR studies, indicating that the CB1
cannabinoid receptor requires a minimum of three cis double
bonds for adequate recognition.60 The results obtained in
this assay suggest that 17 and 18 are devoid of affinity for the
cannabinoid receptors hCB1 and hCB2, which makes them
suitable for labeling and imaging of FAAH.
Radiosynthesis. [11C]-17 and [11C]-18 were prepared by
methylation of the appropriate O-desmethyl precursor (25
and 26, respectively) with [11C]-CH3I under basic conditions

4618 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
wyffels et al.
Figure 4. Uptake (%ID/g) of radioactivity in selected mice organs at various time points after iv injection of [11C]-17 (A) or [11C]-18 (B).
in a homemade synthesis module (Scheme 4). Initially, the
use of NaH and TBAH under various reaction conditions
including different molarities, reaction times, and tempera-
tures were investigated (data not shown). These experiments
indicated that highest yields could be obtained using a
combination of 3 μmol of 25, 1 μL of TBAH, and 250 μL
of DMF in a 10 min reaction at 55 °C for [11C]-17 and using
3 μmol of 26, 10 μL of NaH, and 240 μL of DMF in a 10 min
reaction at 50 °C for [11C]-18. Using the optimized reaction
conditions, [11C]-17 and [11C]-18 were successfully obtained
with decay corrected radiochemical yield of 39.6 ( 0.7%
(n = 5) and 27.5 ( 2.6% (n = 5), respectively (based on
[11C]CH3I trapped in reaction vial). The overall radiosynth-
esis time from end-of-bombardment until end-of-synthesis
was about 40 min. Both tracers were obtained in high
radiochemical purity (>97%) and were free of labeling
precursors. Specific radioactivities were normally between
60 and 100 GBq/μmol. Product identity was confirmed by
coelution with reference compounds 17 or 18 after coinjec-
tion on HPLC.
ity in blood dropped from 24.33 ( 1.76%ID/g at 1 min to
0.78 ( 0.07%ID/g at 60 min for [11C]-17 and from 20.67 (
3.87%ID/g at 1 min to 0.63 ( 0.18%ID/g at 60 min for [11C]-
18. A high initial uptake was observed in heart, lungs, liver,
and kidneys, followed by continuous decrease of radioactiv-
ity until the end of the study. The uptake in intestine
remained rather constant and low throughout the study
(1.79 ( 0.57%ID/g and 1.04 ( 0.17%ID/g in small intestines
and large intestines respectively at 30 min pi for [11C]-17 and
2.85 ( 0.45% ID/g and 1.22 ( 0.66% ID/g in small intestines
and large intestines, respectively, at 30 min pi for [11C]-18),
while the uptake in bladder/urine continuously increased
over time (data not shown), indicating that renal elimination
is the primary excretory pathway. Binding to plasma pro-
teins was found to be 94% reversible.
Conclusions
The aim of this study was the development of a set of
compounds based on the anandamide template that might be
useful for in vivo molecular imaging of FAAH. We synthe-
sized a total of 18 novel aryl anandamide analogues in reason-
able togoodyields. The introduction ofa methoxyoriodine in
the phenyl provided an opportunity for labeling with 11C or
123I and 124I, respectively. We provided novel information on
the interactionbetweenAEA analogues and FAAH, although
the main objective of this study was the discovery and selec-
tion of potential metabolic trapping tracers. In vitro interac-
tion studies with recombinant FAAH revealed that the iodo
substituted aryl anandamide analogues displayed only weak
to no interaction with the enzyme, while the methoxy sub-
stituted aryl anandamide analogues displayed good to very
good interactions with FAAH. Further research identified 17,
18, 19, and 20 as competing substrates, making them potential
metabolic trapping tracers. Two analogues (17 and 18) stand
out as potential candidate metabolic trapping tracers for PET
molecular imaging of brain FAAH. They were successfully
labeled with 11C in satisfactory yields and a good radio-
chemical purity and preliminary in vivo evaluation in mice
indicated brain uptake for both tracers. Both compounds
needfurther exploration invivotoassesstheir suitability for in
vivo molecular imaging of brain FAAH. They can serve as an
entry point to the preparation of FAAH imaging agents.
Ex Vivo Evaluation. Metabolization of [11C]-17 and [11C]-
18 by cerebral FAAH was studied ex vivo using brain
homogenates as source of FAAH. Incubation of [11C]-17
or [11C]-18 with C57BL/6J mice brain homogenates for
30 min, followed by HPLC analysis of radioactivity in brain
extracts, revealed metabolization of both tracers with for-
mation of a polar metabolite. The polar metabolite was
identified as 4-methoxyphenethylamine and thus mediated
by hydrolysis of the amide bond, possibly through FAAH.
Preincubation of the brain homogenates with the potent and
selective FAAH inhibitor URB597 efficiently inhibited the
metabolization of [11C]-17 and [11C]-18, confirming the
metabolization to be FAAH mediated. The results obtained
in this assay endorse the identity of 17 and 18 as FAAH
substrates.
In Vivo Evaluation. To investigate the uptake of [11C]-17
and [11C]-18 in the brain, a general biodistribution in mice
was conducted. Adult male C75BL/6J mice were injected
intravenously in the tail vein with approximately 3.7 MBq of
[11C]-17 or [11C]-18. The radioactivity concentrations in
various tissues as a function of time following administration
of [11C]-17 or [11C]-18 are represented in Figure 4.
Both [11C]-17 (1.44 ( 0.14%ID/g at 1 min post injection)
and [11C]-18 (0.84 ( 0.09%ID/g at 1 min post injection)
displayed brain uptake followed by a continuous decrease
until the end of the study. Considering that the maximal
contribution of blood vessels to the whole brain is 3%, both
compounds demonstrate moderate brain uptake. The activ-
Experimental Section
Materials. All chemical reagents were obtained from com-
mercial sources (Sigma-Aldrich Fluka, Acros Organics,
Belgium) and used without further purification. Solvents were

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4619
purchased from Lab-Scan Analytical Sciences (Dublin,
Ireland). Radiolabeled arachidonoylethanolamide ([3H]-AEA,
labeled in its ethanolamine moiety, specific activity of 60 Ci/
mmol) was obtained from American Radiolabeled Chemicals,
Inc. (St Louis, MO), [3H]-SR141716A (52 Ci/mol) from Amer-
sham (Roosendaal, The Netherlands), and [3H]-CP-55,940 (101
Ci/mol) from NEN Life Science (Zaventem, Belgium). Fatty-
acid-free bovine serum albumin (BSA) was obtained from
Sigma Chemical Co. (Belgium).
2H), 3.79 (s, 3H), 5.35 (m, 4H), 5.55 (brs, 1H), 6.85 (d, J = 8.70
Hz, 1H), 7.10 (d, J = 9.00 Hz, 2H). 13C NMR (75 MHz, CDCl3):
14.30, 22.80, 25.85, 25.96, 27.42, 29.36, 29.48, 29.49, 29.57,
29.85, 31.75, 35.03, 37.07, 40.88, 55.48, 114.26, 128.13, 128.27,
129.91, 130.27, 130.45, 131.12, 158.50, 173.26. MS (ESI) m/z
(% rel int.): 414.5 (100.0 [M þ H]þ).
4-Methoxyphenethylarachidonoylamide (18). Synthesis fol-
lowed the same procedure described for 17, using arachidonic
acid (1 g, 3.29 mmol) as fatty acid and 4-methoxyphenetylamine
(1 mL, 6.58 mmol) as amine. Purification by flash chromato
graphy (8:2 hexane-ethyl acetate) gave 18 as a colorless oil
(1.2 g, 83%). 1H NMR (300 MHz, CDCl3): δ 0.88 (t, J = 6.60 Hz,
3H), 1.30 (m, 6H), 1.68 (qu, J=7.20 Hz, 2H), 2.07 (q, J=6.90 Hz,
4H), 2.12 (t, J = 7.80 Hz, 2H), 2.79 (t, J = 6.60 Hz, 6H), 2.82 (t,
J = 6.30 Hz, 2H), 3.48 (q, J = 5.40 Hz, 2H), 3.79 (s, 3H), 5.35
(m, 8H), 6.84 (d, J = 8.70 Hz, 2H), 7.10 (d, J = 8.70 Hz, 2H).
13C NMR (75 MHz, CDCl3): 14.21, 22.71, 25.66, 25.78, 26.81,
27.36, 29.46, 31.65, 34.95, 36.27, 40.83, 55.40, 114.19, 127.66,
127.99, 128.32, 128.36, 128.74, 128.85, 129.28, 129.81, 130.66,
130.99, 158.42, 172.85. MS (ESI) m/z (% rel int.): 438.7 (100.0
[M þ H]þ).
General Methods. All chemical reactions were conducted
under N2 atmosphere with dry solvents under anhydrous con-
ditions. The thin-layer chromatographic analyses were per-
formed using 200 μm of silica gel with fluorescent indicator
(UV254) coated on plastic plates (Macherey-Nagel, Germany).
Visualization of the plates was done by UV (λ = 254 nm) and
iodine vapors. Purification of compounds was achieved by flash
column chromatography with silica gel (Sigma-Aldrich,
230-400 mesh), using solvent systems indicated in the text.
For mixed solvent systems, ratios are given with respect to
1
volumes. The H and 13C nuclear magnetic resonance (NMR)
spectra were measured with a Varian 300 MHz FT-NMR
spectrometer (Department of Medicinal Chemistry, Ghent Uni-
versity). Signals are quoted as s (singlet), brs (broadened
singlet), d (doublet), t (triplet), q (quartet), qu (quintet), or
m (multiplet). Coupling constants (J) are expressed in Hz.
Chemical shifts were recorded in ppm (δ) from an internal
tetramethylsilane standard in chloroform-d3. Mass spectrome-
try was performed on a Waters Micromass ZMD mass spectro-
meter with an electrospray-ionization probe. Samples were
dissolved in a mixture of MeOH:H2O:HCOOH (50:50:0.1,
v/v). All tested compounds were at least 95% pure on the basis
of HPLC analysis. Radiosyntheses were performed in a home-
made remotely controlled system. Radiotracer purifica-
tions were performed with HPLC on a reversed phase column
(Grace Discovery Econosphere C18 column, 250 mm ꢀ 10 mm,
particle size 10 μm þ Grace Discovery Econosphere C18 guard
33 mm ꢀ 7 mm, particle size 10 μm). [11C]-17 was eluted with a
mixture of MeCN:H2O:HCOOH (90:10:0.1, v/v) and [11C]-18
with a mixture of MeCN:H2O:HCOOH (93:7:0.1, v/v), both at a
flow rate of 4 mL/min. The column outlet was connected with a
Knauer Smartline 2500 UV detector (λ = 254 nm) in series
with a homemade radiodetector with photodiode. The radio-
chemical purities and specific radioactivities of both products
were determined with reversed phase HPLC (GraceSmart RP 18
column, 250 mm ꢀ 4.6 mm, particle size 5 μm) eluted
with MeCN:H2O:HCOOH (90:10:0.1 v/v) as mobile phase.
Eluate was monitored with an absorbance detector (λ = 254
nm) in series with a Ludlum 220 scaler ratemeter equipped with
a GM probe for radiation detection. Specific radioactivity
(GBq/μmol)) were determined with analytical HPLC calibrated
for absorbance (λ = 254 nm) response per mass of ligand.
The radioactivity of the radioligand peak (decay corrected)
(GBq) was divided by the mass of the associated carrier
peak (μmol).
4-Hydroxyphenethyllinoleoylamide (25). Synthesis followed
the same procedure as described for 17 using linoleic acid
(500 mg, 1.67 mmol) as the fatty acid and 4-hydroxyphenethy-
lamine (458 mg, 3.34 mmol). Purification by silica gel flash
chromatography (8:2 hexane-ethyl acetate) gave 25 as a white
1
solid (515 mg) in a 77% yield. H NMR (300 MHz, CDCl3):
δ 0.89 (t, J = 7.20 Hz, 3H), 1.28 (m, 14H), 1.58 (qu, J = 7.20 Hz,
2H), 2.04 (q, J = 6.90 Hz, 4H), 2.11 (t, J = 7.20 Hz, 2H), 2.72 (t,
J = 7.20 Hz, 2H), 2.78 (t, J = 5.70 Hz, 2H), 3.48 (q, J = 6.90 Hz,
2H), 5.34 (m, 4H), 6.78 (d, J = 8.70 Hz, 1H), 7.02 (d, J = 8.70
Hz, 2H). 13C NMR (75 MHz, CDCl3): 14.41, 22.80, 25.85, 25.96,
27.42, 29.43, 29.46, 29.56, 29.83, 29.92, 31.74, 35.00, 37.07,
41.05, 115.82, 128.13, 128.27, 129.94, 130.26, 130.28, 130.47,
155.26, 173.81. MS (ESI) m/z (% rel int.): 400.2 (100.0 [M þ H]þ);
422.5 (10 [M þ Na]þ).
4-Hydroxyphenethylarachidonoylamide (26). The same pro-
cedure described for 17 was followed, using arachidonic acid
(500 mg, 1.64 mmol) and 4-hydroxyphenethylamine (450 mg,
3.28 mmol). Purification by silica gel flash chromatography
(85:15 hexane-ethyl acetate) gave 26 as a clear colorless oil (522
mg) in a 75% yield. 1H NMR (300 MHz, CDCl3): δ 0.88 (t, J =
6.60 Hz, 3H), 1.29 (m, 6H), 1.68 (qu, J = 7.20 Hz, 2H), 2.07 (q,
J = 6.60 Hz, 4H), 2.14 (t, J = 7.20 Hz, 2H), 2.72 (t, J = 7.20 Hz,
6H), 2.80 (t, J = 6.30 Hz, 2H), 3.48 (q, J = 6.90 Hz, 2H), 5.35
(m, 8H), 6.79 (d, J = 8.40 Hz, 2H), 7.01 (d, J = 9.00 Hz, 2H).
13C NMR (75 MHz, CDCl3): 14.22, 22.72, 25.66, 25.78, 26.77,
27.37, 29.46, 31.66, 34.92, 36.30, 41.05, 115.75, 127.67, 128.00,
128.30, 128.39, 128.76, 128.95, 129.18, 129.87, 130.25, 130.68,
155.31, 173.44. MS (ESI) m/z (% rel int.): 424.2 (100.0 [M þ
1]þ); 446 (6.0 [M þ Na]þ).
Compound 2-16 and 19-24. The description of compounds
2-19 and 19-24 synthesis can be found in the Supporting
Information.
Chemistry. 4-Methoxyphenethyllinoleoylamide (17). To a so-
lution of linoleic acid (500 mg, 1.67 mmol) in dry dichloro-
methane (CH2Cl2, 15 mL) was added carbonyldiimidazole
(CDI, 270.54 mg, 1.67 mmol). The reaction mixture was stirred
at room temperature for 1 h. 4-Methoxyphenetylamine (489 μL,
3.34 mmol) 1 was added dropwise, and the reaction mixture was
stirred for 2 h at room temperature. The reaction mixture was
diluted with dichloromethane (30 mL) and washed two times
with water and with saturated brine. The organic layer was dried
over MgSO4, and the solvent was evaporated under reduced
pressure to give the crude product. Purification by flash chro-
matography (8:2 hexane-ethyl acetate) gave 17 as a white
Pharmacology. Fatty Acid Amide Hydrolase Assay. The
method used was based on that of Omeir et al. using [14C]-
AEA as substrate and has been previously described.55,57
Briefly, recombinant rat FAAH fused to maltose binding pro-
tein (rFAAH)61 was diluted to the appropriate assay protein
concentration (1.2 μg per assay) in Tris-HCl buffer (10 mM, pH
7.6) containing 1 mM EDTA. Aliquots (165 μL) were added to
glass tubes containing 10 μL of test compound. Blanks con-
tained assay buffer instead of rFAAH. [3H]-AEA (25 μL, final
concentration 2 μM) was added to the test tubes, and
the samples were incubated for 10 min at 37 °C. After the
incubation, the reaction was stopped by adding 400 μL of
chloroform/methanol (1:1, v/v), followed by vortex mixing
and centrifugation (5 min, 2500 rpm) for phase separation.
Aliquots (200 μL) of the methanol/buffer phase containing the
water-soluble reaction products (containing [3H]-ethanolamine)
1
powder (257 mg, 37%). H NMR (300 MHz, CDCl3): δ 0.89
(t, J = 7.20 Hz, 3H), 1.33 (m, 14H), 1.58 (qu, J = 6.60 Hz, 2H),
2.04 (q, J = 6.90 Hz, 4H), 2.11 (t, J = 7.20 Hz, 2H), 2.75 (t, J =
6.60 Hz, 2H), 2.77 (t, J = 3.90 Hz, 2H,; 3.48 (q, J = 6.90 Hz,

4620 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
wyffels et al.
were measured for tritium content by liquid scintillation spec-
troscopy with quench correction. Experiments were performed
3-fold.
For the preincubation test, 10 μL of test compound in Tris-
HCl buffer (10 mM, pH 7.6) containing 1 mM EDTA was
preincubated with rFAAH (1.2 μg per assay) for 1 h, 2 h, or 3 h
prior to addition of [3H]-AEA (25 μL, final concentration 2
μM). Samples were analyzed as described above, and the
significance of the differences obtained was assessed by One-
way ANOVA test (followed by Dunnett post-test).
through I2. In a quartz tube heated to 600 °C, the [11C]-CH4 was
converted to [11C]-CH3I, which was collected on a second
Porapak N trap. The remaining [11C]-CH4 was circulated con-
tinuously to improve the yield.
Radiosynthesis of [11C]-17 and [11C]-18. The generated [11C]-
CH3I was released from the Porapak N trap by heating and
was bubbled into a reaction vial containing DMF (250 μL), base
(1.5 M Bu4NOH (aq), 1 μL), and O-desmethyl precursor 25
(3 μmol) for [11C]-17 or DMF (240 μL), base (NaH, 10 μL, 1M,
0.5 g/20 mL DMF, kept in freezer prior to use), and O-desmethyl
precursor 26 (3 μmol) for [11C]-18. The reaction mixture
was heated for 10 min at 55 °C for [11C]-17 and at 50 °C for
[11C]-18. HPLC mobile phase (200 μL) was added to the reaction
vial, and the solution was injected remotely controlled on
a semipreparative HPLC system (see General Methods)
to isolate the radiolabeled compounds [11C]-17 (tR = 9.8 min)
and [11C]-18 (tR = 9.3 min). The isolated fraction was diluted
with Dulbecco’s phosphate buffered saline (DPBS, 0.0095 M
(PO4), pH 7.4, 45 mL) and passed over a C18 cartridge (Alltech
Maxi-Clean SPE Prevail C18, previously activated with ethanol
and water). The cartridge was rinsed with water (10 mL), and the
radiolabeled compound was eluted with ethanol (1 mL). A
sample (∼100 μL) was analyzed by HPLC for radiochemical
purity and measurement of specific radioactivity (see General
Methods).
HPLC Analysis of Metabolization. For the assay of metabo-
lization, 0.09 mM of test compound 17 or 18 was incubated with
80 μg of rFAAH in Tris-HCl buffer (10 mM, pH 7.6) containing
1 mM EDTA and 0.1% BSA (final volume of 500 μL) at 37 °C
for 30 min with shaking. Blanks contained assay buffer instead
of rFAAH. The reaction was terminated by adding 500 μL of
cold MeCN, followed by vortex and centrifugation (2 min, 6000
rpm) to remove the proteins. Supernatant (100 μL) was injected
into the HPLC system for analysis (GraceSmart C18 column,
250 mm ꢀ 4.6 mm, particle size 5 μm, MeCN:H2O:HCOOH
(90:10:0.1, v/v), flow rate 1 mL/min, tR 6.3 min for arachidonic
acid, 7.2 min for linoleic acid, 9.1 min for 17, and 8.1 min for 18
using UV detection, 204 nm). An identical incubation with
anandamide (Rt 5.5 min) was carried out to validate the method.
For the FAAH inhibition assay, 150 μg of rFAAH was
preincubated for 15 min with 10nM of MAFP in Tris-HCl
buffer (10 mM, pH 7.6) containing 1 mM EDTA, and 0.1%
BSA (final volume of 500 μL) at 37 °C with shaking. Next, test
compound 17 or 18 was added (final concentration 0.09 mM),
incubated for another 30 min at 37 °C and analyzed as above.
CB1 and CB2 Receptor Binding Assay. The assay was carried
out as previously described using CHO cells expressing selec-
tively the hCB1 or hCB2 cannabinoid receptor.62 Briefly, the
competitive binding experiments were performed using [3H]-
SR141716A (1 nM) or [3H]-CP-55,940 (1 nM) as radioligands
for the hCB1 and hCB2 cannabinoid receptors, respectively, at
30 °C in plastic tubes, and 40 μg of membranes per tube
resuspended in 0.5 mL (final volume) binding buffer (50 mM
Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 0.5% bovine serum
albumine, pH 7.4). The test compounds were present at a 10 μM
concentration, and the nonspecific binding was determined in
the presence of 10 μM HU-210. After 1 h, the incubation was
stopped, and solutions were rapidly filtered through 0.5% PEI
pretreated GF/B glass fiber filters (Whatman, Maidstone, UK)
on a M-48T Brandell cell harvester and washed twice with 5 mL
of ice-cold binding buffer without serum albumin. The radio-
activity on the filters was measured in a Pharmacia Wallac 1410
β-counter using 10 mL of Aqualuma (PerkinElmer, Schaesberg,
The Netherlands) after 10 s of shaking and 3 h of resting. Assays
were performed at least in triplicate. Final DMSO concentra-
tions in the assay were less than 0.1%.
Reference Control. An aliquot of [11C]-17 or [11C]-18 was
coinjected with 50 μg of compound 17 or compound 18 respec-
tively into the HPLC system to confirm its identity. Coelution of
radiolabeled compounds and the nonradioactive references
confirms their identity.
Preparation of Brain Homogenates. Fresh dissected C57BL/6J
mice brain was homogenized in PBS using a glass homogenizer
and subsequently centrifuged at 4 °C for 20 min at 18000g. The
pellet was resuspended in PBS and centrifuged again at 4 °C for
20 min at 18000g. The latter operation was performed twice. The
resulting pellet was resuspended in buffer (10 mM Tris-HCl, 1
mM EDTA). Protein content of the preparation was determined
according to the method of Bradford, using bovine serum
albumin as standard, and aliquots were stored at -80 °C until
used for assay.
Ex Vivo Evaluation. [11C]-17 or [11C]-18 was incubated with
brain homogenates (400-700 μg protein assay-1) in Tris-HCl
buffer (10 mM, pH 7.6) containing 1 mM EDTA, and 0.1% BSA
(final volume of 500 μL) at 37 °C for 30 min with shaking. The
reaction was terminated by adding 500 μL of cold MeCN,
followed by vortex and centrifugation (2 min, 6000 rpm) to
remove the proteins. Supernatant (500 μL) was injected into the
HPLC system for analysis (Grace Discovery Econosphere C18
column, 250 mm ꢀ 10 mm, particle size 10 μm þ Grace
Discovery Econosphere C18 guard 33 mm ꢀ 7 mm, particle size
10 μm, 90:10:0.1% MeCN:H2O:HCOOH, flow rate 4 mL/min).
The HPLC eluate was collected in fractions of 30 s, and their
radioactivity was counted with an automated γ-counter. For the
FAAH inhibition assay, brain homogenates were preincubated
for 10 min with 3 mM URB597 in Tris-HCl buffer (10 mM, pH
7.6) containing 1 mM EDTA, and 0.1% BSA (final volume of
500 μL) at 37 °C with shaking. Next, [11C]-17 or [11C]-18 was
added, incubated for another 30 min at 37 °C, and analyzed as
described above.
Data Analysis. The pooled data expressed as percentage of
control activity containing the same carrier concentration were
analyzed using the built-in equation “sigmoidal dose-response
(variable slope)” of the GraphPad Prism computer program
(GraphPad Software Inc., San Diego, CA). Binding assays on
the cannabinoid receptors (CB1 and CB2) were performed at
least three times in duplicate. Results are expressed as mean (
SEM.
Radiochemistry. [11C]-MeI Production. [11C]-CH4 was pro-
duced in a Cyclone 18 twin cyclotron (IBA, Louvain-la-Neuve,
Belgium) using a 18 MeV proton beam and the 14N(p,R)11C
reaction on N2 containing 5.5% H2. The target gas was irra-
diated for 20 min with a beam intensity of 14 μA. The [11C]-CH3I
was produced from [11C]-CH4 within a lead-shielded hot cell by
iodination using an automated homemade synthesis module.
Briefly, [11C]-CH4 was transferred from the cyclotron target to
the hot cell, where it was trapped on a loop filled with Porapak N
cooled in liquid argon. The loop was allowed to warm to RT.
[11C]-CH4 was swept of with a stream of helium and was passed
In Vivo Evaluation. A biodistribution study of [11C]-17 and
[11C]-18 was performed in male C75BL/6J mice weighing 20-25
g (Charles River Laboratories, Belgium). Mice were injected iv
in the tail vein with approximately 3.7 MBq of [11C]-17 or [11C]-
18 dissolved in 100 μL of ethanol/sterile water (1:9). At 1, 10, 30,
or 60 min post injection (n = 3 for each time point), mice were
sacrificed by cervical dislocation under isoflurane anesthesia.
Blood was collected and brain and organs to be examined were
rapidly removed and weighed. Radioactivity in the dissected
organs and blood was measured using an automatic γ counter.
The uptake of radioactivity in blood and organs was expressed

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4621
as percentage of the injected dose per gram of tissue plus or
minus the standard deviation (%ID/g ( SD).
All animal experiments were conducted according to the
regulations of the Belgian law and the Ghent University local
ethical committee (ECP 08/33).
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Fan, P.; Abouzid, K.; Meng, Z.; Biegel, D.; Makriyannis,
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Acknowledgment. We sincerely thank Line Coucke and
Kristof Dhaenens for their great technical assistance. We are
grateful to Thomas Verbrugghen for acquiring the NMR
spectra and to Geoffray Labar for preparing rat recombinant
FAAH-MBP fusion protein.
(22) Cravatt, B. F.; Demarest, K.; Patricelli, M. P.; Bracey, M. H.;
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Supporting Information Available: Chemistry and spectro-
scopic data for compounds 2-19 and 19-24. This material is
available free of charge via the Internet at http://pubs.acs.org.
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