Development and characterization of a promising fluorine-18 labelled radiopharmaceutical for in vivo imaging of fatty acid amide hydrolase

Article abstract of DOI:10.1016/j.bmc.2013.04.077

Fatty acid amide hydrolase (FAAH), the enzyme responsible for terminating signaling by the endocannabinoid anandamide, plays an important role in the endocannabinoid system, and FAAH inhibitors are attractive drugs for pain, addiction, and neurological di

Full text of DOI:10.1016/j.bmc.2013.04.077

Bioorganic & Medicinal Chemistry 21 (2013) 4351–4357
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development and characterization of a promising fluorine-18
labelled radiopharmaceutical for in vivo imaging of fatty acid amide
hydrolase q
Oleg Sadovski a, Justin W. Hicks a, Jun Parkes a, Roger Raymond a, José Nobrega a,b, Sylvain Houle a,b
,
Mariateresa Cipriano c, Christopher J. Fowler c, Neil Vasdev d, Alan A. Wilson a,b,
⇑
a Research Imaging Centre, Centre for Addiction and Mental Health, 250 College St., Toronto, ON M5T 1R8, Canada
b Dept. of Psychiatry, University of Toronto, Toronto, ON M5T 1R8, Canada
c Dept. of Pharmacology and Clinical Neuroscience, Umeå University, SE90187 Umeå, Sweden
d Division of Nuclear Medicine and Molecular Imaging and Dept. of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Fatty acid amide hydrolase (FAAH), the enzyme responsible for terminating signaling by the endocannab-
inoid anandamide, plays an important role in the endocannabinoid system, and FAAH inhibitors are
attractive drugs for pain, addiction, and neurological disorders. The synthesis, radiosynthesis, and evalu-
ation, in vitro and ex vivo in rat, of an 18F-radiotracer designed to image FAAH using positron emission
tomography (PET) is described.
Received 15 March 2013
Revised 15 April 2013
Accepted 22 April 2013
Available online 7 May 2013
Fluorine-18 labelled 3-(4,5-dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate, [18F]5, was synthe-
sized at high specific activity in a one-pot three step reaction using a commercial module with a radio-
chemical yield of 17–22% (from [18F]fluoride). In vitro assay using rat brain homogenates showed that 5
inhibited FAAH in a time-dependent manner, with an IC50 value of 0.82 nM after a preincubation of
60 min. Ex vivo biodistribution studies and ex vivo autoradiography in rat brain demonstrated that
Keywords:
PET
FAAH
Radiosynthesis
Fluorine-18
Rat
Endocannabinoid
Anandamide
[
18F]5 had high brain penetration with standard uptake values of up to 4.6 and had a regional distribution
which correlated with reported regional FAAH enzyme activity. Specificity of binding to FAAH with [18F]5
was high (>90%) as demonstrated by pharmacological challenges with potent and selective FAAH inhib-
itors and was irreversible as demonstrated by radioactivity measurements on homogenized brain tissue
extracts.
We infer from these results that [18F]5 is a highly promising candidate radiotracer with which to image
FAAH in human subjects using PET and clinical studies are proceeding.
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
1. Introduction
hydrocannabinol (THC) as the active psychotropic component of
Cannabis sativa.3 However, subsequent elucidations in the endo-
The identification1 and cloning2 of the cannabinoid 1 (CB1)
receptor took some time following the discovery of delta-9-tetra-
cannabinoid system have been much more rapid4–7 and the endo-
cannabinoid system has emerged as an important target for basic
neuroscience studies, as well as providing targets for therapeutic
drugs.5,7–12
Abbreviations: FAAH, fatty acid amide hydrolase; PET, positron emission
One of the problems associated with cannabinoids as therapeu-
tic agents is their propensity to cause central psychotropic effects,
and a proposed way around this has been the targeting of the en-
zymes regulating endocannabinoid levels. The enzyme fatty acid
amide hydrolase (FAAH), which regulates the levels of the endog-
enous signaling molecule anandamide (AEA) may be useful in this
respect.13 Unlike ‘classical’ hydrophilic neurotransmitters lipo-
philic AEA is not stored in vesicles but rather is produced on
demand and is rapidly degraded by FAAH to terminate signal-
ing.14,15 FAAH is found in many tissues, in particular the brain, li-
ver, and kidney, and within the brain the activity varies across
tomography; CB1, cannabinoid
1 receptor; THC, delta-9-tetrahydrocannabinol;
AEA, anandamide; SUV, standard uptake value; IC50, concentration required to
inhibit 50% of enzyme activity; HPLC, high performance liquid chromatography;
NMR, nuclear magnetic resonance; HRMS, high resolution mass spectometry; DCM,
dichloromethane; MeOH, methanol; CDCl3, deuterated chloroform; EtOAc, ethyl
acetate; DMSO, dimethylsulphoxide; CH3CN, acetonitrile; EDTA, ethylenediamine-
tetraacetic acid.
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding author. Tel.: +1 416 979 4286; fax: +1 416 979 4656.
E-mail address: alan.wilson@camhpet.ca (A.A. Wilson).
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.077

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O. Sadovski et al. / Bioorg. Med. Chem. 21 (2013) 4351–4357
regions, with the highest activity being found in the hippocampus
and cortex, and the lowest in the brain stem.14,16 Selective inhibi-
tors of FAAH have been actively pursued as a method to increase
AEA levels and activate CB1 receptors in a focused manner, with
the aim of providing therapeutic effects in a variety of disorders
including pain, addiction, and obesity.10,12,16–24 Such compounds
do not produce the ‘cannabis-like’ behaviors seen with CB1 recep-
tor agonists.17,18
In vivo imaging of the endocannabinoid system has also been
pursued using positron emission tomography (PET) and a variety
of radiotracers for the CB1 receptor have been successfully devel-
oped and translated into human PET studies.19–23 For FAAH, a num-
ber of positron emitting radiotracers have been reported by us and
others,24–28 but only one has been validated for use in imaging FAAH
in humans, namely [11C]CURB.29 While this radiotracer shows much
promise, it is labelled with the short-lived radionuclide 11C (t1/
2 = 20.4 min) and thus its use is confined to sites that have an on-site
cyclotron for the production of 11C. Fluorine-18 is the other com-
monly used radionuclide in PET and, with a half-live of 109.8 min,
can be shipped and used at remote locations, thereby enabling mul-
ti-center trials.30 We describe here the synthesis and radiosynthesis
of a novel and potent FAAH inhibitor, 3-(4,5-dihydrooxazol-2-yl)-
phenyl (5-fluoropentyl)carbamate, 5, radiolabelled at high specific
activity with 18F. Evaluation in vitro and ex vivo in rats shows that
Scheme 2. Radiosynthesis of [18F]5.
at 80 °C in acetonitrile to give the t-Boc protected [18F]-fluoropen-
tylamine with incorporation yields of 70–80%. Removal of the t-Boc
group was effected by addition of dilute sulfuric acid with 85–95%
efficiency, yielding [18F]4. After neutralising the acid with phos-
phate buffer, [18F]4 was coupled with carbonate 6 at 80 °C to gen-
erate [18F]5 in 70–80% radiochemical yield.
Reverse-phase HPLC purification (see SI, Fig. S1), followed by so-
lid-phase formulation and sterile filtration, gave [18F]5 suitable for
animal studies in an isolated uncorrected radiochemical yield (from
[
18F]fluoride) of 17–22% (n = 7). The synthesis took 52–55 min from
end-of-bombardment while the radiochemical purity was >97%
with specific activities of 74–144 GBq/ mol at end-of-synthesis.
[
18F]5 is a potent FAAH inhibitor with excellent brain penetration,
l
appropriate regional distribution, and high specific binding to FAAH.
2.3. Inhibition of FAAH by compound 5
2. Results
The ability of 5 to inhibit FAAH activity was measured using rat
brain homogenates and [3H]anandamide as substrate50 (Fig. 1).
Following a 60 min preincubation phase, the compound inhibited
FAAH with a pI50 value of 9.09 0.04, corresponding to an IC50 va-
lue of 0.82 nM (Fig. 1A). Its inhibitory potency was dependent upon
the pre-incubation time used, a characteristic of irreversible en-
zyme inhibitors (Fig. 1B).
2.1. Chemistry
Compound 5 was synthesised in four steps from 5-amino-1-
pentanol (Scheme 1). Protection of the amino group of the amino
alcohol was effected with t-Boc anhydride to form 1, followed by
fluorination with DAST, yielding 3. Acid catalysed removal of the
t-Boc group of 3 gave 5-fluoropentylamine 4 as the hydrochloride
salt. Coupling of this fluoroamine with the p-nitrophenylcarbonate
of 3-(4,5-dihydrooxazol-2-yl)phenol, 6, provided 5 in an overall
yield of 14% (four steps). The t-Boc protected tosylate of 5-ami-
no-1-pentanol, 2, which was required for radiolableling, was syn-
2.4. Biodistribution of [18F]5 in rat brain
Upon tail vein injection of high specific activity [18F]5 (<1 nmol/
kg) into conscious rats, brain uptake of radioactivity was rapid and
high as measured by ex vivo dissection and counting of radioactivity
in specific brain regions (Fig. 2). Based on previous studies, the cal-
culated occupancy of the enzyme is less than 2% at these mass
doses.{Wilson, 2011 #4101} At 2 min post-injection radioactivity
in cortex (a FAAH rich region)16 was 3.9 0.70 SUV, which increased
modestly to 4.55 0.57 SUV at 40 min post-injection. The hypothal-
amus(a comparatively FAAH-poorregion)had radioactivitylevels of
1.74 0.18 SUV and 1.59 0.24 SUV at 2 and 40 min post-injection,
respectively. To demonstrate that radiotracer uptake was mediated
thesised from
1
using p-toluenesulphonyl chloride in
dichloromethane while the activated carbonate, 6, was obtained
by acylation of 3-(4,5-dihydrooxazol-2-yl)phenol with p-nitrophe-
nylchloroformate in DMSO.
2.2. Radiochemistry
The radiosynthesis of [18F]5 is outlined in Scheme 2. Displace-
ment of the tosyl group of 2 by [18F]fluoride proceeded smoothly
Scheme 1. Synthesis of 5 and precursors required for radiosynthesis of [18F]5 and the activated carbonate, 6. Reagents and conditions: (a) t-Boc anhydride, K2CO3, 74%; (b)
TsCl, TEA, 82%; (c) DAST, DCM, 43%; (d) HCl in MeOH, 94%; (e) compound 6, KHCO3, 39%.

O. Sadovski et al. / Bioorg. Med. Chem. 21 (2013) 4351–4357
4353
Figure 1. Inhibition of rat brain FAAH-catalysed [3H]anandamide hydrolysis by compound 5. Panel A shows a concentration–response curve using a preincubation time of
60 min, and Panel B shows the inhibition produced by three concentrations of 5 following different preincubation times. Given that the incubation time was 10 min, the
inhibition seen at preincubation time zero is likely to have a time-dependent component. Data are means SEM, n = 3.
Figure 2. Biodistribution of [18F]5 in rat brain at 2 and 40 min post tail vein
Figure 3. Amount of radioactivity irreversibly bound to rat brain tissue at 2, 10, and
40 min post tail vein injection of [18F]5 (n = 4–6/group, mean SD). The blocked
injection (n = 4/group, mean SD). The blocked groups received URB597 (2 mg/kg,
group received URB597 (2 mg/kg, ip) 30 min before radiotracer. ⁄p <0.05.
ip) or PF-04457845 (0.5 mg/kg, ip) 30 min before radiotracer. ⁄p <0.05.
ible bound by >98% compared with the control group and only 24%
of total brain radioactivity was bound to tissue.
by FAAH binding, groups of rats were pretreated with either the pro-
totypical carbamate FAAH inhibitor URB597 (2 mg/kg, ip) or the re-
cently developed urea FAAH inhibitor, PF-04457845 (0.5 mg/kg, ip),
30 min prior to radiotracer administration.31–35 Both inhibitors
emphatically reduced uptake of radioactivity in all brain regions be-
tween 90% (hypothalamus) and 96% (cortex) with >90% reduction in
uptake in the whole brain compared with controls (Fig. 2). Radioac-
tivity levels in plasma were significantly elevated in pre-treated ani-
mals, an effect seen previously for FAAH radiotracers.27,36
2.6. Metabolism of [18F]5
Reverse-phase radio-HPLC analysis of rat plasma 10 min post-
radiotracer injection showed that almost complete (>95%) metab-
olism had occurred, with the radioactivity associated with hydro-
philic metabolites. However this analysis was compromised as
we found the radiotracer to be unstable in rat plasma. Incubation
of [18F]5 in vitro in rat plasma at ambient temperature resulted
in >50% decomposition within 30 min (Fig. 4). In contrast, [18F]5
was stable in human plasma during the same time-period, with
<5% decomposition, perhaps suggesting that decomposition
in vitro in rat plasma was mediated by carboxylesterases as these
enzymes are not present in human plasma.35,38
Radio-HPLC analysis of rat brain extracts, 10 min post-radio-
tracer injection showed that 50% of the extractable radioactivity
was metabolized [18F]5 (data not shown). However it must be
noted that extractable radioactivity represents <10% of the total
radioactivity present in the brain (vide supra). In addition, as
brains were non-perfused, the vasculature compartment of the
rat brain (5%) represents a significant source of extractable radio-
activity under these conditions.
2.5. Irreversible binding of [18F]5 to rat brain parenchyma
Upon injection of [18F]5 to groups of conscious rats, whole
brains were excised, homogenized, and exhaustively extracted
with aqueous acetonitrile to differentiate irreversibly bound radio-
tracer from unbound.37 Following centrifugation, radioactivities in
the washed pellet (bound) and in the combined supernatant (un-
bound) were counted (Fig. 3). Even at 2 min post-injection, most
(91%) of the radioactivity was irreversibly bound to brain paren-
chyma, an amount which did not change significantly at 10 min
(94%) or 40 min (90%) post-injection, although the absolute
amount of radioactivity bound did increase over this time period.
Pretreatment of rats with URB597 (2 mg/kg, ip) 30 min prior to
radiotracer injection reduced the amount of radioactivity irrevers-

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O. Sadovski et al. / Bioorg. Med. Chem. 21 (2013) 4351–4357
18F-nucleophilic substitution reactions, such aryl carbamates un-
dergo a facile and rapid E2 elimination reaction which precluded
introduction of [18F]fluoride directly.42 However, judicious choice
of reaction conditions that efficiently generated a synthetically
versatile [18F]fluoroamine, [18F]4, allowed [18F]5 to be prepared
in an efficient one-pot three-step reaction (Scheme 1), which was
easily automated using a commercial apparatus.
The suitability of [18F]5 as a radiotracer for imaging FAAH by
PET was demonstrated in several ways:
(1) Ex vivo biodistribution studies showed that brain penetra-
tion of [18F]5 was high and mediated by FAAH as pre-treat-
ment with established and selective FAAH inhibitors
URB597 or PF-04457845 blocked >90% of brain uptake
(Fig. 2).
(2) The brain regional uptake of radioactivity 40 min post
administration of [18F]5 correlated well with the reported
regional FAAH enzyme activity in rat brain. A similar corre-
lation was found with the clinically used 11C-radiotracer
Figure 4. Radio-HPLC chromatograms of [18F]5 upon incubation in rat and human
plasma for 30 min at ambient temperature.
[
11C]CURB and related analogs,26 as well as the closely
2.7. Ex vivo autoradiography of [18F]5 in rat brain
related congener of 5, [11C]8 reported elsewhere (Fig. 6).28
This is direct evidence that regional uptake of [18F]5 is sen-
sitive to FAAH activity.
Figure 5 shows autoradiographic images of [18F]5 at the level of
the forebrain (A) and cerebellum (B). As illustrated, considerable
labelling was present in cortex, hippocampus, thalamic and amyg-
daloid nuclei (A) as well as cerebellar cortex (B). Signal was also
clearly seen in the ventricular space, which is consistent with pre-
viously reports of expression of FAAH in the choroid plexus.39–41
Pretreatment with 0.5 mg/kg of PF-04457845 (C and D) completely
abolished binding in all brain regions, indicating exceptional spec-
ificity of the radiotracer for its target sites ex vivo.
(3) FAAH is inactivated by N-alkyl-O-arylcarbamates by car-
bamoylation of the Ser241 amino acid, resulting in ejection
of the O-aryl residue.43,44 Thus it was anticipated that intro-
duction of the radiolabel on the N-alkyl chain of 5 would
result in the irreversible tagging of FAAH with 18F upon
binding. To demonstrate this, excised rat brain was homog-
enized and repeatedly extracted with solvent to measure the
amount of radiolabel irreversibly bound to rat brain paren-
chyma following radiotracer injection (Fig. 3).37 Less than
10% of total radioactivity could be extracted, demonstrating
the irreversible nature of the binding. This irreversible bind-
ing was mediated by FAAH as demonstrated by its almost
complete abolishment by pre-treatment of animals with
URB597. Scheme 3 depicts the putative mechanism of action
of [18F]5.
3. Discussion
Our target compound, 3-(4,5-dihydrooxazol-2-yl)phenyl (5-flu-
oropentyl)carbamate, 5, (log D, pH 7.4 of 2.33) was chosen on the
basis of previous preclinical studies with 11C-labelled FAAH inhib-
itors which showed that N-alkyl-3-(4,5-dihydrooxazol-2-yl)phenyl
carbamates possess excellent properties as FAAH imaging radiotra-
cers, including high brain uptake, selectivity, and fast kinetics.28 In
vitro enzyme assays showed that 5 was also a potent and irrevers-
ible inhibitor of FAAH with an IC50 of 0.8 nM, akin to its non-fluo-
rinated analogues.28 While one-step radiolabelling is always
desirable, especially when dealing with short-lived radioisotopes,
direct introduction of 18F into 5 using arylsulphonate esters as pre-
cursors proved unfruitful. Under the basic conditions required for
Figure 6. Correlation between regional brain uptake of FAAH radiotracers and
regional FAAH enzyme activity in rat brain. Data taken from this work ([18F]5
uptake); Wilson et al., ([11C]CURB and compound [11C]8 uptake);26,28 and Thomas
et al., (FAAH enzyme activity).16 Error bars represent standard deviation from the
mean.
Figure 5. Ex vivo autoradiography of [18F]5 in rat brain; control (A and B) and
blocked (C and D). See Figure S2 for more slices.

O. Sadovski et al. / Bioorg. Med. Chem. 21 (2013) 4351–4357
4355
methanol (25 mL) was stirred at ambient temperature for 2 h.
The reaction mixture was quenched with water (25 mL) and ex-
tracted with EtOAc (2 Â 25 mL). Combined extracts were washed
with water and brine, dried (MgSO4), and filtered. Solvent removal
gave the product as a viscous colourless oil (4.4 g, 74%). 1H NMR
(CDCl3): d ppm 1.3–1.63 (m, 15H) 3.07–3.18 (m, 2H) 3.65 (t,
J = 6.44 Hz, 2H) 4.55 (br s, 1H). 13C NMR (CDCl3): d ppm 22.89,
28.38, 29.80, 32.20, 40.39, 62.53. 79.07, 156.08. HRMS (ESI+) m/z
calcd for [M+H]+ C10H22NO3 204.15997, found 204.16070.
5.3. 5-((tert-Butoxycarbonyl)amino)pentyl 4-
methylbenzenesulfonate (2)
Scheme 3. Putative mechanism of irreversible binding of [18F]5 to FAAH. Carbam-
oylation of the serine241 residue results in FAAH tagged with the [18F]fluoropen-
tylcarbamoyl group.
A
mixture of tert-butyl (5-hydroxypentyl)carbamate (1.5 g,
7.4 mmol), p-toluenesulfonyl chloride (1.5 g, 7.9 mmol), triethylamine
(100 L, 0.72 mmol), and potassium carbonate (2 g, 14.5 mmol) in
(4) The distribution of radioactivity after injection of [18F]5 as
depicted by ex vivo autoradiography studies are in close
agreement with previous autoradiography, immunohisto-
chemical, and immunoreactivity studies which measured
the patterns of FAAH activity, mRNA, and expression in rat
l
dichloromethane (10 mL) was stirred at ambient temperature for
3 days. Water (100 mL) was added and the mixture extracted with
ethyl acetate (2 Â 50 mL). The combined organic extracts were washed
with brine, dried (Na2SO4), filtered and solvent removed to leave a light
brown oil (2.41 g). Flash chromatography (hexane/EtOAc, 30:70) gave
a colourless oil from heart fractions which crystallised upon trituration
with pentane to give the product as a white solid (0.97 g, 37%). A further
brain,
including
high
levels
in
the
ventricles
(Fig. 5).16,39,41,45 Specificity of uptake was again demon-
strated by the complete disappearance of binding following
pretreatment with a selective FAAH inhibitor.
1
1.2 g (45%) was isolated from other fractions. Mp 47–48 °C. H NMR
(CDCl3): d ppm 1.30–1.38 (m, 2H) 1.40–1.48 (m, 11H) 1.61–1.71 (m,
2H) 2.45 (s, 3H) 3.06 (q, J = 6.56 Hz, 2H) 4.02 (t, J = 6.33 Hz, 2H) 4.50
(br s, 1H) 7.35 (m, J = 7.99 Hz, 2H) 7.79 (m, 2H). 13C NMR (CDCl3): d
ppm 21.85, 22.88, 28.62, 29.65, 40.44, 70.55, 79.36, 128.10, 130.05,
133.35, 144.94, 156.14. HRMS (ESI+) m/z calcd for [M+H]+ C17H28NO5S
358.1683, found 358.1679.
4. Conclusions
We have identified and characterized, in vitro and ex vivo in rats,
a novel 18F-labelled radiotracer for PET imaging of FAAH. [18F]5
shows high brain uptake upon intravenous administration with
binding which is sensitive to FAAH regional activity. As demon-
strated with dissection studies and ex vivo autoradiography, [18F]5
binds to FAAH with a high degree of selectivity and specificity. These
properties suggest that the radiotracer is eminently suitable for neu-
roimaging of FAAH in the living human brain using PET. Clinical
studies with [18F]5 are being pursued in our laboratory.
5.4. tert-Butyl (5-fluoropentyl)carbamate (3)
To a stirred solution of DAST (1.87 ml, 19 mmol) in DCM (5 mL) at
À70 °C was added a solution of tert-butyl (5-hydroxypentyl) carba-
mate (3 g, 14 mmol) in DCM (6 mL) over 30 min. The mixture was
stirred for 12 h at room temperature, quenched with saturated NaH-
CO3 (25 mL) and the organic layer separated. The aqueous phase was
extracted with DCM (10 mL) and the combined organic extracts
dried (MgSO4), filtered, and volatiles evaporated. The product was
obtained as a colourless oil upon purification by silica column chro-
matography with DCM/hexane 1/1 as mobile phase. (1.3 g, 43%) 1H
NMR (CDCl3): d ppm 1.39–1.57 (m, 13H) 1.62–1.78 (m, 2H) 3.12
(q, J = 6.24 Hz, 2H), 4.43 (dt, J = 47.9 Hz, 6.05 Hz, 2H). 13C NMR
(CDCl3): d ppm 22.47 (d, J = 5.37 Hz) 28.37, 29.67, 30.00 (d,
J = 19.94 Hz), 40.35, 79.03, 83.87 (d, J = 164.09 Hz,) 155.96. 19F
NMR (CDCl3) d ppm) 47.59 (tt, J = 47.30, 25.94 Hz). HRMS (ESI+) m/
z calcd for [M+H]+ C10H21FNO2 206.15563, found 206.15582.
5. Experimental methods
5.1. Chemistry
A Scanditronix MC 17 cyclotron was used for radionuclide pro-
duction. Purifications and analyses of radioactive mixtures were
performed by high performance liquid chromatography (HPLC) with
an in-line UV (254 nm) detector in series with a NaI crystal radioac-
tivity detector (purifications) or a Bioscan Flowcount coincidence
radioactivity detector (analyses). Isolated radiochemical yields were
determined with a dose-calibrator (Capintec CRC-712M). Unless
stated otherwise, all radioactivity measurements were normalised
for radioactive decay. Proton and carbon-13 NMR spectra were re-
corded at 25 °C on a Varian Mercury 400 MHz spectrometer. Electro-
spray ionization mass spectrometry was conducted with MDS Sciex
QStar mass spectrometer to obtain the HRMS. All tested compounds
had a purity of >95% as determined by reverse-phase HPLC. All ani-
mal experiments were carried out under humane conditions, with
approval from the Animal Care Committee at the Centre for Addic-
tion and Mental Health, and in accordance with the guidelines set
forth by the Canadian Council on Animal Care. Rats (male, Spra-
gue–Dawley, 300–350 g) were kept on a reversed 12-h light/12-h
dark cycle and allowed food and water ad libitum.
5.5. 5-Fluoropentylamine hydrochloride (4)
A solutionof HCl in MeOH (3 M, 6 mL) was slowly added to a solu-
tion of tert-butyl (5-fluoropentyl)carbamate (0.100 g, 0.49 mmol) in
CHCl3 (5 mL) at room temperature. After 12 h, volatiles were evapo-
rated to give 5-fluoropentylamine hydrochloride (0.065 g, 94%) as a
light brown hygroscopic solid. 1H NMR (MeOH-d4): d ppm 1.43–1.58
(m, 2H), 1.65–1.84 (m, 4H), 2.95 (t, J = 7.61 Hz, 2H), 4.45 (dt, J = 6.05,
47 .4 Hz, 2H) 4.51. 13C NMR (MeOH-d4): d ppm 22.02 (d, J = 5.37 Hz),
26.75, 29.56 (d, J = 19.94 Hz), 39.27, 83.18 (d, J = 164.09 Hz) 19F NMR
(MeOH-d4) d ppm) 45.69 (m). HRMS (ESI+) m/z calcd for [M+H]+
C5H13F 106.10320, found 106.10296.
5.6. 3-(4,5-Dihydrooxazol-2-yl)phenyl (5-fluoropentyl)
carbamate (5)
5.2. tert-Butyl (5-hydroxypentyl)carbamate (1)
A mixture of 5-amino-1-pentanol (3.0 g, 29.1 mmol), t-Boc
anhydride (6.1 g, 34.9 mmol), and K2CO3 (4 g) in 50/50 water/
A
solution of 5-fluoropentylamine hydrochloride (95 mg,
0.67 mmol) in CH3CN/H2O (4:1, 5 mL) was added to a stirred mix-

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O. Sadovski et al. / Bioorg. Med. Chem. 21 (2013) 4351–4357
ture of 3-(4,5-dihydrooxazol-2-yl)phenyl (4-nitrophenyl) carbon-
ate (170 mg, 0.52 mmol) and KHCO3 (200 mg, 2.0 mmol) in CH3CN
(5 mL). The mixture was stirred at room temperature for 3 h then
DCM (10 mL) and satd aqueous NaHCO3 (50 mL) were added. The
aqueous layer was extracted with DCM and the combined organic
layers filtered and solvent removed to leave a light brown oil
(143 mg). Flash chromatography (hexane/EtOAc; 50:50) on silica
gel gave a colourless oil which upon trituration with 20 mL hexane
gave the desired product as a white solid (72 mg, 47% yield); mp
74.5–77 °C. 1H NMR (CDCl3): d ppm 1.46–1.54 (m, 2H), 1.60–1.81
(m, 4H), 3.28 (q, J = 6.63 Hz, 2H), 4.05 (t, J = 9.56 Hz, 2H), 4.37–
4.46 (m, 3H), 4.52 (t, J = 5.85 Hz, 1H), 5.19 (br s, 1H), 7.23–7.29
(m, 1H), 7.39 (t, J = 8.00 Hz, 1H), 7.70 (s, 1H), 7.78 (d, J = 7.80 Hz,
1H). 13C NMR (CDCl3): d ppm 22.30 (d, J = 5.37 Hz), 29.27, 29.81
(d, J = 19.94 Hz), 40.89, 54.74, 67.53, 83.67 (d, J = 164.86 Hz),
121.30, 124.42, 124.82, 128.84, 129.05, 150.77, 154.15, 163.79.
19F NMR (CDCl3): d ppm 47.39 (m). HRMS (ESI+) m/z calcd for
[M+H]+ C15H20FN2O3 295.14580, found 295.14450.
ual acetonitrile, it was eluted with ethanol (1 mL), chased with iso-
tonic citrate buffer (10 mL, pH 6), prepared by dissolving trisodium
citrate dehydrate (0.588 g) and disodium hydrogen citrate 1.5 hy-
drate (0.263 g) in water (100 mL), and passed through a sterile
0.2 lm filter into a sterile, pyrogen-free bottle. Co-injection of
the radioactive product with an authentic standard of 5 under sev-
eral reverse-phase conditions, varying solvents (methanol, acetoni-
trile, THF) pH, (7.0, 5.0, 4.0) with several analytical HPLC columns
(Waters, Grace, Phenomenex, ThermoScientific, C18 and C8) estab-
lished the identity of the radiotracer. Under all conditions [18F]5
co-chromatographed with authentic 5. The formulated radiotracer
displayed <5% radiolysis 120 min post formulation and proved to
be sterile and pyrogen-free, suitable for animal studies.
5.9. Log P measurements
The partition coefficients of [18F]5, between 1-octanol and 0.02 M
phosphate buffer at pH 7.4, was determined to be 2.33 0.01 by a
previously described method, replicated eight times.49
5.7. 3-(4,5-Dihydrooxazol-2-yl)phenyl (4-nitrophenyl)
carbonate (6)
5.10. FAAH assays in vitro
A solution of p-nitrophenylchloroformate (120 mg, 0.6 mmol) in
FAAH activity was assayed in rat brain homogenates as described
CH3CN (2 mL) was added to a stirred solution of 3-(4,5-dihydrooxa-
previously.50 Briefly, 5 (in ethanol) was added to homogenates of
zol-2-yl)phenol46 (100 mg, 0.6 mmol) and DIPEA (110
lL, 0.63 mmol))
frozen brains (minus cerebellum; 0.5 lg per assay) from adult rats
in CH3CN (2 mL) and DMSO (0.8 mL). The mixture was stirred at ambi-
ent temperature for 20 min then quenched with 5% citric acid (17 mL).
The resultant precipitate was collected by vacuum filtration and
washed with copious amounts of water. Drying in vacuo gave the prod-
uct as a pale yellow powder which was purified by flash chromatogra-
phy (hexane/EtOAc, 80:20 to 30:70) yielding a white solid (77 mg,
39%); mp 150–152 °C. 1H NMR (CDCl3): d ppm 4.10 (t, J = 9.65 Hz,
2H), 4.47 (t, J = 9.65 Hz, 2H), 7.38–7.43 (m, 1H), 7.47–7.53 (m, 3H),
7.88–7.93 (m, 2H), 8.30–8.36 (m, 2H). 13C NMR (CDCl3): d ppm 55.02,
67.90, 120.73, 121.74, 123.52, 125.43, 126.44, 129.69, 129.78, 145.69,
150.52, 150.87, 155.20, 163.45. HRMS (ESI+) m/z calcd for [M+H]+
C16H13N2O6 329.0768, found 329.0763.
(Wistar or Sprague–Dawley) in 50 mM Tris–HCl buffer, pH 7.4 con-
taining 1 mM EDTA and 3 mM MgCl2 and preincubated at 37 °C for
the times shown in Figure 1. [3H]Anandamide, labelled in the etha-
nolamine part of the molecule (American Radiolabeled Chemicals,
Inc., St. Louis, MO) in 10 mM Tris–HCl, 1 mM EDTA, pH 7.4, contain-
ing 1% w/v fatty acid-free bovine serum albumin was then added to
give a final substrate concentration of 0.5
200 l. The assay tubes were then incubated for 10 min at 37 °C after
which reactions were stopped by placing the tubes on ice and adding
activated charcoal (80 + 320 l 0.5 M HCl). After mixing and phase
lM and assay volume of
l
l
separation, aliquots of the supernatants were analysed for tritium
content by liquid scintillation spectroscopy with quench correction.
Blanks contained assay buffer in place of enzyme homogenate.
5.8. Radiosynthesis of [18F]5
5.11. Biodistribution studies in rats
Procedures were initially carried out by hand using <1 MBq of
[
18F]fluoride to optimize reaction conditions. Subsequently the
Rats, in a restraining box, received 3–4 MBq of high specific activity
radiosynthesis was carried out using a radiosynthesis module
[
18F]5 (0.2–1 nmol/kg) in 0.3 mL of 10% ethanol in citrate buffer (pH 6)
(for details see47) using 14–20 GBq. [18F]Fluoride was produced
via the tail vein which had been vasodilated in a warm water bath.
Groups of rats (n = 4–6) were sacrificed by decapitation at various time
intervals after radiotracer administration, the brain surgically removed
from the skull and stored on ice. Brain regions were excised, blotted,
and weighed while blood was collected (from the trunk). Radioactivity
in tissues was assayed in an automated gamma counter, back corrected
to time of injection, using diluted aliquots of the initial injected dose as
standards. Values are reported as standard uptake values (SUVs,
mean standard deviation) defined as % injected dose/g of tissue di-
vided by rat weight in kg. Treated groups were compared to those of
the vehicle-treated group by Student’s t test. Statistical comparisons
were considered significant when p <0.05. For pharmacological chal-
lenge studies in rats, groups of rats (n = 4) were pre-treated with solu-
tions of the challenge drug (2 mL/kg in 5% Tween 80 in saline ip) or
vehicle 40 min prior to radiotracer injection. Animals were sacrificed
40 min post radiotracer administration and radioactivity in tissues
measured as describe above.
by the 18O(p, n)18
F
nuclear reaction using >95% enriched
[
18O]H2O. The [18F]fluoride was trapped on a Chromafix ion-ex-
change 30-PS-HCO3 resin, and eluted into a glass reactor with a
solution consisting of 14.4 mg of 4,7,13,16,21,24-hexaoxa-1,10-
diazabicyclo[8.8.8]hexacosane (Kryptofix 222,) and 3 mg of K2CO3
in 1 mL of 4% (v/v) water in acetonitrile.48 The solution was dried
by a combination of heat (90 °C), vacuum, and N2 flow (350 mL/
min) for 5 min. After cooling the reactor to 60 °C, a solution of 2,
(5 mg. 14 lmol) in CH3CN (0.5 mL) was added with stirring and
heated to 80 °C for 7 min. Then 2% aq H2SO4 (0.5 mL) was added,
heating and stirring continued for a further 7 min, and the solution
neutralized with phosphate buffer (0.5 mL, prepared by dissolving
K2HPO4 (34.84 g) and KOH (2.81 g) in water (250 mL)). A solution
of 6 (7 mg, 21.3 lmol) in 70% aq. CH3CN (1 mL) was added, with
heating at 80 °C and stirring continued for a further 7 min. The
mixture was cooled to <45 °C, quenched with water (2 mL), and
purified by HPLC using
a Phenomenex Gemini C18 5 lm,
250 Â 10 mm column with 35:65 CH3CN/H2O + 0.1 N ammonium
formate as eluent at 7 mL/min. The fraction containing [18F]5 was
collected, diluted with 25 mL of water, and trapped on a pre-con-
ditioned reverse-phase cartridge (Waters Sep-Pak tC18+). After
washing the cartridge with water (5 mL) to remove salts and resid-
5.12. Irreversible binding of [18F]5 to FAAH and metabolite
studies
Following tail-vein injection of [18F]5 groups of 4–6 rats were
sacrificed at various timepoints and the whole brain was surgically

O. Sadovski et al. / Bioorg. Med. Chem. 21 (2013) 4351–4357
4357
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Acknowledgments
This work was supported by NIH Grant # 1R21MH094424 to
A.A.W., a University of Toronto Institute of Medical Science Open
Fellowship award to J.W.H., and by a Grant from the Swedish Re-
search Council (Grant no. 12158, medicine) to C.J.F. We would like
to thank Armando Garcia, Winston Stableford, Min Wong, and Al-
vina Ng for their assistance with the radiochemistry and animal
dissection experiments.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.04.077.
These data include MOL files and InChiKeys of the most important
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