Synthesis, ex vivo evaluation, and radiolabeling of potent 1,5-diphenylpyrrolidin-2-one cannabinoid subtype-1 receptor ligands as candidates for in vivo imaging

Article abstract of DOI:10.1021/jm800416m

We have reported that [methyl-¹¹C] (3R,5R)-5-(3-methoxyphenyl)- 3-[(R)-1-phenylethylamino]-1-(4-trifluoromethylphenyl)pyrrolidin-2-one ([ ¹¹C]8, [¹¹C]MePPEP) binds with high selectivity to cannabinoid type-1 (CB₁

Full text of DOI:10.1021/jm800416m

J. Med. Chem. 2008, 51, 5833–5842
5833
Synthesis, Ex Vivo Evaluation, and Radiolabeling of Potent 1,5-Diphenylpyrrolidin-2-one
Cannabinoid Subtype-1 Receptor Ligands as Candidates for In Vivo Imaging
Sean R. Donohue,†,‡ Joseph H. Krushinski,§ Victor W. Pike,*,† Eyassu Chernet,§ Lee Phebus,§ Amy K. Chesterfield,§
Christian C. Felder,§ Christer Halldin,‡ and John M. Schaus§
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, Karolinska Institutet,
Department of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, S-17176 Stockholm, Sweden, and Lilly Research Laboratories,
Eli Lilly and Company, Indianapolis, Indiana 46285
ReceiVed April 10, 2008
We have reported that [methyl-11C] (3R,5R)-5-(3-methoxyphenyl)-3-[(R)-1-phenylethylamino]-1-(4-trifluo-
romethylphenyl)pyrrolidin-2-one ([11C]8, [11C]MePPEP) binds with high selectivity to cannabinoid type-1
(CB1) receptors in monkey brain in vivo. We now describe the synthesis of 8 and four analogues, namely,
the 4-fluorophenyl (16, FMePPEP), 3-fluoromethoxy (20, FMPEP), 3-fluoromethoxy-d2 (21, FMPEP-d2),
and 3-fluoroethoxy analogues (22, FEPEP), and report their activity in an ex vivo model designed to identify
compounds suitable for use as positron emission tomography (PET) ligands. These ligands exhibited high,
selective potency at CB1 receptors in vitro (Kb < 1 nM). Each ligand (30 µg/kg, iv) was injected into rats
under baseline and pretreatment conditions (3, rimonabant, 10 mg/kg, iv) and quantified at later times in
frontal cortex ex vivo with liquid chromatography-mass spectrometry (LC-MS) detection. Maximal ligand
uptakes were high (22.6-48.0 ng/g). Under pretreatment, maximal brain uptakes were greatly reduced
(6.5-17.3 ng/g). Since each ligand readily entered brain and bound with high selectivity to CB1 receptors,
we then established and here describe methods for producing [11C]8, [11C]16, and [18F]20-22 in adequate
activities for evaluation as candidate PET radioligands in vivo.
Introduction
Cannabis satiVa (marijuana) and associated plant-derived
phytocannabinoids are considered the world’s oldest therapeu-
tics. Smoking or ingesting the resin from marijuana leaves or
flowers provides medicinal properties such as analgesia, anti-
emesis, anti-spasmodic, and appetite stimulation, but also less
desirable side effects such as immunosuppression, psychoactive
“high”, drowsiness, and memory impairment.1,2 Only over the
Figure 1. Structures of some CB1 receptor agonists. Asterisks denote
positions of the radiolabel.
past several decades have the biological mechanisms of phy-
tocannabinoid exposure become better understood. A major
advancement was the correct structural identification of (-)-
(6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-
benzo[c]chromen-1-ol [1, ∆9-THCa (Figure 1)],3 which was
identified as the major psychoactive phytocannabinoid. Subse-
quent attempts to improve the potency of 1 through rational
design led to the identification of 2-[(1S,2R,5S)-5-hydroxy-2-
(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol (2,
CP-55,940) as a considerably higher affinity agonist.4 The use
of [3H]2 (Figure 1) in vitro helped to provide direct evidence
of cannabinoid receptors which are now known to belong to
the superfamily of G protein-coupled receptor proteins.5 Two
cannabinoid receptor family members have been cloned and
sequenced (CB1 and CB26,7), but others subtypes may exist.8
CB1 receptors, which have two splice variants (CB1A and
CB1B),9,10 are localized in peripheral tissues (e.g., heart, lung,
prostate, testis, bone, marrow, tonsils, and spleen)8 and central
tissues with highest densities in the brain’s globus pallidus, the
substania nigra, the molecular layer of the cerebellum, the
cerebral cortex, the striatum, and the hippocampus. The pons,
thalamus, and brain stem are almost devoid of CB1 receptor
expression.11,12 CB2 receptors are located mainly in the periph-
eral immune system tissues such as macrophages of spleen, bone
marrow, and pancreas but are also localized at peripheral nerve
terminals.7,13,14 Low levels of CB2 receptors have been found
in brain in both neurons and glia, though their physiological
significance in the brain has not been fully elucidated.15,16
Abnormalities in regional brain CB1 receptor densities or
function may contribute to an array of neuropsychiatric and/or
neurodegenerative disorders. Noninvasive quantitative imaging
of brain CB1 receptors with positron emission tomography (PET)
could help to clarify the role of these receptors in many CNS
disorders. However, an effective PET radioligand, labeled with
either 11C (t1/2 ) 20.4 min) or 18F (t1/2 ) 109.7 min), is required
for such imaging. Such a radioligand would need to show
* To whom correspondence should be addressed: Molecular Imaging Branch,
National Institute of Mental Health, National Institutes of Health, Building 10,
Room B3 C346A, 10 Center Dr., Bethesda, MD 20892-1003. Telephone: (301)
594-5986. Fax: (301) 480-5112. E-mail: pikev@mail.nih.gov.
†
National Institutes of Health.
Karolinska Hospital.
Eli Lilly and Company.
‡
§
a Abbreviations: CB1, cannabinoid subtype-1; CB2, cannabinoid subtype-
2; CHO, Chinese hamster ovary; CNS, central nervous system; D2, dopamine
subtype-2; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
EOS, end of synthesis; EtOAc, ethyl acetate; GDP, guanosine diphosphate;
GTPγS, guanosine 5′-(γ-thio)triphosphate; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; HRMS, high-resolution mass spectrometry;
HPLC, high-performance liquid chromatography; 5-HT2A, serotonin-2A;
IND, investigational new drug; LC-MS, liquid chromatography-mass
spectrometry; mp, melting point; MTBE, methyl tert-butyl ether; NK1,
neurokinin-1; PET, positron emission tomography; RCY, decay-corrected
radiochemical yield; SUV, standardized uptake value; TFA, trifluoroacetic
acid; THC, tetrahydrocannabinol.
10.1021/jm800416m CCC: $40.75
2008 American Chemical Society
Published on Web 08/23/2008

5834 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Donohue et al.
Figure 2. Structures of 3 and candidate CB1 receptor PET radioligands. Asterisks denote positions of the radiolabel.
suitable in vivo characteristics, including adequate maximal
brain uptake, selectivity for binding to CB1 receptors, insig-
nificant brain-penetrating radiometabolite(s), and a low level
of nonspecific binding.17,18
[18F]fluoromethoxy-d2 analogue to take advantage of this kinetic
isotope effect. Another potential metabolic route is aromatic
hydroxylation of the (R)-1-phenylethylamino moiety of 8.
Incorporation of a fluorine atom at the para position of an
aromatic ring is known to increase the biological half-life by
inhibiting metabolic aromatic hydroxylation.28 Hence, a 4-fluo-
rophenyl analogue of 8 was also considered.
Much progress has been made recently in the development
of CB1 receptor PET radioligands. Past attempts at CB1 PET
radioligand development have focused on modifying the sub-
stituents of the 1,5-diarylpyrazole core of rimonabant [3 (Figure
2)].19 This approach led to the development of two promising
radioligands, [11C]4 [[11C]JHU75528 (Figure 2)]20,21 and [11C]5
[[11C]JHU75575 (Figure 2)].21 Merck recently reported [18F]6
[[18F]MK-9470 (Figure 2)]22,23 as a suitable PET radioligand
for in vivo quantification of brain CB1 receptors in humans.
Further promising radioligands are [11C]7 [[11C]PipISB (Figure
2)],24 [18F]7 [[18F]PipISB (Figure 2)],24 and [11C]8 [[11C]Me-
PPEP (Figure 2)].25 [18F]7 is somewhat superior to [11C]7 and
has an in vivo PET time-activity profile similar to that of [18F]6
but exhibits a stronger specific signal, when expressed as
receptor-specific to nonspecific binding (9:1 vs 5:1). In rhesus
monkey, [11C]8 exhibits excellent maximal brain uptake and
binds selectively to CB1 receptors.25 Furthermore, the binding
of [11C]8 appears in some cases to reach equilibrium during
the scan time (120 min after injection), which would allow for
equilibrium-based pharmacokinetic analysis.25 However, the
time of apparent equilibrium is variable and in some cases is
not observed which then requires complex biomathematical
modeling. To surmount this issue, analogues of 8 labeled with
18F are desirable for increasing the allowable scan time.
The structure and pharmacology of 8 suggested [18F]fluo-
ro(m)ethoxy analogues as plausible candidate radioligands.
Direct incorporation of [18F]fluoride ion into the F3C group of
the N-1 aryl ring of 8 was not considered because of the risk of
achieving only low specific radioactivity when high specific
radioactivity would be required in an effective radioligand.18
The in vivo metabolism of [11C]8 in rhesus monkey is very
rapid and can obfuscate accurate measurement of unchanged
radioligand in plasma at late scan times. Such measurements
are required for biomathematical compartmental modeling of
acquired PET data.18,25 A more metabolically stable analogue
of 8 with similar potency and lipophilicity is desirable for
allowing more accurate measurement of plasma radioligand. It
has been reported that replacement of a fluoromethoxy group
with a dideutero-fluoromethoxy group can lead to a decrease
in the rate of defluorination.26,27 Hence, we considered a
The discovery of radioligands suitable for in vivo PET
imaging is a challenging task. PET radioligands need to satisfy
several criteria, such as high potency for the molecular target,
facile penetration into the brain, and a low level of nonspecific
binding.18 In many cases, the properties necessary for PET
ligands are different from those found in therapeutic agents.
While compound potency is relatively straightforward to
measure and optimize, the evaluation of nonspecific binding is
particularly difficult and is generally done using a radiolabeled
compound. The necessity of preparing radiolabeled material has
been an impediment to the rapid evaluation and discovery of
new PET ligands. Consequently, investigators have used cLogP
and cLogD7.4 as surrogate measures of nonspecific binding;
however, the correlation of these parameters with nonspecific
binding in in vivo experiments is often not satisfactory.29 The
development of an ex vivo method for determining the brain
penetration and nonspecific binding of a non-radiolabeled
compound would greatly facilitate the discovery of new PET
ligands.
Highly sensitive LC-MS techniques have been developed for
the quantification of drug levels in tissues.30 We reasoned that
this methodology could be used to measure the concentration
of candidate PET ligands in brain samples following administra-
tion of the compound at very low, tracer doses.30 Not only would
this methodology provide a measure of brain penetration, it also
could be used to assess nonspecific binding. In the case
presented here, brain penetration was determined by administra-
tion to rats of a tracer dose (30 µg/kg, iv) of the study compound.
Nonspecific binding was determined by pretreatment of rats with
a large dose of 3 (10 mg/kg, iv) prior to administration of the
tracer dose. Under these conditions, the vast majority of central
CB1 receptors are occupied by 3 and the tissue levels of the
tracer represent nonspecifically bound compound. Compounds
which displayed low brain levels of tracer in pretreated rats and
high levels in nonpretreated rats were selected for radiolabeling
with 11C or 18F and evaluation in vivo.

CB1 Receptor Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5835
Scheme 1. Synthesis of 8, 16, and 20-22a
a Reagents and conditions: (a) 4-aminobenzotrifluoride, ethyl pyruvate, AcOH; (b) concentrated HCl, AcOH; (c) (R)-(+)-1-phenylethylamine, CH2Cl2;
(d) (R)-1-(4-fluorophenyl)ethylamine, CH2Cl2; (e) NaBH3CN, AcOH; (f) pyridinium hydrochloride, heat; (g) Cs2CO3, DMF, fluoromethyl tosylate; (h) Cs2CO3,
DMF, fluoromethyl tosylate-d2; (i) Cs2CO3, DMF, F(CH2)2Br.
The aims of this study were therefore (1) to synthesize 8 and
closely related analogues (16 and 20-22), (2) to determine their
in vitro potencies at CB1 and CB2 receptors, (3) to quantify 8,
16, 20, and 22 in the CB1-rich frontal cerebral cortex of rat
brains at times after injection under baseline and CB1 preblocked
conditions, and (4) to radiolabel the promising candidate
radioligands with 11C or 18F, to make them available for detailed
evaluation in vivo.
a 20:1 mixture of 8 and 15 that favored the desired diastereomer
8. The absolute stereochemical configuration of 8 was confirmed
by single-crystal X-ray crystallography (unpublished data).
Demethylation of 8 in a pyridinium hydrochloride melt gave
the desired phenol 18 in 60% yield accompanied by a small
amount (12%) of the C-3 epimeric product. The 4-fluorophenyl
analogue (16) and the corresponding phenol (19) were prepared
in an analogous manner. Fluoroalkyl analogues 20-22 were
prepared by alkylation of phenol 18 with fluoromethyl tosylate,
fluoromethyl tosylate-d2, and 1-bromo-2-fluoroethane in 53, 43,
and 89% yields, respectively. No epimerization was observed
during the course of these alkylations.
Results and Discussion
Chemistry. Synthesis of the potential PET ligands is outlined
in Scheme 1. The pyrolidinone ring system was assembled in a
single step using the four-component coupling method devel-
oped by Andreichikov et al.31 Reaction of ethyl pyruvate with
a solution of 4-aminobenzotrifluoride and 3-methoxybenzalde-
hyde in acetic acid led to the formation of pyrolidone 9 in 85%
yield. Acid-catalyzed hydrolysis of the enamine using a mixture
of acetic acid and aqueous hydrochloric acid yielded ketolactam
10 in 80% yield. Condensation with (R)-R-methylbenzylamine
provided a diastereomeric mixture of 11 and 12, which was
readily separated by silica gel chromatography. Reduction of
enamine 11 with sodium cyanoborohydride in acetic acid gave
Lipophilicity Calculation. Calculated lipophilicity (cLogD7.4)
values can be useful for predicting the relative ability of ligands
within the same structural class to pass the blood-brain
barrier.18,29 Moderate lipophilicity, in the cLogP range of
2.5-3.5, is usually considered desirable for adequate brain entry
without a high level of nonspecific binding to brain tissue.18,29
Many exceptions to this guideline are, however, known.29
Although [11C]8 has a high cLogD7.4 value (5.7) (Table 1), it
passes the blood-brain barrier very readily, reaching almost
600% of the standardized uptake value (% SUV) within 10-20

5836 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Donohue et al.
Table 1. In Vitro Potency at CB1 and CB2 Receptors, CB1 Selectivity
over CB2 Receptors, and cLogD7.4 Values for 3, 8, 16, 20, and 22
be related to a reduced degree of metabolic hydroxylation caused
by the introduction of the (R)-1-(4-fluorophenyl)ethylamino
moiety. 8 and 20 exhibited similar brain uptake levels over time.
22 exhibited the lowest level of brain uptake over time, probably
because its cLogD7.4 value is higher than those of the other
ligands. Under conditions in which the rats were pretreated with
3 (Figure 3A-D), maximal brain uptake levels in CB1-rich
frontal cortex were reduced to 11.5 ( 1.9, 17.3 ( 2.0, 9.7 (
0.1, and 6.5 ( 1.1 ng/g, respectively, 0.25 h after injection. 8
and 20 were not quantifiable 4 h after injection; 22 could not
be quantified 2 h after injection, and 0.8 ( 0.0 ng/g of 16
remained 8 h after injection. The reduced brain uptake levels
in CB1-rich cerebral cortex under pretreatment conditions
demonstrate that the vast majority of each ligand in brain was
specifically bound to CB1 receptors.
CB1 Kb
CB2 Kb CB1 vs CB2
(nM)a
selectivity cLogD7.4
ligand R1
R2
OCH3
OCH3
OCH2F
(nM)a
b
3
8
6
0.698 ( 0.200 >1977
>2830
769
7.0
5.7
5.7
5.7
5.8
H
F
0.472 ( 0.160 363 ( 87
0.216 ( 0.004 466 ( 136
0.187 ( 0.018 669 ( 137
2160
20
22
H
H
3580
The excellent brain penetration of these ligands and also their
high ratios of CB1 receptor-specific to nonspecific (nonblock-
able) binding in rats, as measured ex vivo with mass spectrom-
etry, strongly suggested that these ligands would be promising
PET radioligands when labeled with a positron-emitter. We
therefore set out to prepare such candidate PET radioligands
for future more detailed evaluation with PET.
Labeling with 11C. [11C]8 and [11C]16 were prepared by
treating the appropriate O-desmethyl precursor (18 and 19,
respectively) with [11C]iodomethane under basic conditions in
an Autoloop apparatus33 (Scheme 2). After reverse phase HPLC,
the final formulated products ([11C]8 and [11C]16, respectively)
were obtained in 2.50 ( 1.15%25 or 16.5% overall decay-
corrected radiochemical yields (RCY), respectively, from
cyclotron-produced [11C]carbon dioxide with high specific
radioactivity (>78.1 or >261 GBq/µmol at EOS for [11C]8 or
[11C]16, respectively) in a radiosynthesis time of 40 min. The
radiochemical purities of [11C]8 and [11C]16 were >95%. [11C]8
and [11C]16 were thus obtained in activities and purities adequate
for further investigation in animal or human subjects.
OCH2CH2F 0.424 ( 0.021 >8260
>19500
a All values represent means ( the standard error of the mean of at least
three determinations. b cLogD7.4 data calculated using Pallas 3.0 for
Windows.
min of being injected into monkey.25 Candidate ligands 16, 20,
and 22 have a cLogD value similar to or slightly above that of
8, suggesting they would have a similar or slightly weaker ability
to penetrate the blood-brain barrier.
% injected activity
brain tissue (g)
% SUV )
× body weight (g)
Ligand Potencies and Selectivities at the CB1 and CB2
Receptors. The in vitro potencies (Kb values) and selectivities
of 8, 16, 20, and 22 for CB1 versus CB2 receptors compare
well with those of other promising PET radioligands (Table 1).
All the new ligands exhibited high potency for CB1 receptors
in vitro and were >769-fold selective for binding to CB1 over
CB2 receptors. These data showed that all four ligands have
adequate potency and selectivity to be considered for develop-
ment as PET radioligands.
Labeling with 18F. [18F]20-22 were prepared by reaction
of the O-desmethyl precursor (18) with the appropriate [18F]flu-
oroalkyl halide under basic conditions in a modified version of
the automated TRACERlab FXF-N module.34 The RCY of an
isolated labeling agent is an important factor affecting the overall
RCY of a formulated radiopharmaceutical. We therefore
compared radiochemical yields for two methods for preparing
[18F]fluoromethyl bromide as a labeling agent in the radiosyn-
thesis of [18F]20 (Scheme 3). The first method was based on
nucleophilic displacement in dibromomethane with the [18F]flu-
oride ion-K2.2.2-K+ complex in acetonitrile. This method is
well-established35 and has been shown to be useful for the
routine production of radiopharmaceuticals, such as [18F]-
SPA-RQ34,36 and [18F]FMeNER-d2.27 However, there is one
major disadvantage with this method, namely the difficult
separation of the generated labeling agent (BrCH218F or
BrCD218F) from the starting dibromomethane. Unseparated
dibromomethane can act as a competing electrophile in the
product labeling reaction and also lead to difficult separation
of the labeled compound. To circumvent this problem, gas
chromatography has been used to isolate only the desired
[18F]fluoromethyl bromide.37 Alternatively, the labeling agent
can be purified by being passed over a series of four silica plus
SepPak columns, which can be troublesome.35 With the latter
method, we achieved a RCY of 27.5 ( 4.5% (n ) 57) for
isolated [18F]fluoromethyl bromide.34 We also explored the
alternative use of bromomethyl tosylate as a more attractive
precursor for generating the [18F]fluoromethyl bromide (Scheme
3). This nonvolatile precursor obviates the need for GC or
Ex Vivo Experiments. The coupling of single (LC-MS) or
triple (LC-MS/MS) quad mass spectral detectors with liquid
chromatography has produced analytical instrumentation with
a sufficiently high sensitivity and selectivity to be a useful tool
for quantifying the brain distribution of trace doses of new
candidate drugs and candidate radioligands in rats ex vivo over
time. This technique, although not yet widely applied, has been
reported with rat brain receptor occupancy experiments targeting
the dopamine-2 (D2), serotonin-2A (5-HT2A), and neurokinin-1
(NK1) receptors.30,32 This approach to assessing the potential
of new ligands for development as PET radioligands is attractive;
useful definitive information about ligand brain entry and
receptor-specific signal versus nonspecific signal can be gained
without first having to label the ligand with either a ꢀ-emitter
3
(e.g., H) or positron emitter (e.g., 11C or 18F).
In this study, we applied the mass spectrometry technique to
quantify levels of administered 8, 16, 20, and 22 (each at 30
µg/kg, iv) in the frontal cerebral cortex of rat brains under
baseline conditions and also under conditions in which the rats
were pretreated with a selective CB1 receptor inverse agonist
(3; 10 mg/kg, iv) 15 min before injection of ligand (Figure
3A-D). Under baseline conditions, each ligand exhibited a high
level of brain tissue uptake and reached concentrations of 39.0
( 5.0 ng/g (at 0.25 h), 48.0 ( 2.8 ng/g (at 0.5 h), 39.3 ( 8.1
ng/g (at 0.5 h) and 22.0 ( 2.6 ng/g (at 0.25 h) for 8, 16, 20,
and 22, respectively. The levels of uptake then were reduced
to 8.5 ( 0.6, 10.5 ( 0.0, 8.2 ( 0.5, and 0.7 ( 0.1 ng/g 8 h
after injection for 8, 16, 20, and 22 respectively. Ligand 16
exhibited the highest level of brain tissue uptake, which may

CB1 Receptor Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5837
Figure 3. Time course evaluation in the frontal cerebral cortex of male Sprague-Dawley rats after administration (30 µg/kg, iv) of 8 (A), 16 (B),
20 (C), and 22 (D) under baseline conditions and after pretreatment with 3 (10 mg/kg, iv) 15 min before ligand injection: (light stippling) baseline
and (dark stippling) pretreatment.
Scheme 2. Radiosynthesis of [11C]8 and [11C]16a
Scheme 3. Radiosynthesis of [18F]20-22a
a Reagents, conditions, and yields: (a) “loop”, precursor (18 or 19), DMF,
0.5 M (n-Bu)4NOH in MeOH, [11C]MeI, room temperature, ∼3 min, RCY
) 2.5 ( 1.1% (8, n ) 57) and 16.5% ([11C]16, n ) 2) decay-corrected
from [11C]carbon dioxide.
SepPak chromatography, as this precursor does not cotransfer
during isolation of the labeling agent. After optimization of this
method, we achieved a maximal RCY of 14.4% for isolated
[18F]fluoromethyl bromide, a yield inferior to that from the first
method. Therefore, we routinely produced [18F]fluoromethyl
bromide from dibromomethane with SepPak isolation. Reaction
of 18 with [18F]fluoromethyl bromide under basic conditions
in DMF for 10 min at 110 °C gave [18F]20 in 5.92 ( 1.34% (n
) 3) isolated (formulated) RCY with high radiochemical purity
(>95%) and high specific radioactivity (>57 GBq/µmol at
EOS). The radiosynthesis time was ∼120 min.
a Reagents, conditions, and yields: (a) [18F]fluoride ion-K 2.2.2-K+
complex, solvent [MeCN for CH2Br2 and CD2Br2 and o-DCB for bro-
mo(m)ethyl tosylate], ∆; (b) precursor (18), Cs2CO3, DMF, ∆; (c) precursor
(18), DMF, (n-Bu)4NOH in MeOH, decay-corrected RCYs of 5.92 ( 1.34%
([18F]20, n ) 3), 7.93 ( 2.48% ([18F]21, n ) 6), and 7.92 ( 2.16% ([18F]22,
n ) 6) from [18F]fluoride ion.
[18F]21 was prepared in the same way as [18F]22, but with
[18F]fluoromethyl bromide-d2 as the isolated labeling agent
(Scheme 3), and was obtained in 7.93 ( 2.48% (n ) 6) RCY,
in >95% radiochemical purity, and with high specific radioac-
tivity (>68 GBq/µmol at EOS).
methyl bromide under basic conditions in DMF for 5 min at
110 °C gave formulated [18F]22 in 7.92 ( 2.16% (n ) 6) RCY,
in high radiochemical purity (>95%), and with high specific
radioactivity (>86 GBq/µmol at EOS). Overall, the radiosyn-
thesis time was ∼about 120 min.
The radiosynthesis of [18F]22 used [18F]-2-fluoroethyl bro-
mide as the isolated labeling agent. [18F]-2-Fluoroethyl bromide
was itself generated from 2-bromoethyl tosylate as described
previously (Scheme 3).38 Reaction of 18 with [18F]-2-fluoro-

5838 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Donohue et al.
5-(3-Methoxyphenyl)-1-(4-trifluoromethylphenyl)-3-(4-trifluo-
romethylphenylamino)-1,5-dihydropyrrol-2-one (9). 4-Ami-
nobenzotrifluoride (34.0 mL, 270.7 mmol) was added to a solution
of 3-methoxybenzaldehyde (11.0 mL, 90.2 mmol) in glacial acetic
acid (80 mL). Ethyl pyruvate (9.9 mL, 90.2 mmol) was added and
the mixture stirred at ambient temperature for 18 h. The precipitate
was filtered and washed with a mixture of 20% MTBE in heptane
and dried under vacuum to afford 9 (44.4 g, 85%) as an off-white
powder: mp 177-179 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.70
(s, 1H), 7.86 (d, 2H, J ) 8.4 Hz), 7.68 (d, 2H, J ) 8.8 Hz), 7.54
(d, 2H, J ) 8.4 Hz), 7.44 (d, 2H, J ) 8.8 Hz), 7.19 (dd, 1H, J )
7.8, 7.8 Hz), 6.88 (d, 1H, J ) 1.8 Hz), 6.78 (dd, 2H, J ) 7.9, 1.8
Hz), 6.59 (d, 1H, J ) 2.6 Hz), 6.14 (d, 1H, J ) 2.2 Hz), 3.67 (s,
3H); LC-MS ESI m/z 491 (M - H)-; tR ) 5.44 min, method 1.
5-(3-Methoxyphenyl)-1-(4-trifluoromethylphenyl)pyrrolidine-
2,3-dione (10). A slurry of 9 (89.7 g, 182 mmol), glacial acetic
acid (400 mL) and concentrated HCl. (500 mL) was stirred at
ambient temperature for 22 h. The heterogeneous mixture was
heated to 60 °C for 1 h. The mixture was poured onto ice (1 L),
was stirred, and stood for 1 h. The precipitate was filtered, washed
with water, and dried under vacuum to afford a solid. The solid
still contained starting material. The solid was slurried with glacial
acetic acid (500 mL) and concentrated HCl. (500 mL) and stirred
at ambient temperature for 22 h. The mixture was poured onto ice
and water (2 L), was stirred, and stood for 1 h. The solid was
filtered, washed with water, and dried under vacuum to afford 10
Conclusions. Ligands 8, 16, and 20-22 were synthesized
efficiently. Each ligand exhibited high potency (Kb values) in
vitro. Ex vivo experiments in rats using LC-MS and LC-MS/
MS showed that 8, 16, 20, and 22 (and hence 21) adequately
pass the blood-brain barrier after low-dose administration and
bind with high specificity to CB1 receptors in rat brain tissue.
Radiosynthesis of [11C]8, [11C]16, and [18F]20-22 gave ad-
equate decay-corrected RCYs and purities to allow their further
evaluation as promising radioligands with PET imaging. Our
evaluation of [11C]8 in monkey has already been reported,
showing this to be a highly promising PET radioligand.25
Evaluations of the other radioligands will be reported elsewhere.
Both [11C]8 and [18F]21 are being or will be studied in human
subjects with PET under obtained exploratory INDs.
Experimental Section
Materials. All chemicals were purchased from commercial
sources and used as received. The chemicals and solvents were of
ACS or HPLC quality. 3 was synthesized at Eli Lilly and Co.
Fluoromethyl tosylate was prepared at Eli Lilly and Co. as
previously reported.39 Fluoromethyl tosylate-d2 was prepared at
SYNCOM AB (Groningen, Netherlands) using the same procedure,
but substituting dideutero-diiodomethane for diiodomethane. Bro-
mo(m)ethyl tosylate was synthesized at the National Institute of
Mental Health.40
1
(50.8 g, 80%). H NMR showed the material to be a mixture of
enol-keto tautomers: mp 134-144 °C; 1H NMR (400 MHz, DMSO-
d6) δ 10.19 (s, 1H, enol), 7.80 (d, 2H, J ) 8.8 Hz, enol), 7.75 (d,
2H, J ) 8.7 Hz, keto), 7.70 (d, 2H, J ) 8.7 Hz, keto), 7.63 (d, 2H,
J ) 8.8 Hz, enol), 7.19-7.14 (m, 1H enol, 1H keto), 7.02 (dd, 1H,
J ) 2.0, 2.0 Hz, keto), 6.93 (d, 1H, J ) 7.9 Hz, keto), 6.81 (dd,
1H, J ) 2.2, 2.2 Hz, enol), 6.77-6.71 (m, 2H enol, 1H keto), 5.97
(d, 1H, J ) 2.6 Hz, enol), 5.91 (d, 1H, J ) 2.6 Hz, enol), 5.73 (dd,
1H, J ) 7.5, 4.0 Hz, keto), 3.66 (s, 3H, enol), 3.65 (s, 3H, keto),
3.35 (dd, 1H, J ) 19.3, 7.5 Hz, keto), 2.59 (dd, 1H, J ) 19.3, 4.0
Hz, keto); LC-MS ESI m/z 349.8 (M + H)+, 347.8 (M - H)-; tR
) 3.68 min, method 1.
Methods. Column chromatography was performed on silica gel
columns (35-65 µm; Isco Inc., Lincoln, NE). 1H NMR spectra (400
MHz) were recorded on an INOVA spectrometer (Varian, Palo Alto,
1
CA). H NMR (500 MHz) and 13C NMR (125 MHz) spectra were
acquired on a Varian System 500 equipped with a 5 mm direct cold
probe, and the sample temperature was maintained at 25 °C. Ab-
breviations s, d, dd, ddd, m, and br denote singlet, doublet, double
doublet, double double doublet, multiplet, and broad, respectively.
Melting points (mp) were determined on a Thomas-Hoover capillary
melting point apparatus and are uncorrected. Elemental analyses were
acquired from Midwest Microlabs LLC (Indianapolis, IN). Mass
detection was performed with an 1100 Series LC-MSD single
quadrupole spectrometer (Agilent, Santa Clara, CA) with an ESI
interface. Samples were analyzed by method 1 or 2. In method 1, LC-
MS analyses were performed with a heated (50 ( 10 °C) reverse phase
column (Gemini, C18, 2.0 mm × 50 mm, 3.0 µm; Phenomenex,
Torrance, CA) eluted at 1 mL/min with a gradient of MeCN (A) and
MeCN with 0.1% HCO2H (B), with A increased linearly from 5 to
100% (v/v) over 7 min and then held for 1 min. In method 2, LC-MS
analyses were performed with a heated (50 ( 10 °C) reverse phase
column (Xterra C18, 2.1 mm × 50 mm, 3.5 µm; Waters Corp.,
Milford, MA) eluted at 1 mL/min with a gradient of MeCN (A) and
MeOH with 0.2% aqueous HCO2NH4 (B), with A increased linearly
from 5 to 100% (v/v) over 7 min and then held for 1 min. After
electrospray ionization of the eluted test sample, ions between m/z 150
and 750 were captured. High-resolution mass spectrometry (HRMS)
was performed with a Thermo LTQFT instrument (Thermo Electron
Corp., San Jose, CA). Data were acquired in FTMS +ESI mode with
a data range of 98-980 amu. The sample (10 µL) was injected in
loop mode. The solvent was methanol with 0.1% formic acid.
γ-Radioactivity from 11C and 18F was measured with a dose
calibrator (Atomlab 300, Biodex Medical Systems) calibrated with
137Cs and 57Co sources. Low levels of radioactivity (<40 kBq)
were measured with a well type γ-counter (model 1080 Wizard,
Perkin-Elmer) having an electronic window set between 360 and
1800 keV.
Specific radioactivities (gigabecquerels per micromole) were
determined with analytical HPLC methods, described later, cali-
brated for absorbance (λ ) 254 nm) response per mass of ligand.
The radioactivity of the radioligand peak (decay-corrected) (giga-
becquerels) was divided by the mass of the associated carrier peak
(micromoles).
(R)-5-(3-Methoxyphenyl)-3-[(R)-1-phenylethylamino]-1-(4-trif-
luoromethylphenyl)-1,5-dihydropyrrol-2-one (11) and (S)-5-(3-Me-
thoxyphenyl)-3-[(R)-1-phenylethylamino]-1-(4-trifluoromethylphenyl)-
1,5-dihydropyrrol-2-one (12). (R)-(+)-1-Phenylethylamine (22.4
mL, 176 mmol) was added to a solution of 10 (30.8 g, 88.2 mmol)
in CH2Cl2 (225 mL). The solution was stirred at ambient temper-
ature for 18 h. The solution was then poured onto a silica gel
column, and the CH2Cl2 was evaporated off with a stream of
nitrogen. The material was purified by silica gel chromatography
(5-15% EtOAc/hexanes) to afford 12, the first eluting isomer, as
a white foam (9.6 g, 24%) and 11, the second eluting isomer, as a
yellow foam (6.7 g, 17%). A mixture of 11 and 12 (11.5 g, 29%)
was also obtained. 11: 1H NMR (400 MHz, DMSO-d6) δ 7.77 (d,
2H, J ) 8.4 Hz), 7.61 (d, 2H, J ) 8.8 Hz), 7.33 (d, 2H, J ) 7.0
Hz), 7.23 (dd, 2H, J ) 7.6, 7.6 Hz), 7.15-7.10 (m, 1H), 7.03 (dd,
1H, J ) 7.9, 7.9 Hz), 6.64 (ddd, 1H, J ) 8.4, 2.6, 0.9 Hz), 6.57 (s,
1H), 6.53 (d, 1H, J ) 7.9 Hz), 5.84-5.80 (m, 2H), 5.15 (d, 1H, J
) 2.6 Hz), 4.34-4.25 (m, 1H), 3.56 (s, 3H), 1.42 (d, 3H, J ) 6.6
Hz); LC-MS ESI m/z 453 (M + H)+; tR ) 6.06 min, method 2.
1
12: H NMR (400 MHz, DMSO-d6) δ 7.75 (d, 2H, J ) 8.8 Hz),
7.61 (d, 2H, J ) 8.8 Hz), 7.35 (d, 2H, J ) 7.5 Hz), 7.28 (dd, 2H,
J ) 7.4, 7.4 Hz), 7.21-7.11 (m, 2H), 6.75-6.70 (m, 2H), 6.65 (d,
1H, J ) 7.5 Hz), 5.92 (d, 1H, J ) 7.5 Hz), 5.76 (d, 1H, J ) 2.6
Hz), 5.15 (d, 1H, J ) 2.6 Hz), 4.28-4.19 (m, 1H), 3.65 (s, 3H),
1.40 (d, 3H, J ) 7.0 Hz); LC-MS ESI m/z 453 (M + H)+; tR
6.16 min, method 2.
)
(R)-3-[(R)-1-(4-Fluorophenyl)ethylamino]-5-(3-methoxyphe-
nyl)-1-(4-trifluoromethylphenyl)-1,5-dihydropyrrol-2-one (13)
and (S)-3-[(R)-1-(4-Fluorophenyl)ethylamino]-5-(3-methoxyphe-
nyl)-1-(4-trifluoromethylphenyl)-1,5-dihydropyrrol-2-one (14).
A slurry of 10 (6.6 g, 18.9 mmol) in CH2Cl2 (40 mL) was added
to a solution of (R)-1-(4-fluorophenyl)ethylamine (5.24 g, 37.6
mmol) in CH2Cl2 (20 mL). The homogeneous solution was stirred

CB1 Receptor Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5839
at ambient temperature for 18 h. The solution was then poured onto
a silica gel column, and the CH2Cl2 was evaporated off with a
stream of nitrogen. The material was purified by silica gel
chromatography (5-20% EtOAc/hexanes) to afford 14, the first
eluting isomer, as a white foam (3.34 g, 38%) and 13, the second
) 241.9 Hz), 159.3, 142.4, 142.1, 141.3, 129.8, 128.6 (2C, d, J )
7.9 Hz), 125.3 (2C, q, J ) 3.9 Hz), 124.5 (q, J ) 32.0 Hz), 124.1
(q, J ) 271.4 Hz), 122.9 (2C), 118.8, 114.9 (2C, d, J ) 21.1 Hz),
112.7, 112.6, 58.6, 56.8, 55.3, 54.9, 38.6, 24.6; LC-MS ESI m/z
473 (M + H)+; tR ) 5.82 min, method 2; HRMS-FT (m/z) [M +
H]+ calcd for C26H25F4N2O2 473.1847, found 473.1842. Anal.
(C26H24F4N2O2) C, H, N.
1
eluting isomer, as a white foam (3.26 g, 37%). 13: H NMR (400
MHz, DMSO-d6) δ 7.75 (d, 2H, J ) 8.4 Hz), 7.59 (d, 2H, J ) 8.8
Hz), 7.36 (dd, 2H, J ) 8.8, 5.7 Hz), 7.06-6.99 (m, 3H), 6.64 (dd,
1H, J ) 8.1, 2.4 Hz), 6.55-6.53 (m, 1H), 6.51 (d, 1H, J ) 7.9 Hz)
5.87 (d, 1H, J ) 7.5 Hz), 5.80 (d, 1H, J ) 2.6 Hz), 5.16 (d, 1H,
J ) 2.2 Hz), 4.34-4.25 (m, 1H), 3.56 (s, 3H), 1.40 (d, 3H, J )
6.6 Hz); LC-MS ESI m/z 471 (M + H)+; tR ) 6.53 min, method
2. 14: 1H NMR (400 MHz, DMSO-d6) δ 7.74 (d, 2H, J ) 8.4 Hz),
7.59 (d, 2H, J ) 8.8 Hz), 7.38 (dd, 2H, J ) 8.4, 5.7 Hz), 7.15-7.05
(m, 3H), 6.73-6.69 (m, 2H), 6.63 (d, 1H, J ) 7.9 Hz), 5.95 (d,
1H, J ) 7.5 Hz), 5.76 (d, 1H, J ) 2.6 Hz), 5.16 (d, 1H, J ) 2.6
Hz), 4.28-4.20 (m, 1H), 3.63 (s, 3H), 1.37 (d, 3H, J ) 6.6 Hz);
LC-MS ESI m/z 471 (M + H)+; tR ) 6.62 min, method 2.
(3R,5R)-5-(3-Methoxyphenyl)-3-[(R)-1-phenylethylamino]-1-
(4-trifluoromethylphenyl)-pyrrolidin-2-one (8) and (3S,5R)-5-
(3-Methoxyphenyl)-3-[(R)-1-phenylethylamino]-1-(4-trifluorom-
ethylphenyl)pyrrolidin-2-one (15). Sodium cyanoborohydride (780
mg, 12.4 mmol) was added to a solution of 11 (2.8 g, 6.19 mmol)
in glacial acetic acid (31 mL). The reaction mixture was stirred at
ambient temperature for 1 h and concentrated in vacuo. The residue
was dissolved in EtOAc and washed with a saturated NaHCO3
solution, water, and brine, dried (Na2SO4), and concentrated in
vacuo. The material was purified by silica gel chromatography
(10-30% EtOAc/hexanes) to afford 8, the first eluting isomer, as
a white solid (2.26 g, 80%) and 15, the second eluting isomer, as
a clear colorless oil (120 mg, 4.3%). 8: mp 126-127.5 °C; 1H NMR
(400 MHz, DMSO-d6) δ 7.57 (d, 2H, J ) 8.8 Hz), 7.50 (d, 2H, J
) 8.8 Hz), 7.34 (dd, 2H, J ) 8.4, 1.3 Hz), 7.27 (dd, 2H, J ) 7.5,
7.5 Hz), 7.20-7.15 (m, 1H), 7.12 (dd, 1H, J ) 7.9, 7.9 Hz), 6.79
(dd, 1H, J ) 2.0, 2.0 Hz), 6.76 (d, 1H, J ) 7.9 Hz), 6.69 (ddd, 1H,
J ) 8.4, 2.6, 0.9 Hz), 5.16 (dd, 1H, J ) 9.2, 6.6 Hz), 4.33-4.26
(m, 1H), 3.63 (s, 3H), 3.45-3.36 (m, 1H), 2.72-2.67 (m, 1H),
2.37 (ddd, 1H, J ) 13.5, 6.9, 5.6 Hz), 1.49 (ddd, 1H, J ) 16.3,
6.8, 5.5 Hz), 1.28 (d, 3H, J ) 6.6 Hz); LC-MS ESI m/z 455 (M +
H)+; tR ) 2.81 min, method 1; 13C NMR (125 MHz, DMSO-d6)
δ 175.3, 159.3, 146.0, 142.5, 141.3, 129.8, 128.2 (2C), 126.8 (2C),
126.7, 125.3 (2C, q, J ) 3.8 Hz), 124.5 (q, J ) 32.0 Hz), 124.1 (q,
J ) 271.3 Hz), 122.9 (2C), 118.8, 112.7, 112.6, 58.6, 57.0, 56.1,
(3R,5R)-5-(3-Hydroxyphenyl)-3-[(R)-1-phenylethylamino]-1-
(4-trifluoromethylphenyl)pyrrolidin-2-one (18). A mixture of 8
(1.03 g, 2.27 mmol) and pyridinium hydrochloride (20 g) was heated
in a 185 °C oil bath for 2 h under a N2 atmosphere. The reaction
mixture was cooled to ambient temperature. The reaction mixture
was dissolved in water, and the precipitate was filtered and the
filtrate saved. The precipitate was dissolved in EtOAc, and a
concentrated NH4OH. solution was added until the mixture was
basic. The layers were separated, and the organic portion was
washed with water and brine, dried (Na2SO4), and concentrated in
vacuo to give an oil (660 mg). A concentrated NH4OH solution
was added to the filtrate until it was basic. The basic solution was
extracted with EtOAc. The organic extracts were washed with water
and brine, dried (Na2SO4), and concentrated in vacuo give a foam
(430 mg). The oil and the foam were combined and purified by
silica gel chromatography (0-25% MTBE/CH2Cl2) to yield 18 (600
mg, 60%) as a white foam: 1H NMR (400 MHz, DMSO-d6) δ 9.31
(s, 1H), 7.57 (d, 2H, J ) 8.8 Hz), 7.47 (d, 2H, J ) 8.4 Hz), 7.32
(dd, 2H, J ) 8.1, 1.5 Hz), 7.25 (dd, 2H, J ) 7.4, 7.4 Hz), 7.18-7.13
(m, 1H), 6.98 (dd, 1H, J ) 7.9, 7.9 Hz), 6.59 (d, 1H, J ) 7.9 Hz),
6.54 (dd, 1H, J ) 2.0, 2.0 Hz), 6.50 (ddd, 1H, J ) 8.0, 2.5, 1.0
Hz), 5.08 (dd, 1H, J ) 9.2, 6.6 Hz), 4.28-4.21 (m, 1H), 3.42-3.35
(m, 1H), 2.65 (br s, 1H), 2.30 (ddd, 1H, J ) 13.5, 7.1, 5.4 Hz),
1.42 (dd, 1H, J ) 21.8, 10.8 Hz), 1.26 (d, 3H, J ) 6.6 Hz); LC-
MS ESI m/z 441 (M + H)+; tR ) 4.66 min, method 2.
(3R,5R)-3-[(R)-1-(4-Fluorophenyl)ethylamino]-5-(3-hydrox-
yphenyl)-1-(4-trifluoromethylphenyl)pyrrolidin-2-one (19). A
mixture of 16 (1.22 g, 2.58 mmol) and pyridinium hydrochloride
(24 g) was heated in a 185 °C oil bath for 2 h under a N2
atmosphere. The reaction mixture was cooled but while still warm
was dissolved in water. A concentrated NH4OH solution was added
until the mixture was basic. The basic solution was extracted with
EtOAc. The organic extracts were washed with water and brine,
dried (Na2SO4), and concentrated in vacuo. The crude product was
purified by silica gel chromatography (5-15% MTBE/CH2Cl2) to
yield 19. The crude 19 was purified further by silica gel chroma-
tography (0.5-1.5% MeOH/CH2Cl2) to give a white foam (790
54.9, 38.7, 24.6; HRMS-FT (m/z) [M
+
H]+ calcd for
1
C26H26F3N2O2 455.1941, found 455.1936. Anal. (C26H25F3N2O2)
C, H, N.
mg, 67%): H NMR (400 MHz, DMSO-d6) δ 9.34 (s, 1H), 7.58
(d, 2H, J ) 8.8 Hz), 7.49 (d, 2H, J ) 8.4 Hz), 7.37 (dd, 2H, J )
8.4, 5.7 Hz), 7.09 (dd, 2H, J ) 8.8, 8.8 Hz), 7.00 (dd, 1H, J ) 7.8,
7.8 Hz), 6.62 (d, 1H, J ) 7.5 Hz), 6.56 (s, 1H), 6.52 (dd, 1H, J )
7.9, 1.7 Hz), 5.10 (dd, 1H, J ) 9.2, 6.6 Hz), 4.33-4.25 (m, 1H),
3.42-3.34 (m, 1H), 2.74-2.67 (m, 1H), 2.39-2.30 (m, 1H), 1.44
(dd, 1H, J ) 22.0, 10.5 Hz), 1.26 (d, 3H, J ) 6.6 Hz); LC-MS ESI
m/z 459 (M + H)+, 457 (M - H)-; tR ) 2.50 min, method 1.
(3R,5R)-5-(3-Fluoromethoxyphenyl)-3-[(R)-1-phenylethylami-
no]-1-(4-trifluoromethylphenyl)pyrrolidin-2-one (20). Cesium
carbonate (1.19 g, 3.6 mmol) was added to a solution of 18 (267
mg, 0.61 mmol) in DMF (4 mL). A solution of fluoromethyl tosylate
(149 mg, 0.73 mmol) in DMF (2 mL) was added, and the reaction
mixture was stirred at ambient temperature for 26 h. The mixture
was diluted with water and extracted with EtOAc. The EtOAc
extracts were washed with water and brine, dried (Na2SO4), and
concentrated in vacuo. The crude material was purified by silica
gel chromatography (0-25% EtOAc/hexanes) to afford the 20 as
a white solid (153 mg, 53%): mp 116-118 °C; 1H NMR (400 MHz,
DMSO-d6) δ 7.56 (d, 2H, J ) 8.8 Hz), 7.48 (d, 2H, J ) 8.8 Hz),
7.32 (d, 2H, J ) 7.0 Hz), 7.26 (dd, 2H, J ) 7.5, 7.5 Hz), 7.21-7.14
(m, 2H), 6.97-6.91 (m, 2H), 6.85 (dd, 1H, J ) 8.4, 2.2 Hz), 5.79
(dd, 1H, J ) 16.9, 3.3 Hz), 5.66 (dd, 1H, J ) 16.7, 3.4 Hz), 5.19
(dd, 1H, J ) 9.2, 6.6 Hz), 4.33-4.25 (m, 1H), 3.43-3.35 (m, 1H),
2.69 (br s, 1H), 2.38 (ddd, 1H, J ) 13.6, 6.8, 5.5 Hz), 1.48 (dd,
1H, J ) 21.8, 10.8 Hz), 1.26 (d, 3H, J ) 6.6 Hz); 13C NMR (125
MHz, DMSO-d6) δ 175.3, 156.1, 145.9, 143.0, 141.2, 130.1, 128.2
15: 1H NMR (400 MHz, DMSO-d6) δ 7.74 (d, 2H, J ) 8.8 Hz),
7.64 (d, 2H, J ) 8.8 Hz), 7.32 (d, 2H, J ) 7.5 Hz), 7.27 (dd, 2H,
J ) 7.5, 7.5 Hz), 7.20-7.11 (m, 2H), 6.73 (dd, 1H, J ) 8.2, 2.1
Hz), 6.69 (s, 1H), 6.63 (d, 1H, J ) 7.9 Hz), 5.52 (d, 1H, J ) 7.5
Hz), 3.86-3.79 (m, 1H), 3.63 (s, 3H), 3.43-3.37 (m, 1H), 2.56
(br s, 1H), 2.43-2.34 (m, 1H), 2.19-2.11 (m, 1H), 1.27 (d, 3H, J
) 7 0.0 Hz); LC-MS ESI m/z 455 (M + H)+; tR ) 3.02 min,
method 1.
(3R,5R)-3-[(R)-1-(4-Fluorophenyl)ethylamino]-5-(3-methox-
yphenyl)-1-(4-trifluoromethylphenyl)pyrrolidin-2-one (16). So-
dium cyanoborohydride (846 mg, 13.5 mmol) was added to a
solution of 13 (3.16 g, 6.73 mmol) in glacial acetic acid (10 mL)
and CH2Cl2 (10 mL). The reaction mixture was stirred at ambient
temperature for 1 h and concentrated in vacuo. The residue was
partitioned between EtOAc and 1 N NaOH. The organic extract
was washed with brine, dried (Na2SO4), and concentrated in vacuo.
The material was purified by silica gel chromatography (20-30%
EtOAc/hexanes) to afford 16 as a white solid (2.5 g, 79%): mp
1
98-100 °C; H NMR (400 MHz, DMSO-d6) δ 7.56 (d, 2H, J )
8.8 Hz), 7.48 (d, 2H, J ) 8.4 Hz), 7.38-7.33 (m, 2H), 7.13-7.04
(m, 3H), 6.79-6.73 (m, 2H), 6.70-6.66 (m, 1H), 5.15 (dd, 1H, J
) 9.2, 6.6 Hz), 4.31 (q, 1H, J ) 6.6 Hz), 3.61 (s, 3H), 3.37 (dd,
1H, J ) 11.0, 8.4 Hz), 2.72 (br s, 1H), 2.37 (ddd, 1H, J ) 13.6,
7.0, 5.3 Hz), 1.48 (ddd, 1H, J ) 16.3, 6.8, 5.5 Hz), 1.25 (d, 3H, J
) 6.6 Hz); 13C NMR (125 MHz, DMSO-d6) δ 175.3, 161.1 (d, J

5840 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Donohue et al.
(2C), 126.8 (2C), 126.7, 125.4 (2C, q, J ) 3.8 Hz), 124.6 (q, J )
31.7 Hz), 124.1 (q, J ) 271.3 Hz), 123.0 (2C), 121.5, 114.9, 114.7,
100.2 (d, J ) 215.4 Hz), 58.4, 56.9, 56.1, 38.6, 24.6; LC-MS ESI
m/z 473 (M + H)+; tR ) 5.23 min, method 2; HRMS-FT (m/z) [M
+ H]+ calcd for C26H25F4N2O2 473.1847, found 473.1842. Anal.
(C26H24F4N2O2) C, H, N.
Adult male Sprague-Dawley rats (225-250 g; Harlan Sprague-
Dawley, Indianapolis, IN) were used. All studies were performed
in accordance with National Institutes of Health guidelines under
protocols approved by the Animal Care and Use Committee of Eli
Lilly and Co. Rats were pretreated for 15 min with 3 (10 mg/kg,
iv) or its vehicle administered by tail vein injection to groups of
three rats per time point. After the 15 min pretreatment period,
rats received 8, 16, 20, or 22 (30 µg/kg, iv). Rats were sacrificed
at 0.25, 0.5, 1, 2, 4, or 8 h by cervical dislocation. A portion of
each frontal cerebral cortex was dissected, weighed, and placed in
a centrifuge tube on ice. Brain tissues were homogenized using a
cell disrupter in four volumes (w/v) of MeCN containing 0.1%
HCO2H. To extract the ligand (e.g., 8), tissues were centrifuged at
20000g for 14 min. Aliquots of supernatant containing the tracer
were diluted with water to an MeCN content that was lower than
that of the mobile phase and injected with an autosampler onto an
HPLC system employing a Zorbax SB C-18 narrow bore rapid
resolution column (2.1 mm × 50 mm, Agilent Technologies,
Wilmington, DE). Separation was achieved using isocratic condi-
tions, in a mobile phase of 70% MeCN containing 1% HCO2H at
a flow rate of 0.25 mL/min. The amount of eluted 8, 16, 20, or 22
was measured using an Agilent model 1946 single quad mass
spectrometer run in positive mode, fitted with an electrospray ion
source and set to m/z 455, 473, 473, and 487 for each experiment,
respectively. Clearly delineated chromatographic peaks with the
retention time of authentic standards and expected molecular weight
were seen after each injection of sample. Analytes were quantified
on the basis of peak area. Data for 8, 16, 20, and 22 were plotted
with Graphpad Prism version 4.02 (Graphpad, San Diego, CA) and
are presented as nanograms per gram of tissue as means ( the
standard deviation (n ) 3).
(3R,5R)-5-[(3-Fluoromethoxy-d2)phenyl]-3-[(R)-1-phenylethy-
lamino]-1-(4-trifluoromethylphenyl)pyrrolidin-2-one (21). Ce-
sium carbonate (6.42 g, 19.70 mmol) was added to a solution of
18 (1.45 g, 3.29 mmol) in DMF (30 mL). A solution of fluoromethyl
tosylate-d2 (1.02 g, 4.61 mmol) in DMF (10 mL) was added, and
the reaction mixture was stirred at ambient temperature for 26 h.
The mixture was diluted with water and extracted with EtOAc. The
EtOAc extracts were washed with water and brine, dried (Na2SO4),
and concentrated in vacuo. The crude material was purified by silica
gel chromatography [heptane/EtOAc (80:20 to 50:50, v/v)] to afford
the 21 as a white solid (700 mg, 45%): mp 116-118 °C; 1H NMR
(500 MHz, DMSO-d6) δ 7.60 (d, 2H, J ) 8.7 Hz), 7.52 (d, 2H, J
) 8.6 Hz), 7.36 (d, 2H, J ) 7.2 Hz), 7.30 (dd, 2H, J ) 7.5, 7.5
Hz), 7.25-7.18 (m, 2H), 7.00 (s, 1H), 6.97 (d, 1H, J ) 7.8 Hz),
6.89 (dd, 1H, J ) 8.2, 2.3 Hz), 5.23 (dd, 1H, J ) 9.3, 6.6 Hz),
4.36-4.30 (m, 1H), 3.47-3.40 (m, 1H), 2.76-2.70 (m, 1H), 2.42
(ddd, 1H, J ) 13.6, 6.9, 5.6 Hz), 1.52 (dd, 1H, J ) 21.6, 10.8 Hz),
1.30 (d, 3H, J ) 6.6 Hz); 13C NMR (125 MHz, DMSO-d6) δ 175.3,
156.1, 145.9, 143.0, 141.2, 130.1, 128.2 (2C), 126.8 (2C), 126.7,
125.4 (2C, q, J ) 3.8 Hz), 124.6 (q, J ) 31.7 Hz), 124.1 (q, J )
271.3 Hz), 123.0 (2C), 121.5, 114.9, 114.7, 99.6 (d of pentets, J )
213.6, 27.5 Hz), 58.4, 56.9, 56.1, 38.6, 24.6; HRMS-FT (m/z) [M
+ H]+ calcd for C26H23D2F4N2O2 475.1972, found 475.1966. Anal.
(C26H24F4N2O2) C, H, N. (Note that results are based on H because
the instrumentation could not distinguish between H and D.)
(3R,5R)-5-[3-(2-Fluoroethoxy)phenyl]-3-[(R)-1-phenylethy-
lamino]-1-(4-trifluoromethylphenyl)pyrrolidin-2-one (22). Ce-
sium carbonate (426 mg, 1.3 mmol) and 1-bromo-2-fluoroethane
(19 µL, 0.26 mmol) were added to a solution of 18 (96 mg, 0.22
mmol) in DMF (2 mL). The reaction mixture was stirred at ambient
temperature for 22 h. The mixture was diluted with water and
extracted with EtOAc. The EtOAc extracts were washed with water
and brine, dried (Na2SO4), and concentrated in vacuo. The crude
material was purified by silica gel chromatography (0-40% EtOAc/
hexanes) to afford 22 as an oil (94 mg, 89%): 1H NMR (400 MHz,
DMSO-d6) δ 7.56 (d, 2H, J ) 8.8 Hz), 7.48 (d, 2H, J ) 8.4 Hz),
7.32 (dd, 2H, J ) 8.1, 1.5 Hz), 7.25 (dd, 2H, J ) 7.6, 7.6 Hz),
7.18-7.14 (m, 1H), 7.11 (dd, 1H, J ) 7.9, 7.9 Hz), 6.82 (dd, 1H,
J ) 2.0, 2.0 Hz), 6.78 (d, 1H, J ) 7.5 Hz), 6.71 (dd, 1H, J ) 8.3,
2.6 Hz), 5.14 (dd, 1H, J ) 9.4, 6.4 Hz), 4.71-4.67 (m, 1H),
4.59-4.55 (m, 1H), 4.32-4.25 (m, 1H), 4.18-3.99 (m, 2H),
3.43-3.35 (m, 1H), 2.68 (br s, 1H), 2.35 (ddd, 1H, J ) 13.5, 6.9,
5.6 Hz), 1.48 (dd, 1H, J ) 21.7, 10.9 Hz), 1.26 (d, 3H, J ) 6.6
Hz); LC-MS ESI m/z 487 (M + H)+; tR ) 5.16 min, method 2. 22
(94 mg, 0.19 mmol) was dissolved in EtOAc, and HCl (0.97 mL,
0.97 mmol, 1 M in ether) was added. The homogeneous solution
was concentrated to give the hydrochloride salt (103 mg, 100%)
as a yellow solid: mp 137-144 °C; 13C NMR (125 MHz, DMSO-
d6) δ 168.8, 158.3, 141.2, 140.2, 137.0, 129.9, 129.1, 129.0 (2C),
128.1 (2C), 125.6 (q, J ) 32.3 Hz), 125.6 (2C, q, J ) 3.5 Hz),
123.9 (q, J ) 271.7 Hz), 123.6 (2C), 120.0, 113.8, 113.8, 82.0 (d,
J ) 166.6 Hz), 66.9 (d, J ) 19.2 Hz), 59.2, 55.6, 54.0, 33.4, 18.7;
LC-MS ESI m/z 487 (M + H)+; tR ) 5.17 min, method 2; HRMS-
FT (m/z) [M + H]+ calcd for C27H27F4N2O2 487.2003, found
487.1996. Anal. (C27H27ClF4N2O2) C, H, N.
CB1 and CB2 GTPγ35S Potency Assay. The GTPγ35S potency
of 3, 8, 16, 20, and 22 in CHO (Lonza, Walkersville, MD) or Sf9
(PerkinElmer Life Sciences, Boston, MA) cell membranes that
ectopically express the human CB1 or CB2 receptor, respectively,
was measured in a 96-well format with a modified antibody capture
technique as described previously.25,41 The following exceptions
applied to the CB1 CHO GTPγ35S assays: membranes (1 unit/well),
compound, and GTPγ35S (500 pM) were incubated in GTP-binding
assay buffer [20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.5%
fatty acid free bovine serum albumin (Serologicals Corp, Norcross,
GA), and 1 µM GDP (pH 7.4)] at room temperature for 30 min.
Antagonist dose responses were performed in the presence of an
80% efficacious dose of full agonist (methanandamide). A mixture
containing 0.2% Nonidet P40 detergent (Roche, Indianapolis, IN),
anti-Gi antibody (final dilution of 1:362; Covance, Princeton, NJ),
and 1.25 mg of anti-rabbit antibody scintillation proximity assay
beads (GE Healthcare, Piscataway, NJ) was added to all wells.
Assay plates were sealed, vortexed, and incubated for an additional
2 h. Following incubation, plates were centrifuged at 700g for 10
min and counted for 1 min per well (Wallac MicroBeta TriLux
scintillation counter, PerkinElmer). Antagonist Kb values (potency)
were calculated with a modification of the Cheng-Prusoff relation-
ship: Kb ) IC50/(1 + [agonist]/EC50), where IC50 is determined from
a four-parameter fit of displacement curves, [agonist] equals the
EC80 of full agonist, and EC50 is determined from a four-parameter
fit of a full agonist concentration response curve.42 Mean Kb values
were calculated as means of at least three independent determina-
tions ( the standard error of the mean (sem).
Computation of cLogD7.4. cLogD (at pH 7.4) values for 8, 16,
20, and 22 were computed with Pallas 3.0 for Windows (Compu-
Drug, San Francisco, CA).
Ex Vivo Experiments. The time course of uptake of the CB1
receptor ligand into rat brain was studied for 8, 16, 20, and 22.
Ligand levels were measured in the rat frontal cerebral cortex, a
structure which contains a high density of CB1 receptors. 3 (10
mg) was dissolved in 1 mL of vehicle comprised of intralipid
(Sigma-Aldrich Chemical Co., St. Louis, MO) containing 0.2%
AcOH. Ligand 8, 16, 20, or 22 (1 mg) was dissolved in 1 mL of
intralipid containing 1% AcOH, diluted with sterile water to a final
concentration of 30 µg/mL, and administered intravenously to rats
in a volume of 1 mL/kg.
Radionuclide Production. No-carrier-added (NCA) [11C]carbon
dioxide (∼52 GBq) was produced in a target of nitrogen gas (∼225
psi) containing oxygen (1%) via the 14N(p,R)11C reaction induced
by irradiation with a 16 MeV proton beam (45 µA) for 20 min
from a PETtrace cyclotron (GE, Milwaukee, WI). [11C]Iodomethane
was produced within a lead-shielded hot-cell from [11C]carbon
dioxide via reduction to [11C]methane and iodination with a MeI
MicroLab apparatus (GE).

CB1 Receptor Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5841
NCA [18F]fluoride ion was produced with a PETtrace cyclotron
to implement the 18O(p,n)18F reaction on 18O-enriched water (95
at. %, 1.8 mL). Typically, proton bombardments were with 18 MeV
protons at 20 µA for 120 min. Portions of the irradiated water
containing up to 18.5 GBq were used for individual experiments.
Radiosynthesis of [11C]8 and [11C]16. Approximately 3 min
before the end of radionuclide production, the O-desmethyl
precursor 18 or 19 (∼1.5 mg, 3.4 µmol for 18 and 3.3 µmol for
19) dissolved in DMF (80 µL) was treated with tetra-n-butylam-
monium hydroxide in methanol (0.5 M, ∼6 µL). This solution was
then loaded into a loop of stainless steel (internal volume, 2 mL)
of a commercially available radiomethylation apparatus (Bioscan)
housed within a lead-shielded hot-cell. The loop was conditioned
by being flushed with helium (12 mL/min) for ∼3 min.
[11C]Iodomethane was swept into the loop in a stream (12 mL/
min) of helium until radioactivity in the loop was maximized (∼4
min after release of [11C]iodomethane from the MeI Microlab
apparatus). Radiomethylation was allowed to proceed for ∼3 min
at room temperature. Crude [11C]8 or [11C]16 was flushed from
loop the with HPLC mobile phase (2 mL, MeCN/10 mM
HCO2NH4, 67:33, v/v) and purified via HPLC on a Luna C-18
column (10 µM, 10 mm × 250 mm, Phenomenex) eluted with a
mobile phase at 6 mL/min with eluate monitored for radioactivity
and absorbance at 254 nm. [11C]8 (tR ) 12 min) or [11C]16 (tR )
12 min) was collected and concentrated to dryness by rotary
evaporation under reduced pressure and heated at 80 °C, formulated
in sterile physiological saline (0.9%, w/v; 10 mL) containing ethanol
(5% v/v) and Tween 80 (10 mg), and finally filtered through a sterile
filter (0.2 µm pore size, Millex-GV, Millipore) into a sterile,
pathogen free dose vial.
(1 mL) and injected remotely onto on a Luna C-18 column (10
mm × 250 mm, 10 µL) eluted with a MeCN/10 mM aqueous
HCO2NH4 mixture (55:45, v/v) over 40 min at 6 mL/min. Eluate
was monitored for absorbance at 254 nm and radioactivity. The
purified [18F]20, [18F]21, or [18F]22 was collected, diluted in water
(∼85 mL), and passed through a SepPak Plus C-18 cartridge
[preconditioned first with ethanol (10 mL) and then water (10 mL)].
Under TRACERlab FXF-N module control, the product ([18F]20,
[18F]21, or [18F]22) was washed with sterile water (5 mL). The
product ([18F]20, [18F]21, or [18F]22) was eluted from the cartridge
first with ethanol (USP; 9.5 mL) and then with sodium chloride
(0.9% USP; 9 mL), which were then combined in a separate vessel
(20 mL). The mixture was pushed through a sterile filter (0.2 µm
pore size, Millex-GV, Millipore, for [18F]20 or [18F]21; 0.22 µm
pore size, Anatop, for [18F]22) into a sterile dose vial (10 mL).
Specific radioactivities, chemical purities, and radiochemical
purities of [18F]20 or [18F]21 (tR ) 9.5 min) were assayed by HPLC
of a 0.1 mL aliquot of formulated dose on a Luna C-18 column
(4.6 mm × 250 mm) eluted with a MeCN/1% aqueous TFA mixture
(45:55, v/v) at 2 mL/min with eluate monitored for radioactivity
and absorbance at 254 nm. Specific radioactivity, chemical purity,
and radiochemical purity of [18F]22 (tR ) 6.7 min) were assayed
by HPLC of a 0.1 mL aliquot of formulated dose on a Luna C-18
column (4.6 mm × 250 mm) eluted with a MeCN/10 mM aqueous
HCO2NH4 mixture (65:35, v/v) at 2 mL/min with eluate monitored
for radioactivity and absorbance at 254 nm.
Acknowledgment. This research was supported by the
Intramural Research Program of the National Institutes of
Health, specifically the National Institute of Mental Health
(NIMH), and also by a Cooperative Research and Development
Agreement between Lilly Research Laboratories, NIMH, and
the Karolinska Institutet. We are grateful to Ms. Cheryl L. Morse
and Mr. Jinsoo Hong for assistance in radioligand production.
We thank the NIH PET Department for 11C and 18F production.
Specific radioactivity, chemical purity, and radiochemical purity
of [11C]8 and [11C]16 were assessed by HPLC of a 0.1 -mL aliquot
of formulated dose on a Luna C-18 column (4.6 mm × 250 mm,
Phenomenex) eluted with MeCN/0.1% TFA (45:55, v/v) at 2 mL/
min with eluate monitored for radioactivity and absorbance at 235
nM (tR values for [11C]8 and [11C]16 were 5.12 and 6 min,
respectively).
Supporting Information Available: Elemental analyses of target
ligands 8, 16, and 20-22. This material is available free of charge
via the Internet at http://pubs.acs.org.
Synthesis of [18F]Fluoro(m)ethyl Bromide. Cyclotron-produced
[18F]fluoride ion in [18O]water was delivered into a vial containing
Kryptofix 2.2.2 (5.2 mg, 3.6 µmol; Aldrich Chemical Co.) and
potassium carbonate (0.5 mg, 3.6 µmol) in MeCN/H2O (94:4, v/v;
0.1 mL). The [18F]fluoride ion mixture was transferred to a modified
version of the TRACERlab FXF-N module which was then followed
by MeCN (1 mL). The mixture was evaporated to dryness at 90
°C under reduced pressure with a nitrogen flow. MeCN (2 mL)
was again added and then evaporated to dryness. The CH2Br2 (50
µL), CD2Br2 (50 µL), or bromo(m)ethyl tosylate (30 µL) in solvent
[1 mL; MeCN for CH2Br2 and CD2Br2 and o-dichlorobenzene for
bromo(m)ethyl tosylate] was added to the dry [18F]fluoride
ion-K2.2.2-K+ complex which was then heated to 110 °C for 15
min. The reaction vessel was then cooled to 35 °C. Nitrogen gas
(30 mL/min) was used to transfer the volatile [18F]fluoro(m)ethyl
bromide through a series of four silica gel cartridges (SepPak Plus)
for CH2Br2 and CD2Br2 and or one silica gel SepPak Plus for
bromo(m)ethyl tosylate and then swept into a precooled V-vial
(volume, 1 mL) with a crimp-seal silicone-Teflon septum cap.
Radiosynthesis of [18F]20, [18F]21, and [18F]22. A glass re-
action vessel was loaded with 18 (∼0.5 mg, 1.1 µmol), DMF (1
mL), and base [Cs2CO3 (0.5 mg, 1.5 µmol) and 18-crown-6 (5 mg,
19 µmol) for [18F]20 and [18F]21, respectively; (n-Bu)4NOH (0.176
M in MeOH; ∼8 µL) for [18F]22]. [18F]FMeBr, [18F]FMeBr-d2,
or [18F]FEtBr was transferred to a solution under computer control
from the TRACERlab FXF-N module. Radioactivity transfer was
monitored by two external Bioscan detectors and was stopped when
radioactivity in the vessel reached a maximum. The vessel was
heated at 110 °C for 10 min ([18F]20 and [18F]21) or 5 min
([18F]22). The mixture containing [18F]20 or [18F]21 (tR ) 34 min)
was diluted with water (1 mL) and injected remotely onto a Luna
C-18 column (10 mm × 250 mm, 10 µL) eluted with a MeCN/1%
aqueous TFA mixture (35:65, v/v) over 40 min at 6 mL/min. The
fraction containing [18F]22 (tR ) 28 min) was diluted with water
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