Radiosynthesis, in vitro and in vivo evaluation of 123I-labeled anandamide analogues for mapping brain FAAH

Article abstract of DOI:10.1016/j.bmc.2008.11.019

Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for terminating the signaling of endocannabinoids, including anandamide. This paper is the first report of the synthesis, [¹²³I]-labeling and in vitro and in vivo evaluation of anandamide analogues as potential metabolic trapping radioligands for in vivo evaluation of brain FAAH. N-(2-Iodoethyl)linoleoylamide (2) and N-(2-iodoethyl)arachidonylamide (4) were synthesized with good yields (75% and 86%, respectively) in a two steps procedure starting from their respective acids. In vitro analyses, performed using recombinant rat FAAH and [³H]-anandamide, demonstrated interaction of 2 and 4 with FAAH (IC₅₀ values of 5.78 μM and 3.14 μM, respectively). [¹²³I]-2 and [¹²³I]-4 were synthesized with radiochemical yields of 21% and 12%, respectively, and radiochemical purities were >90%. Biodistribution studies in mice demonstrated brain uptake for both tracers (maximum values of 1.23%ID/g at 3 min pi for [¹²³I]-2 and 0.58%ID/g at 10 min pi for [¹²³I]-4). However, stability studies demonstrated the sensitivity of both tracers to dehalogenation.

Full text of DOI:10.1016/j.bmc.2008.11.019

Bioorganic & Medicinal Chemistry 17 (2009) 49–56
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Radiosynthesis, in vitro and in vivo evaluation of 123I-labeled anandamide
analogues for mapping brain FAAH
a
a
b
a
Leonie wyffels a, , Sylvie De Bruyne , Peter Blanckaert , Didier M. Lambert , Filip De Vos
*
a Department of Radiopharmacy, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium
b Unité de Chimie Pharmaceutique et de Radiopharmacie, Université catholique de Louvain, Avenue E. Mounier, 73, UCL-CMFA 73-40, B-1200 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 June 2008
Revised 31 October 2008
Accepted 9 November 2008
Available online 17 November 2008
Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for terminating the signaling
of endocannabinoids, including anandamide. This paper is the first report of the synthesis, [123I]-labeling
and in vitro and in vivo evaluation of anandamide analogues as potential metabolic trapping radioligands
for in vivo evaluation of brain FAAH. N-(2-Iodoethyl)linoleoylamide (2) and N-(2-iodoethyl)arachidonyla-
mide (4) were synthesized with good yields (75% and 86%, respectively) in a two steps procedure starting
from their respective acids. In vitro analyses, performed using recombinant rat FAAH and [3H]-ananda-
Keywords:
FAAH
mide, demonstrated interaction of 2 and 4 with FAAH (IC50 values of 5.78
[
lM and 3.14 lM, respectively).
123I]-2 and [123I]-4 were synthesized with radiochemical yields of 21% and 12%, respectively, and radio-
Anandamide
Metabolic trapping
chemical purities were >90%. Biodistribution studies in mice demonstrated brain uptake for both tracers
(maximum values of 1.23%ID/g at 3 min pi for [123I]-2 and 0.58%ID/g at 10 min pi for [123I]-4). However,
stability studies demonstrated the sensitivity of both tracers to dehalogenation.
Ó 2008 Elsevier Ltd. All rights reserved.
[
123I]
1. Introduction
not only for anandamide but also for the anti-inflammatory com-
pound N-palmitoylethanolamine (PEA),11 the sleep inducing lipid
Since the discovery in the early 1990s of specific membrane
cis-9-octadecenoamide (oleamide),12 and even esters such as 2-
AG (Fig. 1).13
receptors of marijuana’s (Cannabis sativa) psychoactive compound
D
9-tetrahydrocannabinol (
D
9-THC) and the revelation of a whole
The importance of FAAH as a regulatory enzyme for key physi-
ological functions is suggested by studies with transgenic mice
lacking FAAH. They possess high endogenous concentrations of
anandamide and related fatty acid amides in the brain that corre-
late with CB1 dependent behavioral responses including hypomo-
tility, analgesia, catalepsy and hypothermia.14 An increasing
number of reports suggests that the symptoms of several neurolog-
ical and neuropsychiatric disorders could be caused by changes in
the endocannabinoid biosynthesis and degradation, including:
addiction,15–18 schizophrenia,19,20 anxiety,21 depression,22,23 multi-
endogenous signaling system known as the endocannabinoid
system, considerable research has been devoted to the characteri-
zation of the different members of this endocannabinoid system.
Besides the cannabinoid receptors CB1 and CB2, the endocannabi-
noid system also comprises their endogenous ligands (the endo-
cannabinoids, with anandamide (N-arachidonoylethanolamine,
AEA) and 2-arachidonoylglycerol (2-AG) being the most studied
ones) and the proteins for their synthesis and inactivation.1
Fatty acid amide hydrolase (FAAH) is the main enzyme respon-
sible for terminating the signaling of AEA by hydrolysis of the
amide bond after uptake in the cell by the still controversial AEA
membrane transporter (AMT).2,3 (For reviews on AMT see Refs.
4,5). FAAH is a dimeric integral membrane enzyme, found predom-
inantly in microsomal and mitochondrial fractions and is the first
characterized mammalian member of the amidase signature
family.6–8 It is a nonclassical serine hydrolase that utilizes a Ser-
Ser-Lys catalytic triad instead of the more common Ser-His-Asp
catalytic triad with serine-241 as the catalytic nucleophile in the
active site of the enzyme.8–10 FAAH may act as a hydrolytic enzyme
* Corresponding author. Tel.: +32 9 264 80 65; fax: +32 9 264 80 71.
E-mail address: leonie.wyffels@ugent.be (L. wyffels).
Figure 1. Chemical structures of representative FAAH substrates.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.019

50
L. wyffels et al. / Bioorg. Med. Chem. 17 (2009) 49–56
ple sclerosis,24,25 epilepsy,26 Parkinson’s disease,27,28 and Hunting-
ton’s disease.29
and arachidonylethanolamide (3) in a high yield procedure. An-
other, although less common, synthetic route is the direct conden-
sation between in situ pre-activated arachidonic acid or linoleic
acid, employing the coupling agent carbonyldiimidazole (CDI),
and the corresponding amine, thus allowing for a one-pot amide
formation. Using this method, we were able to synthesize 1 and
3 in high yields (78% and 64%, respectively). In practice, the imi-
dazolides of arachidonic acid and linoleic acid were preformed
for 1 h and then ethanolamine was added to form the correspond-
ing acylethanolamide. Attempts to synthesize N-(2-iodoethyl)lin-
oleoylamide (2) and N-(2-iodethyl)arachidonylamide (4) by
reaction of the activated acid with 2-iodoethylamine did not give
the compounds in satisfactory purity nor high yield. The 2-iodoeth-
ylamine was obtained by deprotection of N-trityl-2-iodoethan-
amine with 1-hydroxybenzotriazole (HOBt) in trifluoroethanol or
obtained after deprotection of tert-butyl-2-iodoethyl-carbamate
with HCl (4 M in dioxane) or trifluoroacetic acid. On the other
hand, conversion of compounds 1 and 3 to the iodide by treatment
with triphenylphospine/I2 did result in compounds 2 and 4, respec-
tively, in acceptable purity and high yields (75% and 86%, respec-
tively) (Scheme 1).
To better understand the relationship between the FAAH-endo-
cannabinoid system and neuronal and neuropsychiatric disorders
and to find cause-effect relationships between changes in the
expression/activity of FAAH and pathological conditions, in vivo
molecular imaging using metabolic trapping can be helpful. The
principle of this method is that a radiolabeled substrate analogue
passes the blood–brain-barrier (BBB), is taken up in the cell and
hydrolyzed by FAAH to produce a labeled amine which has limited
permeability through the BBB and becomes trapped in neuronal
cells. In that manner, we can visualize the enzyme and quantify
the amount and functionality of FAAH. Metabolic trapping has
already been successfully applied for evaluation of acetylcholines-
terase activity in the brain for Alzheimer’s disease diagnosis using
[
18F]-, [11C]- and [123I]-labeled acetylcholine analogues.30–33 Other
well-known examples are the use of the glucose analogue [18F]-2-
fluorodeoxyglucose ([18F]-FDG) to assess rates of cerebral glucose
utilization for detection of brain tumours, epileptic foci and Alzhei-
mer’s disease34,35 and the use of [99mTc]-ethylene cysteine dimer
([99mTc]-ECD) for the study of regional cerebral perfusion.36
The visualization of FAAH in vivo with positron emission
tomography (PET) or single photon emission tomography (SPECT)
forms a powerful method for the study of several neuropsychiatric
diseases.
2.2. In vitro evaluation
2.2.1. FAAH assay
Since no tracers for in vivo evaluation of FAAH currently exist,
the purpose of this study was to synthesize and evaluate ligands
based on an anandamide template with a view to discovering met-
abolic trapping ligands for visualization of this enzyme in the brain
using SPECT. Structure–activity relationships studies demon-
strated that anandamide analogues with an electronegative head
group substituent are better FAAH substrates than their unsubsti-
tuted counterparts37; therefore, we chose to develop an ananda-
A prerequisite for a metabolic trapping ligand is that the com-
pound is a substrate of the enzyme under investigation. For the
anandamide analogues synthesized here, it is not known whether
they are substrates for FAAH. The FAAH assay used in this study al-
lows us to investigate the interaction between recombinant rat
FAAH (rFAAH) and compounds 2 and 4 by their ability to prevent
the enzyme from hydrolyzing [3H]-AEA. It is important to empha-
size that using this approach no information is given on the effi-
cacy of the compounds as substrates but rather allows for
determination of their affinity for the enzyme.39 Experiments were
performed using ten concentrations of test compound 2 or 4 in the
mide analogue based
[
123I]-SPECT ligand for visualization of
FAAH. Hereby, we describe the synthesis of N-(2-iodoethyl)linol-
eoylamide (2) and N-(2-iodoethyl)arachidonylamide (4). Both 2
and 4 were tested in vitro using recombinant rat FAAH for evalua-
tion of their interaction with the enzyme. Subsequently, the
123I-derivatives [123I]-2 and [123I]-4 were prepared for biodistribu-
tion studies using NMRI mice to evaluate their potentials as
radioligand for mapping brain FAAH in vivo.
range of 5 lM–1.5 nM. Potencies of 2 and 4 to inhibit the hydroly-
sis of [3H]-AEA by FAAH are expressed as pIC50. Compound 1 is
known to be a substrate for FAAH. Compared to AEA (rate = 100%),
1 is hydrolyzed to an extent of about 75%.37 As a result of this
observation, we used 1 as a reference compound as it inhibits
[3H]-AEA metabolism by acting as a competing substrate. The re-
sults are presented in Figure 2. A pIC50 value of 5.12 0.05 (corre-
2. Results and discussion
2.1. Chemistry
sponding to an IC50 value of 7.58 lM) was obtained for reference
compound 1, thus demonstrating a comparable potency to the
endogenous FAAH substrate palmitoylethanolamide (PEA, pIC50
value of 5.30) reported by Vandevoorde et al.40 Compounds 2
The most often described method for the synthesis of ananda-
mide analogues is by preparation of the acyl chloride followed by
treatment with the appropriate amine or alcohol.38 However, be-
cause of the instability of linoleoyl and arachidonyl chloride, using
this method did not result in N-(2-hydroxyethyl)linoleoylamide (1)
and 4 gave an IC50 value of 5.78 0.69 lM and 3.14 0.30 lM,
respectively. These results are in line with SAR studies showing
that fatty acid ethanolamides containing fewer than four cis non-
conjugated double bonds are not as good substrates as AEA.37,41
Scheme 1. Reagents and conditions: (a) CDI, CH2Cl2, room temperature, 1 h, then NH2CH2CH2OH, room temperature, 3 h (yield: compound 1 78%, compound 3 64%). (b) PPh3,
I2, imidazole, CH2Cl2, room temperature, 3 h (yield: compound 2 75%, compound 4 86%).

L. wyffels et al. / Bioorg. Med. Chem. 17 (2009) 49–56
51
donic acid and ethanolamine, HPLC analysis with UV detection of
test solution containing AEA and rFAAH reveals the generation of
arachidonic acid. Incubation of 2 and 4 with rFAAH resulted in
the formation of linoleic acid and arachidonic acid, respectively.
No formation of fatty acid could be detected in the blank experi-
ments, containing assay buffer instead of rFAAH. To rule out spon-
taneous formation of fatty acid during incubation, the stability of
anandamide, 2 and 4 in assay buffer was studied by incubation
at 37 °C. Hundred microliters of the test solution was injected into
the HPLC system after the first minute and at 15 min intervals for
1 h. The results showed that no formation of fatty acid was de-
tected 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. rFAAH
was preincubated with 10 nM of MAFP in assay buffer for 10 min
at 37 °C followed by addition of AEA, 2 or 4 and another 20 min
incubation at 37 °C. HPLC analysis revealed no formation of fatty
acid, indicating inhibition of conversion of AEA, 2 and 4 by FAAH
with MAFP preincubation.
Although the above described FAAH assay did us assume that
only 4 interacts with FAAH as a substrate while 2 interacts with
FAAH without being efficiently metabolized, these data support
the identity of both 2 and 4 as FAAH substrates.
Figure 2. Inhibition of the hydrolysis by recombinant rat FAAH of 2 l
M [3H]-AEA
by compound 1,
2 and 4. 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.
The results are comparable with the IC50 value of FAAH substrate
PEA and give an indication that compounds 2 and 4 show an inter-
action with FAAH, possibly as substrates.
One way of distinguishing inhibitors from competing substrates
is to preincubate 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.42 Therefore, we
selected the concentrations of test compounds 2 or 4 that gave
20%, 50% and 80% inhibition of [3H]-AEA metabolism in the previ-
ously described experiment and preincubated them with rFAAH for
0, 15, 30 or 60 min before adding [3H]-AEA. The preincubation
showed little effect on the pIC50 values of compound 2 (calculated
IC50 values after 0, 15, 30 and 60 min preincubation with rFAAH:
2.3. Radiochemistry
Radiochemical synthesis of [123I]-2 and [123I]-4 was conducted
by a nucleophilic substitution of the triflate precursor using n.c.a.
(no carrier added) [123I]NaI (Scheme 2). Briefly, each time fresh
2-(linoleoylamido)-ethyl
trifluoromethanesulfonate
(5)
or
2-(arachidonylamido)-ethyl trifluoromethanesulfonate (6) was
prepared and used without further purification. The triflate precur-
sor (4 mg) was dissolved in dry acetone and left to react with
6.20
pound 4 showed a larger decrease in potency with preincubation.
Calculated IC50 values for compound 4 of 4.311 M, 7.93 M,
9.89 M, 10.07 M (0, 15, 30 and 60 min preincubation, respec-
lM, 6.66 lM, 7.64 lM, 7.33 lM, respectively); however, com-
[
123I]NaI for 30 min at elevated temperature (50 °C for [123I]-2
and 40 °C for [123I]-4). [123I]-2 and [123I]-4 were separated from
radioactive impurities by analytical HPLC and formulated in a
0.9% NaCl solution containing ethanol (8%) for in vivo evaluation.
l
l
l
l
tively) suggest metabolization of 4 by rFAAH which results in a de-
crease of potency to inhibit [3H]-AEA metabolism.
Radiochemical yields were 21 3% (n = 6) for
[
123I]-2 and
12 6.3% (n = 4) for [123I]-4. Identification of the collected tracer
was performed by comparing retention times on HPLC between
the radioactive labeled product [123I]-2 and [123I]-4 and the
unlabeled iodinated molecules 2 and 4. Co-elution confirmed the
identity of [123I]-2 and [123I]-4. HPLC analysis showed a chemical
and radiochemical purity of >90%.
From the above experiments it appears that both 2 and 4 show
an interaction with rFAAH. The preincubation experiments demon-
strated that 4 interacts with FAAH as a competing substrate while
compound 2 probably binds to FAAH without being efficiently
metabolized.
2.2.2. HPLC analysis of metabolization
To further identify 2 and 4 as substrates for FAAH, a HPLC meth-
od was used to demonstrate the generation of linoleic acid or ara-
2.4. Uptake of [123I]-2 and [123I]-4 in mouse brain
To investigate the uptake of [123I]-2 and [123I]-4 in the brain, a
chidonic acid as
a result of enzymatic metabolization after
incubation of the compounds with FAAH. An experiment with
the known FAAH substrate AEA was performed to validate the
method. Since AEA is hydrolyzed by FAAH to the fatty acid arachi-
general biodistribution in mice was conducted. Adult male NMRI
were injected iv in the tail vein with 4–5
4. The radioactivity concentrations of various tissues as a function
l
Ci of [123I]-2 or [123I]-
Scheme 2. Reagents and conditions: (a) Tf2O, TEA, CH2Cl2, ꢀ20 to 0 °C, 1h (yield: compound 5 72%, compound 6 88%). (b) [123I]NaI, acetone, 50 °C ([123I]-2), 40 °C ([123I]-4),
30 min.

52
L. wyffels et al. / Bioorg. Med. Chem. 17 (2009) 49–56
of time following administration of [123I]-2 or [123I]-4 are shown in
Figure 4.
lene-iodine bond was found in vivo with iodoethylamides.45 Since
our in vivo results lead us to suspect in vivo dehalogenation of
Both [123I]-2 and [123I]-4 demonstrate uptake in brain, with the
highest uptake at 3 min for [123I]-2 (1.23%ID/g) and at 10 min for
[
123I]-2 and [123I]-4, tracer stability studies were performed using
HPLC and thin-layer chromatographic (TLC) analysis with radioac-
tivity detection. TLC analysis of the radioligands directly after HPLC
purification and concentration demonstrated an acceptable stabil-
ity of the tracer with 95% authentic [123I]-2 and 90% authentic
[
123I]-4 (0.579%ID/g), followed by a continuous decrease until the
end of the study (Fig. 3). The uptake in the intestines remained
rather constant and low throughout the study (1.84%ID/g and
1.33%ID/g in small intestines and large intestines, respectively, at
10 min pi for [123I]-2 and 1.46%ID/g and 1.04%ID/g in small intes-
tines and large intestines, respectively, at 10 min pi for [123I]-4),
while the uptake in bladder/urine continuously increased over
time, indicating that renal elimination is the primary excretory
pathway. Also, an increase in uptake of radioactivity was observed
in stomach (from 2.58%ID/g at 1 min pi to 24.57%ID/g at 1 h for
[
123I]-4. An ex vivo stability experiment in mouse blood and brain
tissue was performed. Radioactivity was extracted with acetoni-
trile from the plasma and brain of mice and submitted to HPLC
and TLC analysis. From blank samples, spiked with [123I]-2, an
extraction efficiency of 94 3.5% (n = 3) for brain tissue and
94 1.5% (n = 3) for plasma was obtained. For [123I]-4, extraction
efficiencies of 89 1.2% (n = 3) for brain tissue and 92 0.6%
(n = 3) were obtained. However, TLC and HPLC analysis of the
supernatant revealed that, in the case of [123I]-2, >45% of the ex-
tracted radioactivity was present in the form of free [123I]Iꢀ, and
even >60% of free [123I]Iꢀ in the case of [123I]-4. Determination of
authentic [123I]-2 in plasma and brain tissue 3 min postinjection,
demonstrated almost complete metabolization of the labeled com-
pound. Besides a small fraction of authentic [123I]-2 (<2%) in plas-
ma, only free [123I]Iꢀ could be observed in brain tissue.
[
[
123I]-2 and from 3.10%ID/g at 1 min to 17.74%ID/g at 1 h for
123I]-4). Since the transport from iodide from the bloodstream into
the gastric lumen is a known phenomenon44, the high uptake in
stomach indicates dehalogenation of [123I]-2 and [123I]-4.
2.5. Tracer dehalogenation
Compounds containing an iodine atom in aliphatic position are
generally not very stable due to the relatively poor biochemical
stability of the C-I bond.44 Nevertheless, a relatively stable methy-
Since these experiments proved the sensitivity of the radioli-
gands for rapid dehalogenation, they will not be of any practical
Figure 3. Time dependency of uptake (%ID/g) of [123I] radioactivity in blood, brain and stomach after iv injection of [123I]-2 or [123I]-4 in mice.
Figure 4. Uptake (%ID/g) of radioactivity in mice organs at various time points after iv injection of [123I]-2 (A) or [123I]-4 (B).

L. wyffels et al. / Bioorg. Med. Chem. 17 (2009) 49–56
53
use for the in vivo visualization of FAAH despite their good in vitro
results. However, since the interest of FAAH in normal and dis-
eased states, the search for radioligands for visualization of the en-
zyme should be continued. Further research is necessary to
develop FAAH radioligands for fast and objective diagnosis of men-
tal illnesses like depression and schizophrenia, as well as for eval-
uation of usefulness of newly developed FAAH inhibitors. Iodinated
endocannabinoid analogues less sensitive to dehalogenation as
well as [18F]- or [11C]-labeled endocannabinoid analogues should
be developed for use as metabolic trapping tracers in SPECT and
PET. Another line of approach is the use of FAAH inhibitors, selec-
tively and covalently binding to the enzyme, allowing visualiza-
tion, and giving a broader spectrum of molecules for labeling
with SPECT or PET isotopes. The drawback of using inhibitors is
that only differences in regional enzyme density can be visualized
without providing information on the functionality of the enzyme.
For mixed solvent systems, ratios are given with respect to vol-
umes. The 1H and 13C nuclear magnetic resonance (NMR) spectra
were measured with a Varian 300 MHz FT-NMR spectrometer
(Department of Medicinal Chemistry, Ghent University). Signals
are quoted as s (singlet), d (doublet), t (triplet), q (quartet), qu
(quintet) or m (multiplet). Chemical shifts were recorded in ppm
(d) from an internal tetramethylsilane standard in chloroform-d3.
Mass spectrometry was performed on a Waters Micromass ZMD
mass-spectrometer with electrospray-ionization probe. Samples
were dissolved in methanol. [123I]-Sodium iodide (in 0.05 M NaOH)
was purchased from GE Healthcare (Belgium). High performance
liquid chromatography (HPLC) purification and analyses of the
radiotracer were performed using a Waters 515 HPLC pump, a
Waters 2487 UV detector (204 nm), and a Ludlum 220 scaler rate-
meter equipped with a GM-probe. Radioactivity of samples was
measured in a Packard Cobra automated c-counter equipped with
five 100 ꢁ 100 NaI(Tl) crystals. Radioactivity on TLC plates was quan-
tified using a Cyclone Storage Phosphor System (Perkin Elmer,
USA). For the animal studies, male NMRI (Naval Medical Research
Institute) mice were used. All animal experiments were conducted
according to the regulations of Belgian law and the local Ethical
Committee (ECP 07/05).
3. Conclusions
The aim of this study was to develop tracers for in vivo evalua-
tion of FAAH in the brain using metabolic trapping. For this
approach two iodinated AEA analogues were synthesized and eval-
uated in vitro and in vivo. Compounds 2 and 4 were synthesized in
good yields of, respectively, 75% and 86%. In vitro evaluation of the
compounds showed that both compounds were able to inhibit the
metabolization of the endogenous FAAH substrate AEA and, conse-
quently, interact with the enzyme possibly as a substrate. Incuba-
tion with the enzyme and HPLC analysis of formed fatty acid
confirmed the identity of both 2 and 4 as FAAH substrates.
4.2. Chemistry
4.2.1. N-(2-Hydroxyethyl)linoleoylamide (1)
To a solution of linoleic acid (500 mg, 1.67 mmol) in dry dichlo-
romethane (CH2Cl2, 15 ml) was added carbonyl diimidazole (CDI,
270.54 mg, 1.67 mmol). The reaction mixture was stirred at room
123I]-2 and [123I]-4 were synthesized in moderate to low yields
with acceptable radiochemical purity. The biodistribution of
123I]-2 and [123I]-4 in NMRI mice demonstrated uptake of both
temperature for 1 h. Ethanolamine (3.34 mmol, 202 ll) was added
[
dropwise, and the reaction mixture was stirred for 3 h at room
temperature. The reaction mixture was diluted with dichlorometh-
ane (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. Puri-
fication by silica gel flash chromatography (8:2 ethyl acetate/hex-
ane) gave 1 as a white solid (420 mg) in a 78% yield. 1H NMR
(300 MHz, CDCl3): d = 0.86 (3H, t, J = 6.6 Hz); 1.32 (14H, m); 1.64
(2H, qu, J = 7.5 Hz); 2.05 (4H, q, J = 6.9 Hz); 2.20 (2H, t,
J = 7.2 Hz); 2.76 (2H, t, J = 6.3 Hz); 3.43 (2H, q, J = 5.4 Hz); 3.72
(2H, t, J = 5.1 Hz); 5.34 (4H, m); 5.9 (1H, br s); 13C NMR (75 MHz,
CDCl3): d = 14.21, 22.71, 25.77, 25.86, 27.33, 27.34, 27.34, 29.27,
29.39, 29.48, 29.75, 31.66, 36.78, 42.63, 62.62, 128.04, 128.21,
130.17, 130.38, 174.75. MS (ESI) m/z (% rel int.): 324.2 (100.0
[M+1]+); 346.1 (28.2 [M+Na]+); 647 (20 [2Mꢀ1]+).
[
radioligands in the brain, but no retention of radioactivity could
be shown. The high stomach values suggest dehalogenation of
the radioligands. Stability studies in vitro and in vivo in mouse
blood and brain confirm the susceptibility of both radioligands to
dehalogenation. In conclusion, we have described for the first time
the synthesis and evaluation of radioligands for the in vivo evalu-
ation of FAAH activity in the brain. The in vitro results support the
potential of [123I]-2 and [123I]-4 as a tracer for visualising brain
FAAH by metabolic trapping. However, stability studies lead us
to suspect that although there is brain uptake, the radioligands will
not be useful for in vivo visualization of FAAH because of rapid
dehalogenation. Nevertheless, they could serve as an entry point
to the preparation of a wide variety of radioligands based on endo-
cannabinoids or on FAAH inhibitors for the in vivo visualization of
FAAH. Work is in progress to design more stable endocannabinoid
derivatives with the aim to develop a suitable radioligand for
mapping brain FAAH in vivo.
4.2.2. N-(2-Iodoethyl)linoleoylamide (2)
To
a
stirred solution of triphenylphosphine (913 mg,
3.48 mmol) and imidazole (237 mg, 3.48 mmol) in dry CH2Cl2
(20 ml) cooled to 0 °C was added in small portions I2 (883 mg,
3.48 mmol) over a period of 5 min. The yellow-orange suspension
was warmed to room temperature and stirred for 10 min. A solu-
tion of amide 1 (930 mg, 2.90 mmol) in dry CH2Cl2 (10 ml) was
added dropwise, and the resulting mixture was stirred at room
temperature for 3 h. The reaction mixture was washed twice with
aqueous Na2S2O3 (5%). The layers were separated and the aqueous
layer extracted with CH2Cl2. The combined organic layers were
dried over MgSO4, and the solvent was evaporated under reduced
pressure to give the crude product. Purification by silica gel flash
chromatography (7:3 hexane/ethyl acetate) gave 2 as a yellow
solid (342 mg) in a 75% yield. 1H NMR (300 MHz, CDCl3): d = 0.88
(3H, t, J = 6.9 Hz); 1.30 (14H, m); 1.64 (2H, qu, J = 7.2 Hz); 2.04
(4H, q, J = 7.2 Hz); 2.20 (2H, t, J = 7.2 Hz); 2.76 (2H, t, J = 6.0 Hz);
3.25 (2H, t, J = 6.0 Hz); 3.60 (2H, q, J = 6.0 Hz); 5.35 (4H, m); 5.80
(1H, br s); 13C NMR (75 MHz, CDCl3): d = 6.10, 14.22, 22.71,
25.77, 25.80, 27.33, 27.34, 29.26, 29.37, 29.38, 29.48, 29.74,
4. Experimental
4.1. General procedures and materials
All chemical reagents were obtained from commercial sources
(Sigma–Aldrich Fluka, Acros Organics, Belgium) and used without
further purification. Solvents were purchased from Lab-Scan Ana-
lytical Sciences (Dublin, Ireland). All reactions were conducted un-
der N2 atmosphere with dry solvents under anhydrous conditions.
The thin-layer chromatographic analyses were performed using
200 lm silica gel with fluorescent indicator (UV254) coated on plas-
tic plates (Machery-Nagel, Germany). The spots were visualized
using iodine vapours. Purification of unlabeled compounds was
achieved by column chromatography with silica gel (Sigma–Al-
drich, 230–400 mesh), using solvent systems indicated in the text.

54
L. wyffels et al. / Bioorg. Med. Chem. 17 (2009) 49–56
31.66, 36.84, 41.76, 128.04, 128.19, 130.17, 130.36, 173.26. MS
(ESI) m/z (% rel int.): 434.3 (5.0 [M+1]+); 306.3 (100 [MꢀI]+;
324.3 (12.2 [(MꢀI)+H2O]+.
ditions were obtained with a mixture of 4 mg of compound 5 or 6
in 0.5 ml acetone at 50 °C for 30 min for [123I]-2 and at 40 °C for
30 min for [123I]-4.
The freshly synthesized triflate precursor
5
(4 mg,
4.2.3. Arachidonylethanolamide (3)
0.00879 mmol) or 6 (4 mg, 0.00835 mmol) was dissolved in dry
acetone (0.5 ml). The conical reaction vial containing the reaction
mixture was flushed with N2, and [123I]NaI in sodium hydroxide
Synthesis followed the same procedure as described for 1 using
arachidonic acid (1 g, 3.29 mmol) as the fatty acid. Purification by
silica gel flash chromatography (6:4 ethyl acetate/hexane) gave 3
as a white solid (730 mg) in a 64% yield. 1H NMR (300 MHz, CDCl3):
d = 0.89 (3H, t, J = 6.6 Hz); 1.30 (6H, m); 1.73 (2H, qu, J = 7.2 Hz);
2.08 (4H, q, J = 6.9 Hz); 2.20 (2H, t, J = 7.5 Hz); 2.80 (6H, m); 3.40
(2H, q, J = 5.4 Hz); 3.72 (2H, t, J = 5.1 Hz); 5.36 (8H, m); 5.95 (1H,
br s); 13C NMR (75 MHz, CDCl3): d = 14.20, 22.71, 25.61, 25.61,
25.77, 26.77, 27.36, 29.45, 31.65, 36.05, 42.60, 62.55, 127.65,
127.98, 128.27, 128.41, 128.76, 129.00, 129.14, 130.68, 174.37.
MS (ESI) m/z (% rel int.): 348.5 (100 [M+1]+); 695.2 (34.0
[2M+1]+); 370.4 (16.4 [M+Na]+); 329.6 (14.0 [MꢀH2O]+.
solution (4 ll, 0.05 M) was added. The reaction proceeded for
30 min at 50 °C for [123I]-2 and at 40 °C for [123I]-4. The acetone
was evaporated under a stream of N2, and the reaction mixture
was redissolved in acetonitrile (100
into a HPLC system (Alltech Alltima C18 column, 150 ꢁ 4.6 mm,
particle size 5
m + Alltech Alltima C18 guard 7.5 ꢁ 4.6 mm, parti-
cle size 5 m) and eluted with a 80:20:0.1% mixture of acetoni-
ll). The solution was injected
l
l
trile:water:formic acid at a flow rate of 1 ml/min to isolate the
radiolabeled compounds [123I]-2 and [123I]-4. The desired fraction
eluted at 24–26 min for [123I]-2 and at 22–24 min for [123I]-4,
was collected and concentrated. The product was redissolved in
0.9% NaCl solution containing ethanol (8%) for biologic evaluation.
Yields: [123I]-2: 21 3% (n = 6), [123I]-4: 12 6.3% (n = 4).
4.2.4. N-(2-Iodoethyl)arachidonylamide (4)
The same procedure described for the synthesis of 2 was ap-
plied, using 3 as the added amide, resulting in the crude product.
Purification by silica gel flash chromatography (7:3 hexane/ethyl
acetate) gave 4 as a yellow-white solid (448 mg) in a 86% yield.
1H NMR (300 MHz, CDCl3): d = 0.89 (3H, t, J = 7.0 Hz); 1.30 (6H,
m); 1.73 (2H, qu, J = 7.6 Hz); 2.05 (2H, q, J = 7.0 Hz); 2.13 (2H, q,
J = 7.0 Hz); 2.20 (2H, t, J = 7.3 Hz); 2.81 (6H, m); 3.27 (2H, t,
J = 6.5 Hz); 3.60 (2H, q, J = 6.1); 5.36 (8H, m); 5.77 (1H, br s); 13C
NMR (75 MHz, CDCl3): d = 6.04, 14.23, 22.72, 25.56, 25.81, 26.80,
27.38, 29.47, 31.67, 36.12, 41.78, 127.67, 128.00, 128.29, 128.41,
128.77, 129.04, 129.15, 130.68, 172.94. MS (ESI) m/z (% rel int.):
457.9 (6.4 [M+1]+); 330.5 (100 [MꢀI]+); 347.9 (25.0 [(MꢀI)+H2O]+).
4.3.2. Radioanalytical data
4.3.2.1. Quality control.
the chemical and radiochemical purity. 500
tion was re-injected into the HPLC column (Alltech Alltima C18 col-
umn, 150 ꢁ 10 mm, particle size 10 m + Alltech Alltima C18 guard
m, 80:20:0.1% acetonitrile:water:for-
HPLC analysis was used to measure
l
l of the product frac-
l
33 ꢁ 7 mm, particle size 10
l
mic acid, flow rate 6 ml/min, tR 19.9 min for [123I]-2 and 19.4 min
for [123I]-4 using ultraviolet (UV) and radioactivity detectors).
The quality control did not show any radioactive impurities, with
the exception of some [123I]Iꢀ (tR 1.2 min, <10%). Only the injection
peak was detectable within the UV-range.
4.2.5. 2-(Linoleoylamido)-ethyl trifluoromethanesulfonate (5)
To a stirred solution of 1 (200 mg, 0.619 mmol) and freshly dis-
4.3.2.2. Reference control.
was co-injected with 50 g of compound 2 or compound 4, respec-
tively, into the HPLC system to confirm its identity. The radiola-
beled compounds and the non-radioactive references co-eluted,
confirming their identity.
An aliquot of [123I]-2 or [123I]-4
tilled triethylamine (96
ꢀ20 °C was added trifluoromethanesulfonic anhydride (112
l
l, 0.681 mmol) in dry CH2Cl2 (10 ml) at
l,
l
l
0.681 mmol) over 30 min. The resulting opaque solution was stir-
red at 0 °C for 1 h in the dark. The reaction mixture was diluted
with CH2Cl2 (30 ml) and washed with water. The organic phase
was isolated and dried over MgSO4, and the solvent was evapo-
rated under reduced pressure. A yellowish oil (204 mg, 72%) was
obtained and used without further purification. 1H NMR
(300 MHz, CDCl3): d = 0.86 (3H, t, J = 7.0 Hz); 1.30 (14H, m); 1.56
(2H, q, J = 7.3 Hz); 1.98 (4H, q, J = 6.7 Hz); 2.28 (2H, t, J = 7.3 Hz);
2.70 (2H, t, J = 5.6 Hz); 3.50 (2H, q, J = 5.3 Hz,); 4.16 (2H, t,
J = 5.0 Hz); 5.29 (4H, m). MS (ESI) m/z (% rel int.) 306.3 (100
[MꢀOTf]+); 324.3 (35.6 [(MꢀOTf)+H2O]+).
4.4. In vitro evaluation
4.4.1. Compounds
Radiolabeled arachidonylethanolamide ([3H]-AEA, labeled in its
ethanolamine moiety, specific activity of 60 Ci/mmol) was ob-
tained from American Radiolabeled Chemicals, Inc. (St. Louis, MO,
USA). Non-radioactive arachidonylethanolamide (AEA) and methyl
arachidonoyl fluorophosphonate (MAFP) were purchased from
Cayman Chemical Company (Ann Arbor, MI, USA). Fatty acid-free
bovine serum albumin (BSA) was obtained from Sigma Chemical
Co. (Belgium).
4.2.6. 2-(Arachidonylamido)-ethyl trifluoromethanesulfonate
(6)
The procedure for the synthesis of 5 was applied to 3 yielding 6
as a yellowish oil (244 mg, 88%) which was used without further
purification. 1H NMR (300 MHz, CDCl3): d = 0.88 (3H, t,
J = 7.0 Hz); 1.28 (6H, m); 1.71 (2H, q, J = 7.03 Hz); 2.08 (4H, dq,
J = 7.03 and 7.62 Hz); 2.36 (2H, t, J = 7.62 Hz); 2.81 (6H, q,
J = 5.85 Hz); 3.54 (2H, q, J = 5.27 Hz); 4.22 (2H, t, J = 4.98 Hz);
5.37 (8H, m). MS (ESI) m/z (% rel int.): 330.3 (100 [MꢀOTf]+);
348.3 (90.0 [(MꢀOTf)+H2O]+).
4.4.2. FAAH assay
The method used was based on that of Omeir et al. using [14C]-
AEA as substrate, and has been previously described.39,42 Briefly,
recombinant rat FAAH fused to Maltose Binding Protein (rFAAH)46
was diluted to the appropriate assay protein concentration (1.2
per assay) in Tris–HCl buffer (10 mM, pH 7.6) containing 1 mM
EDTA. Aliquots (165 l) were added to glass tubes containing
10 l of test compound 2 or 4. Blanks contained assay buffer in-
stead of rFAAH. [3H]-AEA (25
l, final concentration 2 M) was
lg
l
l
4.3. Radiochemistry
l
l
added to the test tubes, and the samples were incubated for
10 min at 37 °C. After the incubation the reaction was stopped by
4.3.1. N-(2-[123I]-iodoethyl)linoleoylamide ([123I]-2) and N-(2-
[
123I]-iodoethyl)arachidonylamide ([123I]-4)
adding 400
mixing and centrifugation (5 min, 2500 rpm) for phase separation.
Aliquots (200 l) of the methanol/buffer phase containing the
water soluble reaction products (containing [3H]-ethanolamine)
ll chloroform/methanol (1:1, v/v), followed by vortex
The radiosynthesis of [123I]-2 and [123I]-4 was performed start-
ing from the triflate precursor (compound 5 and compound 6,
respectively) followed by HPLC purification. Optimal reaction con-
l

L. wyffels et al. / Bioorg. Med. Chem. 17 (2009) 49–56
55
were measured for tritium content by liquid scintillation spectros-
copy with quench correction. Experiments were performed in
threefold.
tion efficiency. The supernatant was analyzed by radio-thin-layer
chromatography (TLC, silica, 4:6 hexane/ethylacetate) and HPLC.
Five hundred microliters of the supernatant was injected into a
HPLC system (Alltech Alltima C18 column, 150 ꢁ 10 mm, particle
4.4.3. Analysis of data
size 10
10
min). The eluate was collected in fractions of 3 ml (=0.5 min) and
l
m + Alltech Alltima C18 guard 33 ꢁ 7 mm, particle size
The pooled data expressed as percentage of control activity con-
taining 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, USA).
lm, 80:20:0.1% acetonitrile/water/formic acid, flow rate 6 ml/
counted for radioactivity in a c-counter.
4.6.2. In vivo tracer stability
Three male white mice (20–25 g) were each injected with
4.4.4. HPLC analysis of metabolization
approximately 200 l of ethanol/
l
Ci of [123I]-2 dissolved in 200
l
For the assay of metabolization, 0.22 mM of test compound 2 or
saline (8:92) and sacrificed 3 min postinjection. Blood and brain
were taken. Blood samples (in EDTA-coated vials) were centrifuged
for 3 min at 3000g and the plasma was separated from the pellet.
4 was incubated with 150
pH 7.6) containing 1 mM EDTA, and 0.1% BSA (final volume of
500 l) at 37 °C for 20 min with shaking. Blanks contained assay
buffer instead of rFAAH. The reaction was terminated by adding
500 l cold acetonitrile, followed by vortex and centrifugation
lg of rFAAH in Tris–HCl buffer (10 mM,
l
To 200
ture was vortexed for 10 s followed by centrifugation for 3 min at
3000g. To the brain tissue, 1500 l of acetonitrile was added. It was
mixed for 30 s and centrifuged for 3 min at 3000g. For both plasma
and brain, 500 l of each supernatant was processed on a HPLC
ll of plasma 800 ll of acetonitrile was added and the mix-
l
l
(2 min, 6000 rpm) to remove the proteins. Hundred microliters of
supernatant was injected into the HPLC system for analysis (All-
l
tech GraceSmart C18 column, 250 ꢁ 4.6 mm, particle size 5
l
m,
system (Alltech Alltima C18 column, 150 ꢁ 10 mm, particle size
90:10:0.1% acetonitrile/water/formic acid, flow rate 1 ml/min, tR
5.7 min for arachidonic acid, 6.2 min for linoleic acid, 7.6 min for
2 and 7.1 min for 4 using UV detection, 204 nm). An identical incu-
bation with anandamide (tR 5.4 min) was carried out to validate
the method.
10
10
l
l
m + Alltech Alltima C18 guard 33 ꢁ 7 mm, particle size
m, 80:20:0.1% acetonitrile/water/formic acid, flow rate 6 ml/
min). The eluate was collected in fractions of 3 ml (=.5 min) and
counted for radioactivity in a -counter.
c
For the FAAH inhibition assay, 150
lg of rFAAH was preincu-
4.6.3. Radio-thin-layer chromatography
bated for 15 min with 10 nM of MAFP in Tris–HCl buffer (10 mM,
The test solutions were spotted at 1 cm from one end of silica
pH 7.6) containing 1 mM EDTA, and 0.1% BSA (final volume of
TLC plates (200 lm silica gel coated on plastic plates, Machery-Na-
500
l
l) at 37 °C with shaking. Next, test compound 2 or 4 was
gel, Germany). The plates were developed in hexane/ethylacetate
(4:6) and activity on the plates was quantified using a Cyclone
Storage Phosphor System. Regions of interest corresponding to
authentic radiotracer, metabolite and chromatographic origin were
drawn manually. Rf values were 0.64 for authentic [123I]-2 and 0.66
for authentic [123I]-4. The percentage of authentic radiotracer was
calculated as the optical density of the authentic tracer region di-
vided by the total signal in all regions.
added (final concentration 0.22 mM), incubated for another
20 min at 37 °C and analyzed as above.
4.5. Brain uptake of [123I]-2 and [123I]-4 in mouse brain
A biodistribution study of [123I]-2 and [123I]-4 was performed in
NMRI mice. Adult male NMRI mice weighing 20–25 g were injected
iv in the tail vein with 4–5
200 l of ethanol/saline (8:92). The mice were sacrificed by cervi-
l
Ci of [123I]-2 or [123I]-4 dissolved in
l
Acknowledgments
cal dislocation under isoflurane anesthesia at selected time points
after injection (n = 3 per time point). Blood and urine were col-
lected, and the brain and main organs to be examined were rapidly
removed and weighed. Radioactivity of the samples was measured
The authors wish to thank Dr. Jeff Lacey for his support and
great help in the organic chemistry part and Thomas Verbrugghen
for acquiring the NMR spectra. As well as Geoffray Labar for pre-
paring rat recombinant FAAH-MBP fusion protein.
in a
c-counter. The uptake of radioactivity in organs and tissues
was expressed as a percentage of the injected dose per gram of tis-
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