Discovery and labeling of high-affinity 3,4-diarylpyrazolines as candidate radioligands for in vivo imaging of cannabinoid subtype-1 (CB1) receptors

Article abstract of DOI:10.1021/jm800329z

Imaging of cannabinoid subtype-1 (CB₁) receptors in vivo with positron emission tomography (PET) is likely to be important for understanding their role in neuropsychiatric disorders and for drug development. Radioligands for imaging with PET are required for this purpose. We synthesized new ligands from a 3,4-diarylpyrazoline platform of which (-)-12a ((-)-3-(4-chlorophenyl)- N′-[(4-cyanophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1- carboxamidine) was found to have high-affinity and selectivity for binding to CBi receptors. (-)-12a and its lower affinity enantiomer ((+)-12a) were labeled with carbon-11 (t_1/2 = 20.4 min) using [¹¹C]cyanide ion as labeling agent and evaluated as PET radioligands in cynomolgus monkeys. After injection of [¹¹C](-)-12a, there was high uptake and retention of radioactivity across brain according to the rank order of CB₁ receptor densities. The distomer, [¹¹C](+)-12a, failed to give a sustained CB₁ receptor-specific distribution. Polar radiometabolites of [¹¹C](-)-12a appeared moderately slowly in plasma. Radioligand [¹¹C](-)-12a is promising for the study of brain CB₁ receptors and merits further investigation in human subjects.

Full text of DOI:10.1021/jm800329z

5608
J. Med. Chem. 2008, 51, 5608–5616
Discovery and Labeling of High-Affinity 3,4-Diarylpyrazolines as Candidate Radioligands for In
Vivo Imaging of Cannabinoid Subtype-1 (CB1) Receptors
Sean R. Donohue,*,†,‡ Victor W. Pike,† Sjoerd J. Finnema,‡ Phong Truong,‡ Jan Andersson,‡ Bala´zs Gulya´s,‡ and
Christer Halldin‡
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
ReceiVed March 22, 2008
Imaging of cannabinoid subtype-1 (CB1) receptors in vivo with positron emission tomography (PET) is
likely to be important for understanding their role in neuropsychiatric disorders and for drug development.
Radioligands for imaging with PET are required for this purpose. We synthesized new ligands from a 3,4-
diarylpyrazoline platform of which (-)-12a ((-)-3-(4-chlorophenyl)-N′-[(4-cyanophenyl)sulfonyl]-4-phenyl-
4,5-dihydro-1H-pyrazole-1-carboxamidine) was found to have high-affinity and selectivity for binding to
CB1 receptors. (-)-12a and its lower affinity enantiomer ((+)-12a) were labeled with carbon-11 (t1/2
)
20.4 min) using [11C]cyanide ion as labeling agent and evaluated as PET radioligands in cynomolgus monkeys.
After injection of [11C](-)-12a, there was high uptake and retention of radioactivity across brain according
to the rank order of CB1 receptor densities. The distomer, [11C](+)-12a, failed to give a sustained CB1
receptor-specific distribution. Polar radiometabolites of [11C](-)-12a appeared moderately slowly in plasma.
Radioligand [11C](-)-12a is promising for the study of brain CB1 receptors and merits further investigation
in human subjects.
Introduction
The psychotropic, analgesic, and healing properties of Can-
nabis satiVa (marijuana) have been known throughout docu-
mented history.1 As for some other plant-derived medications
(e.g., opium), there has been significant abuse of marijuana
mainly because of an accompanying feeling of relaxation and
psychological “high”.2,3 Nevertheless, legitimate medical use
of marijuana may extend to the treatment of chemotherapy-
induced emesis,4,5 appetite stimulation in acquired immune
Figure 1. Structures of CB1 receptor ligands (1-3).
6,7
deficiency syndrome (AIDSa),
caused by multiple sclerosis.8,9
and movement disorders
Efforts to elucidate the biological response to marijuana intake
have identified (-)-(6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-
tetrahydro-6H-benzo[c]chromen-1-ol, 1 (∆9-THC, Figure 1),10,11
as its most abundant active compound. 1 interacts with two main
receptor types, namely cannabinoid subtype-1 (CB1) and can-
nabinoid subtype-2 (CB2) receptors.12,13 Two spliced variants
of the CB1 receptor have also been identified, CB1A and
CB1B.14,15 CB1 receptors are located throughout the body and
have high densities in regions of the brain, such as the
hippocampus, striatum, and basal ganglia.16,17 By contrast, CB2
receptors are located mainly in peripheral tissues and are
associated with the immune system.13,18,19
In 1994, Sanofi-Synthlabo introduced 5-(4-chlorophenyl)-1-
(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-
3-carboxamide, 2 (SR141716A, rimonabant, Figure 1), as a
high-affinity inverse agonist at CB1 receptors.20 2 has recently
gained approval for use in the European Union as a treatment
for morbid obesity. The therapeutic use of 2 may extend to
addiction and neurodegenerative disorders. Consequently, there
has been a considerable effort by pharmaceutical industry to
develop novel CB1 receptor inverse agonist platforms. Solvay
AB succeeded with the development of (4S)-3-(4-chlorophenyl)-
N-methyl-N′-[(4-chlorophenyl)sulfonyl]-4-phenyl-4,5-dihydro-
1H-pyrazole-1-carboxamidine, 3 (SLV319, Figure 1).21-23
Brain CB1 receptors may be involved in several neuropsy-
chiatric disorders. Currently, there is a need for suitable ligands
that are amenable to labeling with positron-emitters for nonin-
* To whom correspondence should be addressed. Phone: 1-301-451-3909.
Fax: 1-301-480-5112. E-mail: donohues@intra.nimh.nih.gov. Address:
Molecular Imaging Branch, National Institute of Mental Health, National
Institutes of Health, Building 10, Rm B3 C342, 10 Center Drive, Bethesda,
MD 20892-1003.
†
Molecular Imaging Branch, National Institute of Mental Health,
National Institutes of Health.
‡
Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry
Section, Karolinska Hospital.
a Abbreviations: ACS, American Chemical Society; AIDS, acquired
immune deficiency syndrome; BSA, bovine serum albumin; CB1, cannab-
inoid subtype-1; CB2, cannabinoid subtype-2; CRADA, Cooperative
Research and Development Agreement; DAT, dopamine transporter; DMSO,
dimethyl sulfoxide; DOR, δ opioid receptor; DTT, dithiothreitol; ee,
enantiomeric excess; EDTA, ethylene diamine tetraacetic acid; fwhm, full-
width at half-maximum; GTPγS, guanosine-5′-(γ-thio)-triphosphate; H,
histamine receptor; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; HPLC, high-performance liquid chromatography; HRMS, high-
resolution mass spectrometry; 5-HT, 5-hydroxytryptamine; K 2.2.2,
4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane; KI, Karolinska
Institutet; KOR, κ opioid receptor; LC-MS, liquid chromatography-mass
spectrometry; MeCN, acetonitrile; MOR, µ opioid receptor; MRI, magnetic
resonance imaging; mp, melting point; MTBE, tert-butyl methyl ether; NET,
norepinephrine transporter; NIH, National Institutes of Health; NIMH,
National Institute of Mental Health; NMR, nuclear magnetic resonance;
PDSP, Psychoactive Drug Screening Program; PET, positron emission
tomography; PipISB, N-(4-fluoro-benzyl)-4-(3-(piperidin-1-yl)-indole-1-
sulfonyl)benzamide; P-gp, permeability-glycoprotein; ppm, parts per million;
SERT, serotonin transporter; SPECT, single-photon emission computed
tomography; SUV, standardized uptake value; TEA, triethylamine; ∆9-THC,
∆9-tetrahydrocannabinol, VOI, volume of interest.
10.1021/jm800329z CCC: $40.75
2008 American Chemical Society
Published on Web 08/28/2008

EValuation of CB1 Receptor Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5609
Figure 2. Structures of [11C]4-6, [18F]6, [18F]7, and [11C]8. Asterisks denote position of radiolabel.
Scheme 1. Synthesis of 3,4-Diarylpyrazoline Derivativesa
a Conditions: (a) methyl chloroformate, TEA, MeCN; (b) 3-(4-chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole, toluene, reflux; (c) chlorobenzene, PCl5;
(d) methanolic NH3; (e) ChiralPak AD, MeCN, 8 mL/min for 12a and 12b and 6 mL/min for 12c.
vasively imaging CB1 receptors in vivo with PET under control
and diseased states. Previous attempts at radioligand develop-
ment have focused on the modification of the 1,5-diarylpyrazole
CB1 receptor class of 2 to allow for labeling with carbon-11
(t1/2 ) 20.4 min), fluorine-18 (t1/2 ) 109.7 min), or iodine-124
(t1/2 ) 4.15 d). Some success has been achieved with this
approach (Figure 2), namely through the development of [11C]4
([11C]JHU75528)24,25 and [11C]5 ([11C]JHU75575).25 Promising
PET radioligands from other structural platforms have recently
been reported (Figure 2) such as [11C]6 ([11C]PipISB),26 [18F]6
([18F]PipISB),26 [18F]7 ([18F]MK-9760),27,28 and [11C]8 ([11C]Me-
PPEP).29
mind, we have synthesized novel analogues of 3 that are
amenable to radiolabeling. The CB1 receptor affinities (Ki
values) and selectivities were determined for the enantiomers
of two ligands that were considered amenable to labeling with
carbon-11 or radioiodine (e.g., iodine-123, t1/2 ) 13.13 h, or
iodine-124). Additionally, one racemic ligand (12a), along with
its eutomer and distomer, were labeled in high-specific radio-
activity with [11C]cyanide ion and investigated in monkeys with
PET imaging. The emergence of radiometabolites in monkey
plasma was measured with HPLC.
Results and Discussion
A 3,4-diarylpyrazoline class of CB1 receptor ligand also
presents favorable physiological and pharmacological attributes
for PET radioligand development. The lead structure (3) shows
high selectivity and potency for CB1 receptors with little to no
substrate behavior for P-glycoprotein (P-gp) efflux pumps.22
Nevertheless, this structural class has remained largely unex-
plored for PET radioligand development. With this purpose in
Chemistry. Ligands (12a-c) were synthesized by modifica-
tions of known general procedures (Scheme 1).22 Briefly, the
appropriate 4-substituted benzenesulfonamides (9a-c) were
treated with methyl chloroformate plus triethylamine in aceto-
nitrile to give the corresponding carbamic acid methyl esters
(10a-c), which were then treated with 3-(4-chlorophenyl)-4,5-
dihydro-4-phenyl-1H-pyrazole in toluene to give 11a-c in good

5610 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Donohue et al.
a
Scheme 2. Radiosynthesis of [11C](()-12a
Table 1. IC50 and Ki Values for the CB1 and CB2 Receptors and ClogP
Data for Ligands 2, (-)-12a, (+)-12a, (-)-12c, and (+)-12c
CB1
CB2
CB1
CB2
ligand R1 IC50 (nM)a IC50 (nM)a Ki (nM)a Ki (nM)a ClogPb
a Conditions, reagents and decay-corrected yield: (a) Pt, NH3 (20-30
mL/min), 990 °C; (b) 50% H2SO4, 90 °C; (c) Pd(PPh3)4, KOH, K2.2.2,
DMSO, 110 °C, 36% (n ) 2).
2
2.2 ( 0.5 4570 ( 410 0.4 ( 0.1 697 ( 63
6.95
3.85
3.85
5.07
5.07
(-)-12a CN
(+)-12a CN
(-)-12c
(+)-12c
2.8 ( 0.3 >33000
100 ( 10 >33000
1.9 ( 0.7 >33000
103.5 ( 9.0 >33000
0.5 ( 0.1 >5000
16.9 ( 2.0 >5000
0.3 ( 0.1 >5000
17.4 ( 2.0 >5000
I
I
barrier despite its very high ClogP value (5.01).22 The ClogP
values of 12a and its enantiomers are substantially lower (3.85)
than that of 3 (Table 1), and hence they may be expected to
enter brain readily. The values for 12c and its enantiomers are
similar to 3, and hence they may also be expected to enter brain
adequately.
Radiosynthesis. Initially, we set out to find an effective and
rapid method for labeling 12 as its racemate. [11C]Cyanide ion
is a useful precursor for 11C-labeling molecules with an aryl
nitrile group.32 The incorporation of [11C]cyanide ion into an
aryl ring is best achieved with copper33,34 or palladium32
catalyzed reactions. We considered each method for labeling
(()-12a. At first glance, [11C]Cu(I)CN appears attractive for
labeling PET radiopharmaceuticals. In general, use of this
labeling agent requires one-pot and is insensitive to H2O or NH3
accompanying the production of [11C]HCN. However, the
overall radiochemical yields can be very low (e.g., 2.5%) and
inferior to those from the palladium-catalyzed method.34 Hence,
the latter method was selected for labeling (()-12a.
a Values are represent the mean ( SD of three determinations. b ClogP
values were calculated by using the Pallas 3.0 software (Compudrug, USA).
yields. Treatment of 11a-c with PCl5 in chlorobenzene gave
the crude imino chlorides, which were readily converted with
methanolic NH3 into the target ligands, 12a-c. These ligands
were then resolved into their enantiomers with chiral HPLC.
CB1 and CB2 in Vitro Binding Assays. High affinity is a
prerequisite in candidate radioligands for PET imaging of
neuroreceptors.30 Generally, the more sparse the receptor the
higher the affinity must be to permit successful imaging. As a
guide, binding potentials represented by Bmax/Kd should well
exceed unity, when Bmax and Kd (or as surrogate, Ki or IC50)
are expressed in nM.30 CB1 receptors are among the most
abundant receptors in brain,16,17 and hence moderately high
affinity (Ki < 10 nM) may be acceptable. The IC50 and Ki values
of rimonabant, (-)-12a, (+)-12a, (-)-12c, and (+)-12c, are
shown in Table 1. The (-)-enantiomers exhibited high affinities
for CB1 receptors with IC50 values and Kis in low or sub nM
range, respectively. These values compare well with other
successful radioligands targeting CB1 receptors, which generally
have potencies or affinities in the nM range (e.g., [18F]7 and
[11C]8). The (+)-enantiomers of 12a and 12c exhibited lower
CB1 receptor affinities than their (-)-enantiomers; eudismic
ratios were found to be about 35 for 12a and 56 for 12c. These
ratios are similar to those of similar CB1 receptor ligands from
the 3,4-diarylpyrazoline class.22 The eutomer of one such ligand
has been shown to have the S configuration by X-ray crystal-
lography.22 Hence, the eutomers of 12a and 12c are also
predicted to have S configuration.
[11C]HCN, which itself was prepared from cyclotron-produced
[11C]methane, was trapped in a DMSO solution of KOH and
4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K
2.2.2), yielding a [11C]CN--K+-K 2.2.2 complex, which was then
added to the bromo precursor ((()-12b) and Pd(PPh3)4 in DMSO
and heated (Scheme 2). [11C](()-12a was separated from the crude
product with reverse phase HPLC. The fraction containing [11C](()-
12a was evaporated to dryness and formulated for safe intravenous
injection. The overall radiosynthesis time was about 30 min. The
nonoptimized decay-corrected yield was 36% (n ) 2). There was
no great improvement in yield when using the iodo compound (-)-
12c as precursor. [11C](()-12a was obtained in high radiochemical
purity (>98%) and was free of labeling precursors. Specific
radioactivities were g56 GBq/µmol. Product identity was con-
firmed by liguid chromatography-mass spectrometry (LC-MS) of
associated carrier and by coinjection with 12 in HPLC analysis
and observation of coelution.
Receptor Screening. Ligand (-)-12a showed <50% inhibi-
tion (n ) 4) for the following receptors and binding sites:
5-HT1A-E, 5-HT2B-C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7, R1A,B
,
R
2A-C, ꢀ1-3, D1-5, DAT, DOR, H1,4, KOR, M1-5, MOR, NET,
SERT, σ1,2. Ki values (n ) 3) of >7710 ( 1110 nM for the
5-HT2A and >10000 nM for H2,3 receptors were found. Hence,
(-)-12a was found to have excellent CB1 receptor selectivity
for development as a PET radioligand.
Lipophilicities. The lipophilicity of a radioligand may
critically influence its ability to penetrate the blood-brain
barrier. Generally, a logP value in the range 2.0-3.5 is
considered desirable for adequate brain entry without excessive
nonspecific binding to brain tissue (i.e., fats, proteins).31 ClogP
is a useful tool for predicting lipophilicity trends among
compounds of the same structural class.30,31 ClogP was com-
puted for (-)-12a, (+)-12a, (-)-12c, and (+)-12c (Table 1).
Ligand 3 has previously been shown to penetrate the blood-brain
We attempted to prepare [11C](-)-12a from precursor (-)-
12b under the reaction conditions used to prepare [11C](()-
12a. However, chiral HPLC analyses of the collected radioactive
products revealed that complete racemization had occurred
during the reactions (Table 2). Racemization was likely
promoted by the strong base (KOH plus K 2.2.2) (Scheme 3).
To try to avoid racemization, several weaker bases were used
in place of the KOH plus K 2.2.2. Interestingly, the use of
NaHCO3 as base gave [11C](-)-12a to [11C](+)-12a in a 9:1
ratio. Serendipitously, we found that the use of KH2PO4 (Table
2) gave [11C](-)-12a in >94% ee (n ) 4). These conditions
were also used with (+)-12c as precursor, and the resulting

EValuation of CB1 Receptor Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5611
Table 2. Enantiomer Composition (%) of [11C]12a after Treating
(-)-12b, (-)-12c, or (+)-12c with [11C]Cyanide Ion in DMSO in the
Presence of Various Bases
By contrast with results from [11C](-)-12a, after intravenous
injection of the lower affinity enantiomer, [11C](+)-12a, into
monkeys, maximal brain radioactivity concentration reached
280% SUV at 1.5 min but then rapidly declined in all regions
to about 95% SUV at 90 min (Figure 4B). These features of
the time-activity curves are consistent with a high proportion
of nonspecific binding in all examined regions and are consistent
with the lower affinity of the radioligand.
precursor
base (a)
[
11C](-)-12a (%)
[
11C](+)-12a (%)
(-)-12b
KOH, K2.2.2
NaHCO3
NaOAc
NaHCO3
KH2PO4
50
90
50
50
10
50
(-)-12c
90
10
>97 (n ) 4)
<3 (n ) 1)
<3 (n ) 4)
>97 (n ) 1)
Horizontal PET images obtained at the level of the striatum
from data acquired between 9 and 93 min after injection of
[11C](-)-12a showed a distribution of radioactivity consistent
with a large proportion of specific binding to CB1 receptors,
whereas corresponding images obtained with [11C](+)-12a were
strikingly homogeneous, indicating little receptor-specific bind-
ing (Figure 5).
(+)-12c
KH2PO4
product, [11C](+)-12a, was obtained in >94% ee. The chemical
identities of [11C](-)-12a and [11C](+)-12a were confirmed with
LC-MS of associated carrier. Thus, [11C](-)-12a and [11C](+)-
12a were obtained in high-chiral purity for evaluation as
radioligands in monkeys with PET.
Emergence of Radiometabolites of [11C](-)-12a in Plasma.
Analysis of venous samples showed that after injection of
[11C](-)-12a into cynomolgus monkeys, three less lipophilic
PET Measurements. After intravenous injection of [11C](()-
12a into cynomolgus monkey, the brain radioactivity distributed
according to the rank order of regional CB1 receptor densities
(Figure 3A). The highest radioactivity uptake was in CB1
receptor-rich striatum, reaching 220% SUV at 30 min after
injection. This slowly diminished to 180% SUV at 90 min after
injection. The lowest maximal brain uptake was in pons reaching
150% SUV at 24 min after injection. The concentration of
radioactivity in this region diminished to 124% SUV at 90 min
after injection. In an experiment in which the CB1 receptor-
selective ligand 6 was given in high dose (1 mg/kg, iv) at 25
min after injection of [11C](()-12a, the regional brain radio-
activity became homogeneous and diminished to about 95%
SUV at 90 min after injection (Figure 3B). When 6 (1 mg/kg,
iv) was given at 20 min before injection of [11C](()-12a, brain
radioactivity became homogeneous and was characterized by a
lower maximal uptake and fast washout, reaching 175% SUV
at 15 min after injection and declining to 85% SUV at 90 min
after injection (Figure 3C). These results demonstrated that a
high proportion of brain radioactivity in the baseline experiment
was reversibly bound to CB1 receptors. The higher affinity
enantiomer in this racemic radioligand was expected to be
responsible for the majority of receptor-specific binding, while
the lower affinity enantiomer was expected to bind mostly
nonspecifically. We therefore set out to inject the homochiral
radioligands, [11C](-)-12a and [11C](+)-12a, to test these
expectations.
radiometabolite fractions (tRs ) 2.3, 5.8 and 8 min, cf. tR
)
8.5 min for [11C](-)-12a) emerged in plasma (Figure 6A).
Unchanged radioligand had declined to 50% of radioactivity in
plasma at 45 min after injection (Figure 6B). The presence of
the three radiometabolite fractions slowly increased as a
percentage of total radioactivity in plasma throughout the scan.
In this study, we did not determine the identities of any of these
radiometabolite fractions nor whether they crossed the blood-
brain barrier.
Finally, (-)-12c was not labeled with radioiodine in this
study, but its properties (high affinity, high selectivity, and
lipophilicity) suggest it has potential for development as a
radioligand for imaging brain CB1 receptors, either for PET or
SPECT.
Conclusions
3,4-Diarylpyrazoline CB1 ligands with high-affinity and
selectivity for CB1 receptors were discovered. One racemic
ligand ((()-12a), its eutomer ((-)-12a), and its distomer ((+)-
12a) were successfully labeled with carbon-11 in high specific
radioactivity. [11C](-)-12a was found to be a promising
radioligand for PET receptor imaging and merits further
exploration in humans.
Experimental Section
After intravenous injection of the higher affinity enantiomer,
[11C](-)-12a, into monkeys, the brain radioactivity again
distributed according to the regional rank order of CB1 receptor
densities. The highest uptake of radioactivity was in striatum,
reaching 200% SUV at 48 min after injection. The lowest
maximal brain uptake was in the pons, reaching and maintaining
∼125% SUV from 24 min after injection (Figure 4A). These
time-activity curves are consistent with a high proportion of
receptor-specific binding in all examined brain regions, as also
seen in the experiment with racemic radioligand (Figure 3A).
In these experiments, as in PET imaging studies of other CB1
receptor radioligands,27,29 no region could be identified to
represent nonspecific binding only. Pons does not serve this
purpose because it contains some CB1 receptors,16,17,27 and
measurements of its radioactivity concentration are contaminated
from other nearby regions (e.g., CB1 receptor-rich cerebellum)
through the partial volume effect. In the absence of a reference
region, ratios of specific to nonspecific binding cannot be
estimated at all accurately by visual inspection of time-activity
curves. Bolus plus constant infusion35 or full kinetic compart-
mental model utilizing an arterial input function would be
required to extract this information.29
Materials. All reagents were of ACS or HPLC quality and
purchased from commercial sources and were used as received.
4-Cyanophenylsulfonamide, 4-bromophenylsulfonamide, and 4-io-
dophenylsulfonamide were synthesized by known procedures.36
3-(4-Chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole was also
synthesized as reported.37 6 was provided by Eli Lilly and Co.
1
General Methods. H (400 MHz) and 13C (100 MHz) NMR
spectra were recorded at room temperature on an Avance-400
spectrometer (Brucker, Billerica, MA). Chemical shifts are reported
in δ units (ppm) downfield relative to the chemical shift for
tetramethylsilane. Signals are quoted as s (singlet), d (doublet), dd
(double doublet), dt (double triplet), t (triplet), q (quartet), or m
(multiplet). High-resolution mass spectra (HRMS) were determined
using a time-of-flight electrospray instrument (University of Illinois
at Urbana, Champaign, IL). Melting points (mp) were determined
using a Mel-temp melting point apparatus (Electrothermal, Fisher
Scientific, USA) and were uncorrected. Chiral HPLC, for the
preparative resolution of racemates to enantiomers, was performed
on a chiral column (ChiralPak AD, 20 mm × 250 mm) eluted with
acetonitrile at 6 or 8 mL/min, as later specified. The enantiomeric
excess (ee) of each resolved compound was measured by HPLC
with the same method as used for resolution. Optical rotations
([R]2D2) were measured with a P-1010 polarimeter (JASCO; Easton,

5612 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Donohue et al.
Figure 3. Regional time-radioactivity curves after iv injection of [11C](()-12a in cynomolgus monkey under baseline condition (A), with 6 (1
mg/kg, iv) administered as a displacing agent at 25 min (B), or pretreatment condition with 6 (1 mg/kg, iv) (C). Key: ×, striatum; 4, cerebellum;
], frontal cortex; 0, lateral temporal cortex; +, thalamus; O, medial temporal cortex; 2, pons.
Figure 4. Regional time-radioactivity curves after iv injection of [11C](-)-12a (100 MBq) (A) or [11C](+)-12a (98 MBq) (B) in cynomolgus
monkeys. Key: ×, striatum; 4, cerebellum; ], frontal cortex; 0, lateral temporal cortex; +, thalamus; O, medial temporal cortex; 2, pons.
Figure 5. Horizontal PET images, obtained at the level of the striatum from data acquired between 9 and 93 min after injection of [11C](-)-12a
(100 MBq, (A)) or [11C](+)-12a (98 MBq, (B)).
Scheme 3. Proposed Mechanism for the Epimerization of [11C](-)-12a under Labeling Conditions
MD). Specific rotations were obtained at room temperature. Mass
spectra (MS) were acquired using a LCQDECA LC-MS instrument
(Thermo Finnigan, San Jose, CA) fitted with a reverse phase LC
column (Luna, C18; 5 µm, 2 mm × 150 mm; Phenomenex).
Radiosyntheses were performed in a custom-made remotely con-
trolled apparatus.38 Radioligand separations were performed with
HPLC on a reverse phase column (µ-Bondapak C-18; 7.8 mm ×
300 mm, 10 µm; Waters). The column outlet was connected to an
absorbance detector (λ ) 254 nm) in series with a GM-tube for
radiation detection. [11C](()-12a, [11C](-)-12a, and [11C](+)-12a
were purified in this system using MeCN-0.01 M H3PO4 (55: 45,
v/v) as mobile phase at 6 mL/min. The radiochemical purities and
specific radioactivities of each product were determined with reverse
phase HPLC on a µ-Bondapak C-18 column (3.9 mm × 300 mm,

EValuation of CB1 Receptor Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5613
Figure 6. Radio-HPLC of plasma of [11C](-)-12a in cynomolgus monkeys at 15 min after injection (A), and time course of radioactivity in
plasma represented by parent radioligand and radiometabolite fractions (B). Key: 0, [11C](-)-12a; 4, metabolite a; 3, metabolite b; O, metabolite
c.
10 µm; Waters) eluted at 3 mL/min with MeCN-H3PO4 (0.01 M;
55: 45 v/v) as mobile phase. Eluate was monitored with an
absorbance detector (λ ) 254 nm) in series with a ꢀ-flow detector
(Beckman) for radiation detection. The enantiomeric excess of each
labeled product was measured by chiral HPLC, as described above;
eluate was monitored for absorbance and radioactivity. Specific
radioactivities (GBq/µmol) were determined with analytical HPLC
calibrated for absorbance (λ ) 254 nm) response per mass of ligand.
The specific radioactivity was calculated as the radioactivity of the
radioligand peak (decay-corrected) (GBq) divided by the mass of
the associated carrier peak (µmol). The metabolism of [11C](-)-
12a was assessed with HPLC on a reverse phase (µ-Bondapak C-18
column; 7.8 mm × 300 mm, 10 µm; Waters) eluted at 6 mL/min
with a gradient of MeCN (A) and aq-H3PO4 (0.01 M) (B), with A
increasing linearly from 35 to 65% v/v for 6 min and then to 35%
v/v over the next 2 min and then held for 4 min. The column outlet
was connected to an absorbance detector (λ ) 270 nm) in series
with a GM-tube for radiation detection.
128.25, 128.74, 129.08, 129.23, 129.61, 132.72, 136.85, 138.93,
143.06, 147.55, 156.82. LC-MS m/z (M+ + H) ) 464.9. HRMS
calcd for C23H18N4O3SCl (M+ + H), 465.0788; found, 465.0774;
error (ppm): -3.0.
3-(4-Chlorophenyl)-N-[(4-bromophenyl)sulfonyl]-4-phenyl-4,5-
dihydro-1H-pyrazole-1-carboxamide (11b). 11b was prepared from
3-(4-chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole and 10b in
1
82% yield by the method described for 11a; mp 214-216 °C. H
NMR (400 MHz, CDCl3): δ 3.92 (1H, dd, J ) 6.2, 5.2 Hz), 4.34
(1H, t, J ) 4.3 Hz), 4.75 (1H, dd, J ) 6.2, 5.2 Hz), 7.12 (2H, dt,
J ) 6.6, 1.6 Hz), 7.33-7.24 (5H, m), 7.55 (2H, dt, J ) 8.6, 1.8
Hz), 7.71 (2H, dt, J ) 8.6, 1.8 Hz), 8.04 (2H, dt, J ) 8.6, 1.8 Hz),
8.76 (1H, s). 13C NMR (100.62 MHz, DMSO-d6): δ 21.02, 49.43,
54.53, 101.86, 125.28, 127.25, 127.48, 128.17, 128.56, 128.87,
129.06, 129.17, 129.26, 134.69, 137.31, 137.92, 138.16, 139.59,
140.15, 148.52, 155.89. LC-MS m/z (M+ + H) ) 519.9. HRMS
calcd for C22H18N3O3SClBr (M+ + H), 517.9941; found 517.9929;
error (ppm): -2.3.
N-[(4-Cyanophenyl)sulfonyl]carbamic acid methyl ester (10a).
Methyl chloroformate (6.34 mL, 82.4 mmol) was slowly added to
a stirred solution of 4-cyanobenzenesulfonamide (10 g, 54.9 mmol)
and triethylamine (23 mL, 165 mmol) in acetonitrile (75 mL). The
reaction was stirred at room temperature for 16 h and then
evaporated to dryness in vacuo. After addition of ethyl acetate and
aq NaHCO3 to the crude residue, the aqueous layer was separated
and acidified. The oily precipitate crystallized on standing and was
filtered off, washed with water, and dried to give 10a (6.9 g, 52%
3-(4-Chlorophenyl)-N-[(4-iodophenyl)sulfonyl]-4-phenyl-4,5-di-
hydro-1H-pyrazole-1-carboxamide (11c). 11c was prepared from
3-(4-chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole and 10c in
74.5% yield by the method described for 11a; mp 212-214 °C.
1H NMR (400 MHz, CDCl3): δ 3.92 (1H, dd, J ) 6.2, 5.2 Hz),
4.34 (1H, t, J ) 4.3 Hz), 4.73 (1H, dd, J ) 6.1, 5.2 Hz), 7.16 (2H,
dt, J ) 6.6, 1.6 Hz), 7.33-7.24 (5H, m), 7.55 (2H, dt, J ) 8.7, 2.0
Hz), 7.71 (4H, qt, J ) 9.8, 8.8, 1.8 Hz), 8.8 (1H, s). 13C NMR
(100.62 MHz, CDCl3): δ 51.5, 54.0, 127.2, 128.1, 128.2, 128.3,
128.7, 129.1, 129.6, 129.9, 132.3, 136.7, 138.1, 139.1, 147.8, 156.4.
LC-MS m/z (M+ + H) ) 565.8. HRMS calcd for C22H18N3O3SClI
(M+ + H), 565.9802; found: 565.9801; error (ppm): -0.2.
3-(4-Chlorophenyl)-N′-[(4-cyanophenyl)sulfonyl]-4-phenyl-4,5-
dihydro-1H-pyrazole-1-carboxamidine (12a). A mixture of 11a (6
g, 12.9 mmol) and PCl5 (2.8 g, 13.5 mmol) was dissolved in
chlorobenzene (80 mL), refluxed for 1 h, and then concentrated in
vacuo. The residue was treated with methanolic NH3 (1M, 35 mL).
The mixture was stirred at room temperature for 1 h and then
concentrated in vacuo. The product was recrystallized from MeOH
to give 12a (2.2 g, 37%); mp 208-210 °C. 1H NMR (400.13 MHz,
CDCl3): δ 4.02 (dd, J ) 12.0, 4.0 Hz, 1H), 4.42 (t, J ) 12.0 Hz,
1H), 4.76 (dd, J ) 12.0, 4.0 Hz, 1H), 7.10 (dt, J ) 6.4, 2.0 Hz,
2H), 7.34-2.25 (m, 3H), 7.55 (dt, J ) 8.7, 2.0 Hz, 2H), 7.6 (d, J
) 8.0 Hz, 2H), 7.75 (dt, J ) 8.6, 1.4 Hz, 2H), 8.05 (dt, J ) 8.6,
1.4 Hz, 2H). 13C NMR (100.62 MHz, CDCl3): δ 51.42, 55.34,
115.29, 117.77, 126.87, 127.18, 128.04, 128.22, 128.69, 129.09,
129.61, 132.58, 136.89, 138.93, 147.60, 152.72, 158.05. LC-MS
1
yield); mp 130-132 °C. H NMR (400 MHz, CDCl3): δ 3.72 (s,
3H), 7.87 (2H, dt, J ) 9.0, 2.0 Hz), 8.19 (2H, dt, J ) 8.8, 2.0 Hz),
NH proton invisible.
N-[(4-Bromophenyl)sulfonyl]carbamic acid methyl ester (10b).
10b was prepared from 4-bromobenzenesulfonamide in 48% yield
by the method described for 10a; mp 120-122 °C. 1H NMR (400
MHz, CDCl3): δ 3.72 (s, 3H), 7.71 (2H, dt, J ) 8.8, 2.0 Hz), 7.93
(2H, dt, J ) 8.8, 2.0 Hz), NH proton invisible.
N-[(4-Iodophenyl)sulfonyl]carbamic acid methyl ester (10c). 10c
was prepared from 4-iodoobenzenesulfonamide in 56% yield by
the method described for 10a; mp 116-118 °C. 1H NMR (400.13
MHz, CDCl3): δ 3.72 (s, 3H), 7.77 (2H, dt, J ) 8.8, 2.0 Hz), 7.93
(2H, dt, J ) 8.8, 2.0 Hz), NH proton invisible.
3-(4-Chlorophenyl)-N-[(4-cyanophenyl)sulfonyl]-4-phenyl-4,5-di-
hydro-1H-pyrazole-1-carboxamide (11a). To a solution of 3-(4-
chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole (6.4 g, 25.4 mmol)
in toluene (100 mL) was added 10a (6.1 g, 25.4 mmol), and the
resulting solution was heated to reflux for 2 h. After cooling to
room temperature, 11a began to crystallize slowly from solution.
The crystals were filtered off and washed twice with MTBE to give
pure 11a (9.2 g, 78% yield); mp 208-210 °C. 1H NMR (400 MHz,
CDCl3): δ 3.92 (1H, dd, J ) 6.2, 5.2 Hz), 4.34 (1H, t, J ) 4.3 Hz),
4.75 (1H, dd, J ) 6.2, 5.2 Hz), 7.12 (2H, dt, J ) 6.6, 1.6 Hz),
7.33-7.24 (5H, m), 7.55 (2H, dt, J ) 8.6, 1.8 Hz), 7.86 (2H, dt, J
) 8.6, 1.8 Hz), 8.30 (2H, dt, J ) 8.6, 1.8 Hz), 8.8 (1H, bs). 13C
NMR (100 MHz, CDCl3): δ 51.54, 54.02, 117.39, 127.21, 127.92,
m/z (M+ + H), 464.0. HRMS calcd for C23H19N5O2SCl (M+
+
H), 464.0948; found, 464.0957; error (ppm): 1.9.
(-)-3-(4-Chlorophenyl)-N′-[(4-cyanophenyl)sulfonyl]-4-phenyl-
4,5-dihydro-1H-pyrazole-1-carboxamidine ((-)-12a). Resolution of
12a by chiral HPLC (see General Methods) gave (-)-12a (tR
)
9.8 min at 8 mL/min, >98% ee); [R]2D2 ) -69.3°, c ) 0.010,
CH2Cl2; mp 208-210 °C. 1H NMR: as found for 12a. MS m/z
(M+ + H), 464.1. HRMS, calcd for C23H19N5O2SCl (M+ + H),

5614 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Donohue et al.
464.0948; found, 464.0962; error (ppm): 3.0. Anal. (C23H18-
ClN5O2S) C, H, N.
(+)-3-(4-Chlorophenyl)-N′-[(4-cyanophenyl)sulfonyl]-4-phenyl-
4,5-dihydro-1H-pyrazole-1-carboxamidine ((+)-12a). Resolution of
[35S]GTPγS was diluted with distilled water to a final concentra-
tion of 0.5 nM. The inhibitory effect of test ligands was measured
against a concentration (EC80) of the CB1r/CB2r agonist CP-55940
(Tocris) added to the solution.
12a by chiral HPLC (see General Methods) gave (+)-12a (tR
)
Membrane solution (100 µL) and radioligand solution (100 µL)
were added to the assay plates together with 2 µL compound
(concentration-response in DMSO) and incubated for 45 min at 30
°C. Bound [35S]GTPγS was separated from free [35S]GTPγS by
rapid filtration under vacuum through Whatman GF/B glass fiber
filters, followed by washes with cold wash solution (final concen-
tration: 50 mM Tris, 5 mM MgCl2, 50 mM NaCl). The filters were
dried for 60 min at 50 °C. Scintillation film was melted onto the
filters, which were then counted in a Microbeta scintillation counter.
Nonspecific binding was determined in the presence of a saturating
concentration of GTPγS (final concentration 20 µM). IC50 values
were converted to Ki values according to the Cheng and Prusoff
equation.39 The data represent Ki ( SD (nM) from triplicate
determinations vs CB1r or CB2r.
12.27 min at 8 mL/min, >98% ee); [R]2D2 ) + 66.3°, c ) 0.011,
CH2Cl2; mp 208-210 °C. 1H NMR: as found for 12a. MS m/z
(M+ + H) 464.1. HRMS calcd for C23H19N5O2SCl (M+ + H)
464.0948; found, 464.0961, error (ppm): 2.8. Anal. (C23H18-
ClN5O2S) C, H, N.
3-(4-Chlorophenyl)-N′-[(4-bromophenyl)sulfonyl]-4-phenyl-4,5-
dihydro-1H-pyrazole-1-carboxamidine (12b). 12b was prepared
from 11b in 43% yield by the method described for 12a; mp
214-216 °C. 1H NMR (400.13 MHz, DMSO-d6): δ 3.80 (1H, dd,
J ) 6.6, 4.7 Hz), 4.37 (1H, t, J ) 11.5 Hz), 5.04 (1H, dd, J ) 6.6,
4.7 Hz), 7.16 (2H, d, J ) 7.1 Hz), 7.25 (1H, t, J ) 7.2 Hz), 7.35
(2H, t, J ) 7.6 Hz), 7.44 (2H, d, J ) 8.6 Hz), 7.76 (2H, d, J ) 8.4
Hz), 7.79-7.71 (2H, m). 13C NMR (100.62 MHz, DMSO-d6) δ
49.62, 55.60, 125.22, 127.20, 127.52, 127.85, 128.60, 128.83,
129.12, 129.21, 131.83, 134.92, 140.05, 142.96, 152.76, 157.60.
LC-MS m/z [M + H]+ 517.0; HRMS calcd for C22H19N4O2SClBr
(M+ + H), 517.0101; found, 517.0117, error (ppm): 3.1.
Receptor Screening. Ligand (-)-12a was screened for binding
to a wide range of receptors and transporters by the National
Institute of Mental Health Psychoactive Drug Screening Program.
Detailed protocols are available online for all binding assays at the
NIMH-PDSP Web site (http://pdsp.med.unc.edu).
(-)-3-(4-Chlorophenyl)-N′-[(4-bromophenyl)sulfonyl]-4-phenyl-
4,5-dihydro-1H-pyrazole-1-carboxamidine ((-)-12b). Resolution of
[11C]HCN Production. [11C]Methane was produced at the
Karolinska Hospital with a GEMS PETtrace cyclotron using 16.4
MeV protons in the 14N(p,R)11C reaction on N2(g) containing 10%
H2(g).40 The target gas was irradiated for 5 min with a beam
intensity of 35 µA. The [11C]methane was isolated from the target
gas in a Porapak Q trap, which was cooled with N2(l). The Porapak
Q trap was warmed and the [11C]methane passed in nitrogen (200
mL/min) with NH3(g) (20-30 mL/min) over heated (990 °C) Pt
wire (1.3 g, 0.127 mm, 99%; Sigma Aldrich) in a quartz tube within
a Carbolite furnace (MTF 10/15). The resulting [11C]NH4CN was
bubbled through aq 50% H2SO4 (2 mL) at 56 °C to generate
[11C]HCN.
12b by chiral HPLC (see General Methods) gave (-)-12b (tR
)
13.78 min at 8 mL/min, >98% ee); [R]2D2 ) -65.7°, c ) 0.010,
1
CH2Cl2; mp 214-216 °C. H NMR: as found for 12b. MS m/z
(M+ + H) 517.0. HRMS calcd for C22H19N4O2SClBr (M+ + H)
517.0101, found: 517.0126, error (ppm): 4.8.
(+)-3-(4-Chlorophenyl)-N′-[(4-bromophenyl)sulfonyl]-4-phenyl-
4,5-dihydro-1H-pyrazole-1-carboxamidine ((+)-12b). Resolution of
12b by chiral HPLC (see General Methods) gave (+)-12b (tR
)
18.14 min at 8 mL/min, >98% ee); [R]2D2 ) 65.2°, c ) 0.010,
1
CH2Cl2; mp 214-216 °C. H NMR: as found for 12b. MS m/z
(M+ + H) 517.0. HRMS calcd for C22H19N4O2SClBr (M+ + H)
Radiosyntheses of [11C](()-12a, [11C](-)-12a, and [11C](+)-
12a. The generated [11C]HCN (∼12.4 GBq) was trapped in a V-vial
(5-mL) containing DMSO (400 µL) base (KOH, 1 mg, 17.8 µmol;
K 2.2.2., 5 mg, 13.3 µmol) for [11C](()-12a or KH2PO4 (1 mg,
7.34 µmol) for [11C](-)-12a or [11C](+)-12a. After trapping of
[11C]HCN was complete, the solution was transferred into another
V-vial (10 mL) which contained DMSO (400 µL), Pd(PPh3)4 (5.5
mg, 4.8 µmol) and the requisite precursor ((()-12b, (-)-12b, (-)-
12c, or (+)-12c; 1 mg). The reaction was heated 135 °C for 4 min
and then cooled to room temperature. HPLC mobile phase (800
µL) was added to the V-vial and the radioactive product separated
with HPLC (see General Methods). The radioactive fraction (tR )
12.3 min) was collected, evaporated to dryness, and taken up in
ethanol-propylene glycol (30:70 v/v, 3 mL) with sterile phosphate
buffer (0.2 M, pH ) 7.4, 5 mL) and filtered through a sterile filter
(Millex GV, 0.22 µm pore size; Millipore Corp., Corrigtwohill,
Co. Cork, Ireland).41 A sample (∼100 µL) was analyzed by HPLC
for radiochemical purity and measurement of specific radioactivity
(see General Methods).
PET Measurements in Monkey. Cynomolgus monkey (Macaca
fascicularis) experiments were performed at the Karolinska Institutet
(KI) according to “Guidelines for Planning, Conducting and
Documenting Experimental Research” (Dnr 4820/06-600) of the
KI as well as the “Guide for the Care and Use of Laboratory
Animals”.42 The study was approved by the Animal Ethics
Committee of the Swedish Animal Welfare Agency. Two cyno-
molgus monkeys (3.4 and 4.8 kg) were used in the PET experi-
ments. Anesthesia was induced and maintained with repeated im
injections of a mixture of ketamine hydrochloride (3.75 mg/kg-1
h-1 Ketalar, Pfizer) and xylazine hydrochloride (1.5 mg/kg-1 h-1
Rompun Vet., Bayer). The head for each monkey was immobilized
in a stereotactic frame for the duration of scans.43 The body
temperature was maintained by a Bair Hugger model 505 (Arizant
Healthcare, MN) and monitored by rectal thermometer (Precision
Thermometer, Harvard Apparatus, MA).
517.0101, found: 517.0110, error (ppm): 1.7.
3-(4-Chlorophenyl)-N′-[(4-iodophenyl)sulfonyl]-4-phenyl-4,5-di-
hydro-1H-pyrazole-1-carboxamidine (12c). 12c was prepared from
11c in 64% yield by the method described for 12a; mp 222-224
°C. 1H NMR (400 MHz, DMSO-d6): δ 3.80 (1H, dd, J ) 6.6, 4.7
Hz), 4.37(1H, t, J ) 11.5 Hz), 5.04 (1H, dd, J ) 6.6, 4.7 Hz), 7.18
(2H, d, J ) 7.1 Hz), 7.26 (1H t, J ) 7.2 Hz), 7.35 (2H, t, J ) 7.6
Hz), 7.44 (2H, d, J ) 8.6 Hz), 7.62 (2H, d, J ) 8.4 Hz), 7.77 (2H,
d, J ) 8.6 Hz), 7.90 (2H, d, J ) 8.4 Hz). 13C NMR (100.62 MHz,
DMSO-d6): δ 49.60, 55.59, 99.28, 127.19, 127.52, 127.59, 128.69,
128.83, 129.11, 129.21, 134.92, 137.65, 140.05, 143.30, 152.74,
157.57. LC-MS m/z (M+ + H) 565.1. HRMS calcd for
C22H19N4O2SClI (M+ + H) 564.9962, found: 564.9974, error
(ppm): 2.1.
(-)-3-(4-Chlorophenyl)-N′-[(4-iodophenyl)sulfonyl]-4-phenyl-4,5-
dihydro-1H-pyrazole-1-carboxamidine ((-)-12c). Resolution of 12c
by chiral HPLC (see General Methods) gave (-)-12c (tR ) 18.18
min at 6 mL/min, >98% ee); [R]2D2) -160°, c ) 0.011, CHCl3;
mp 222-224 °C. 1H NMR: as found for 12c. LC-MS m/z (M+
+
H) 565.1. HRMS calcd for C22H19N4O2SClI (M+ + H): 564.9962,
found: 564.9960, error (ppm): -0.4. Anal. (C22H18ClIN4O2S) C,
H, N.
(+)-3-(4-Chlorophenyl)-N′-[(4-iodophenyl)sulfonyl]-4-phenyl-4,5-
dihydro-1H-pyrazole-1-carboxamidine ((+)-12c). Resolution of 12c
by chiral HPLC (see General Methods) gave (+)-12c (tR ) 24.38
min at 6 mL/min, >98% ee); [R]2D2 ) + 151°, c ) 0.010, CHCl3;
mp 222-224 °C. 1H NMR: as found for 12c. LC-MS m/z (M+
+
H) 565.0. HRMS calcd for C22H19N4O2SClI (M+ + H) 564.9962,
found: 564.9960, error (ppm): -0.4. Anal. (C22H18ClIN4O2S) C,
H, N.
CB1 and CB2 Binding Assays. Frozen cell membranes (recom-
binant hCB1r or hCB2r; Perkin-Elmer) were thawed and diluted
(7 µg/well) in membrane buffer (final concentrations: 100 mM
NaCl, 5 mM MgCl2, 1 mM EDTA, 50 mM HEPES, 10 µM GDP,
100 µM DTT, 0.01% fatty acid free BSA).

EValuation of CB1 Receptor Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5615
In baseline experiments, each radioligand was administered by
bolus injection over about 5 s, with injected activities of 56, 100,
and 98 MBq and specific radioactivities of 78, 65, and 56 GBq/
µmol for [11C](()-12a, [11C](-)-12a, and [11C](+)-12a, respec-
tively. The masses of injected carrier ligand were 0.33 µg (0.72
nmol), 0.71 µg (1.53 nmol), and 0.81 µg (1.75 nmol) for [11C](()-
12a, [11C](-)-12a, and [11C](+)-12a. In the displacement experi-
ment, 6 (1 mg/kg, iv) was infused at 25 min after injection of
[11C](()-12a. For this purpose, compound 6 was formulated in
vehicle solution (8 mL) (saline/alcohol/cremophore EL, 9:1:0.1 by
vol.). The solution was homogenized by vortexing and then passed
through a sterile filter (0.2 µm pore size, Millex-GV, Millipore).
The injected activity was 56 MBq with a specific radioactivity of
80.3 GBq/µmol. The mass of carrier associated with the injected
radioactivity was 0.32 µg (0.70 nmol). The preblock experiment
was performed at 5 h after the baseline experiment. 6 was infused
at 20 min before injection of radioligand. The injected activity was
57 MBq with specific radioactivities of 75.4 GBq/µmol. The mass
of carrier associated with the injected radioactivity was 0.35 µg
(0.76 nmol). In each PET experiment, scans were acquired in 3
frames over 93 min.
PET Imaging. Radioactivity in brain was measured with the
Siemens ECAT EXACT HR system. The three-ring detector block
architecture gives a 15-cm wide field of view. All acquisitions were
acquired in 3D-mode.44 The transversal resolution in the recon-
structed image is about 3.8 mm full-width half-maximum (fwhm)
and the axial resolution, 3.125 mm. The data were corrected for
attenuation with three rotating 68Ge rod sources. Raw PET data
were then reconstructed using standard filtered back projection
consisting of the following reconstruction parameters: 2 mm
Hanning filter, scatter correction, a zoom factor of 2.17, and a 128
× 128 matrix size.44 Emission data were collected continuously
for 93 min, according to a preprogrammed series of 20 frames
starting immediately after iv injection of radioligand. The 3 initial
frames were 1 min each, followed by 4 frames of 3 min each and
13 frames of 6 min each.
members of the PET group at the Karolinska Institutet for
assistance. We are grateful to Eli Lilly and Co. for the provision
of 6 under a CRADA (Cooperative Research and Development
Agreement) with NIMH. We thank the NIMH Psychoactive
Drug Screening Program (PDSP) and Marie Wennerberg at
AstraZeneca for performing binding assays. The PDSP is
directed by Bryan L. Roth, Ph.D., with project officer Jamie
Driscol (NIMH), at the University of North Carolina at Chapel
Hill (contract NO1MH32004).
Supporting Information Available: Table of elemental analyses
for target ligands (-)-12a, (+)-12a, (-)-12c, and (+)-12c. This
material is available free of charge via Internet at http://pubs.acs.org.
References
(1) Vincent, B. J.; McQuiston, D. J.; Einhorn, L. H.; Nagy, C. M.; Brames,
M. J. Review of cannabinoids and their anti-emetic effectiveness.
Drugs 1983, 25, 52–62.
(2) Mackie, K. Cannabinoid receptors as therapeutic targets. Annu. ReV.
Pharmacol. Toxicol. 2005, 46, 101–122.
(3) Lambert, D. M.; Fowler, C. J. The endocannabinoid system: drug
targets, lead compounds, and potential therapeutic applications. J. Med.
Chem. 2005, 48, 5059–5087.
(4) Izzo, A. A.; Coutts, A. A. Cannabinoids and the digestive tract. Handb.
Exp. Pharmacol. 2005, 168, 573–598.
(5) Darmani, N. A.; Crim, J. L. ∆9-Tetrahydrocannabinol differentially
suppresses emesis versus enhanced locomotor activity produced by
chemically diverse dopamine D2/D3 receptor agonists in the least shrew
(Cryptotis parVa). Pharmacol., Biochem. BehaV. 2005, 80, 35–44.
(6) Beal, J. E.; Olson, R.; Laubenstein, L.; Morales, J. O.; Bellman, P.;
Yangco, B.; Lefkowitz, L.; Plasse, T. F.; Shepard, K. V. Dronabinol
as a treatment for anorexia associated with weight loss in patients
with AIDS. J. Pain Symptom Manage. 1995, 10, 89–97.
(7) Beal, J. E.; Olson, R.; Lefkowitz, L.; Laubenstein, L.; Bellman, P.;
Yangco, B.; Morales, J. O.; Murphy, R.; Powderly, W.; Plasse, T. F.;
Mosdell, K. W.; Shepard, K. V. Long-term efficacy and safety of
dronabinol for acquired immunodeficiency syndrome-associated an-
orexia. J. Pain Symptom Manage. 1997, 14, 7–14.
The mean image of the PET measurements (9-93 min) was
transformed into a standard anatomical space using the monkey
version of the Human Brain Atlas developed at the Karolinska
Institutet.45 The transformation matrix generated on this image was
applied to all frames of the corresponding baseline, displacement,
and pretreatment experiments. PET data were subsequently resliced
to a resolution of 1.00, 1.00, 1.00 mm. Volumes of interest (VOIs)
were manually defined on the coronal planes of an average monkey
MRI. Similar VOIs were applied, as reported by Yasuno et al.,29
including cerebellum (1.9 cm3), frontal cortex (7.4 cm3), lateral
temporal cortex (5.0 cm3), medial temporal cortex (2.9 cm3),
striatum (2.1 cm3), thalamus (0.9 cm3), and pons (0.7 cm3). Tissue
radioactivity concentrations were expressed as % standardized
uptake values (%SUV). Tissue radioactivity concentrations were
decay-corrected and, in order to normalize for injected dose and
body weight, expressed as % standardized uptake values (%SUV),
where:
(8) Grundy, R. I. The therapeutic potential of the cannabinoids in
neuroprotection. Expert Opin. InVestig. Drugs 2002, 11, 1365–1374.
(9) Pryce, G.; Ahmed, Z.; Hankey, D. J.; Jackson, S. J.; Croxford, J. L.;
Pocock, J. M.; Ledent, C.; Petzold, A.; Thompson, A. J.; Giovannoni,
G.; Cuzner, M. L.; Baker, D. Cannabinoids inhibit neurodegeneration
in models of multiple sclerosis. Brain 2003, 126, 2191–2202.
(10) Gaoni, Y.; Mechoulam, R. Isolation structure and partial synthesis of
active constituent of hashish. J. Am. Chem. Soc. 1964, 86, 1646–1647.
(11) Mechoulam, R.; Gaoni, Y. The absolute configuration of ∆1-
tetrahydrocannabinol, the major active constituent of hashish. Tetra-
hedron. Lett. 1967, 12, 1109–1111.
(12) Devane, W. A.; Dysarz, F. A.; Johnson, M. R.; Melvin, L. S.; Howlett,
A. C. Determination and characterization of a cannabinoid receptor
in rat-brain. Mol. Pharmacol. 1988, 34, 605–613.
(13) Munro, S.; Thomas, K. L.; Abushaar, M. Molecular characterization
of a peripheral receptor for cannabinoids. Nature 1993, 365, 61–65.
(14) Shire, D.; Carillon, C.; Kaghad, M.; Calandra, B.; Rinaldicarmona,
M.; Lefur, G.; Caput, D.; Ferrara, P. An amino-terminal variant of
the central cannabinoid receptor resulting from alternative splicing.
J. Biol. Chem. 1995, 270, 3726–3731.
(15) Ryberg, E.; Vu, H. K.; Larsson, N.; Groblewski, T.; Hjorth, S.;
Elebring, T.; Sjorgren, S.; Greasley, P. J. Identification and charac-
terisation of a novel splice variant of the human CB1 receptor. FEBS
Lett. 2005, 579, 259–264.
(16) Herkenham, M.; Lynn, A. B.; Little, M. D.; Johnson, M. R.; Melvin,
L. S.; Decosta, B. R.; Rice, K. C. Cannabinoid receptor localization
in brain. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1932–1936.
(17) Herkenham, M.; Lynn, A. B.; Johnson, M. R.; Melvin, L. S.; Decosta,
B. R.; Rice, K. C. Characterization and localization of cannabinoid
receptors in rat brain: a quantitative in vitro autoradiographic study.
J. Neurosci. 1991, 11, 563–583.
(18) Lynn, A. B.; Herkenham, M. Localization of cannabinoid receptors
and nonsaturable high-density cannabinoid binding sites in peripheral
tissues of the rat: implications for receptor-mediated immune modula-
tion by cannabinoids. J. Pharmacol. Exp. Ther. 1994, 268, 1612–1623.
(19) Griffin, G.; Fernando, S. R.; Ross, R. A.; McKay, N. G.; Ashford,
M. L. J.; Shire, D.; Huffman, J. W.; Yu, S.; Lainton, J. A. H.; Pertwee,
R. G. Evidence for the presence of CB2-like cannabinoid receptors
on peripheral nerve terminals. Eur. J. Pharmacol. 1997, 339, 53–61.
% injected activity
brain tissue (g)
%SUV )
× body weight (g)
(1)
[
]
PET Plasma Measurements. For radiometabolite measurements,
venous blood (1 mL) was sampled from monkey at 5, 15, 30, and
45 min after injection of each radioligand. Plasma samples were
measured as described previously.46 Briefly, the supernatant liquid
(0.5 mL) obtained after centrifugation at 2000g for 1 min was mixed
with MeCN (0.7 mL) containing standard (()-12a. The supernatant
liquid (1 mL) after another centrifugation at 2000g for 1 min was
counted in a well counter and subsequently injected onto HPLC.
Acknowledgment. This work was supported by the Intra-
mural Program of the National Institute of Mental Health
(project no. Z01-MH-002795). Mr. S.R. Donohue was also
supported with a studentship under the NIH-Karolinska Institutet
Graduate Training Partnership in Neuroscience. We thank other

5616 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Donohue et al.
(20) Rinaldi-Carmona, M.; Barth, F.; Heaulme, M.; Shire, D.; Calandra,
B.; Congy, C.; Martinez, S.; Maruani, J.; Neliat, G.; Caput, D.; Ferrara,
P.; Soubrie´, P.; Brelie`re, J.-C.; Le Fur, G. SR141716A, a potent and
selective antagonist of the brain cannabinoid receptor. FEBS Lett. 1994,
350, 240–244.
(21) Lange, J. H. M.; Kruse, C. G.; Tipker, J.; Tulp, M. T. M.; Van Vliet,
B. J. 4,5-Dihydro-1H-pyrazole derivatives having CB1-antagonist
activity. PTC Int. Appl. WO 0170700, 2001.
(22) Lange, J. H. M.; Coolen, H.; van Stuivenberg, H. H.; Dijksman,
J. A. R.; Herremans, A. H. J.; Ronken, E.; Keizer, H. G.; Tipker, K.;
McCreary, A. C.; Veerman, W.; Wals, H. C.; Stork, B.; Verveer, P. C.;
den Hartog, A. P.; de Jong, N. M. J.; Adolfs, T. J. P.; Hoogendoorn,
J.; Kruse, C. G. Synthesis, biological properties, and molecular
modeling investigations of novel 3,4-diarylpyrazolines as potent and
selective CB1 cannabinoid receptor antagonists. J. Med. Chem. 2004,
47, 627–643.
brain with positron emission tomography. Mol. Imaging Biol. 2003,
5, 363–375.
(31) Waterhouse, R. N. Determination of lipophilicity and its use as a
predictor of blood-brain barrier penetration of molecular imaging
agents. Mol. Imaging Biol. 2003, 5, 376–389.
(32) Andersson, Y.; Långstro¨m, B. Transition metal-mediated reactions
using [11C]cyanide in synthesis of 11C-labeled aromatic-compounds.
J. Chem. Soc., Perkin Trans. 1 1994, 11, 1395–1400.
(33) Ponchant, M.; Hinnen, F.; Demphel, S.; Crouzel, C. [11C]Copper(I)
cyanide: a new radioactive precursor for 11C-cyanation and function-
alization of haloarenes. Appl. Radiat. Isot. 1997, 48, 755–762.
(34) Mathews, W. B.; Monn, J. A.; Ravert, H. T.; Holt, D. P.; Schoepp,
D. D.; Dannals, R. F. Synthesis of a mGluR5 antagonist using
[
11C]copper(I) cyanide. J. Label. Compd. Radiopharm. 2006, 49, 829–
834.
(35) Carson, R. E. PET physiological measurements using constant infusion.
Nucl. Med. Biol. 2000, 27, 657–660.
(36) Jaffe, H.; Leffler, J. E. Synthesis of benziodathiazoles. J. Org. Chem.
1975, 40, 797–799.
(23) Lange, J. H. M.; Kruse, C. G.; Tipker, J.; Hoogendoorn, J. 4,5-Dihydro-
1H-pyrazole derivatives having CB1-antagonist activity. PTC Int. Appl.
WO 02076949, 2002.
(24) Horti, A. G.; Fan, H.; Kuwabara, H.; Hilton, J.; Ravert, H. T.; Holt,
D. P.; Alexander, M.; Kumar, A.; Rahmim, A.; Scheffel, U.; Wong,
D. F.; Dannals, R. F. 11C-JHU75528: A radiotracer for PET imaging
of CB1 cannabinoid receptors. J. Nucl. Med. 2006, 47, 1689–1696.
(25) Fan, H.; Ravert, H. T.; Holt, D. P.; Dannals, R. F.; Horti, A. G.
Synthesis of 1-(2,4-dichlorophenyl)-4-cyano-5-(4-[11C]methoxyphe-
nyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide ([11C]JHU75528)
and 1-(2-bromophenyl)-4-cyano-5-(4-[11C]methoxyphenyl)-N-(piperi-
din-1-yl)-1H-pyrazole-3-carboxamide ([11C]JHU75575) as potential
radioligands for PET imaging of cerebral cannabinoid receptor.
J. Label. Compd. Radiopharm. 2006, 49, 1021–1036.
(37) Grosscurt, A. C.; Vanhes, R.; Wellinga, K. 1-Phenylcarbamoyl-2-
pyrazolines, a new class of insecticides. 3. Synthesis and insecticidal
properties of 3,4-diphenyl-1-phenylcarbamoyl-2-pyrazolines. J. Agric.
Food Chem. 1979, 27, 406–409.
(38) Airaksinen, A. J.; Andersson, J.; Troung, P.; Karlsson, O.; Halldin,
C. Radiosynthesis of [11C]ximelagatran via palladium catalyzed
[
11C]cyanation. J. Label. Compd. Radiopharm. 2008, 51, 1–5.
(39) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant
(Ki) and the concentration of inhibitor which causes 50% inhibition
(IC50) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, 3099–
108.
(26) Donohue, S. R.; Halldin, C.; Schou, M.; Hong, J.; Phebus, L. A.;
Chernet, E.; Hitchcock, S. A.; Gardinier, K. M.; Ruley, K. M.;
Krushinski, J. H.; Schaus, J. M.; Pike, V. W. Radiolabeling of a high
potency cannabinoid subtype-1 receptor ligand, N-(4-fluoro-benzyl)-
4-(3-(piperidin-1-yl)-indole-1-sulfonyl)benzamide (PipISB), with carbon-
11 or fluorine-18. J. Label. Compd. Radiopharm. 2008, 51, 146–152.
(27) Burns, H. D.; Van Laere, K.; Sanabria-Bohorquez, S.; Hamill, T. G.;
Bormans, G.; Eng, W. S.; Gibson, R.; Ryan, C.; Connolly, B.; Patel,
S.; Krause, S.; Vanko, A.; Van Hecken, A.; Dupont, P.; De Lepeleire,
I.; Rothenberg, P.; Stoch, S. A.; Cote, J.; Hagmann, W. K.; Jewell,
J. P.; Lin, L. S.; Liu, P.; Goulet, M. T.; Gottesdiener, K.; Wagner,
J. A.; de Hoon, J.; Mortelmans, L.; Fong, T. M.; Hargreaves, R. J.
(40) Christman, D. R.; Finn, R. D.; Karlstrom, K. I.; Wolf, A. P. Production
of ultra high activity 11C-labeled hydrogen-cyanide, carbon-dioxide,
carbon-monoxide, and methane via 14N(p,R) 11C reaction. Intl. J. Appl.
Radiat. Isot. 1975, 26, 435–442.
(41) Foged, C.; Halldin, C.; Hiltunen, J.; Braestrup, C.; Thomsen, C.;
Hansen, H. C.; Suhara, T.; Pauli, S.; Swahn, C. G.; Karlsson, P.;
Larsson, S.; Farde, L. Development of 123I-labelled NNC 13-8241 as
a radioligand for SPECT visualization of benzodiazepine receptor
binding. Nucl. Med. Biol. 1996, 23, 201–209.
(42) Clark, J. D.; Baldwin, R. L.; Bayne, K. A.; Brown, M. J.; Gebhart,
G. F.; Gonder, J. C.; Gwathmey, J. K.; Keeling, M. E.; Kohn, D. F.;
Robb, J. W.; Smith, O. A.; Steggerda, J.-A. D.; VandeBer, J. L. Guide
for Care and Use of Laboratory Animals; National Academies Press:
Washington, DC, 1996.
(43) Karlsson, P.; Farde, L.; Halldin, C.; Swahn, C. G.; Sedvall, G.; Foged,
C.; Hansen, K. T.; Skrumsager, B. PET examination of [11C]NNC-
687 and [11C]NNC-756 as new radioligands for the D1-dopamine
receptor. Psychopharmacology 1993, 113, 149–156.
(44) Wienhard, K.; Dahlbom, M.; Eriksson, L.; Michel, C.; Bruckbauer,
T.; Pietrzyk, U.; Heiss, W. D. The ECAT Exact HR: Performance of
a new high-resolution positron scanner. J. Comput. Assist. Tomogr.
1994, 18, 110–118.
(45) Roland, P. E.; Graufelds, C. J.; Wåhlin, J.; Ingelman, L.; Andersson,
M.; Ledberg, A.; Pedersen, J.; Åkerman, S.; Dabringhaus, A.; Zilles,
K. Human brain atlas: for high-resolution functional and anatomical
mapping. Human Brain Mapping 1994, 1, 173–184.
(46) Halldin, C.; Swahn, C. G.; Farde, L.; Sedvall, G. Radioligand
disposition and metabolism. In PET for Drug DeVelopment and
EValuation; Comar, D. Ed.; Kluwer Academic Publishers: Dordrecht,
Netherlands, 1995; pp 55-65.
[
18F]MK-9470, a positron emission tomography (PET) tracer for in
vivo human PET brain imaging of the cannabinoid-1 receptor. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 9800–9805.
(28) Liu, P.; Lin, L. S.; Hamill, T. G.; Jewell, J. P.; Lanza, T. J.; Gibson,
R. E.; Krause, S. M.; Ryan, C.; Eng, W. S.; Sanabria, S.; Tong, X. C.;
Wang, J. Y.; Levorse, D. A.; Owens, K. A.; Fong, T. M.; Shen, C. P.;
Lao, J. L.; Kumar, S.; Yin, W. J.; Payack, J. F.; Springfield, S. A.;
Hargreaves, R.; Burns, H. D.; Goulet, M. T.; Hagmann, W. K.
Discovery of N-{(1S,2S)-2-(3-Cyanophenyl)-3-[4-(2-[18F]fluoroethox-
y)phenyl]-1-methylpropyl}-2-methyl-2-[(5-methylpyridin-2-yl)oxy]pro-
panamide, a cannabinoid-1 receptor positron emission tomography
tracer suitable for clinical use. J. Med. Chem. 2007, 50, 3427–3430.
(29) Yasuno, F.; Brown, A. K.; Zoghbi, S. S.; Krushinski, J. H.; Chernet,
E.; Tauscher, J.; Schaus, J. M.; Phebus, L. A.; Chesterfield, A. K.;
Felder, C. C.; Gladding, R. L.; Hong, J.; Halldin, C.; Pike, V. W.;
Innis, R. B. The PET radioligand [11C]MePPEP binds reversibly and
with high specific signal to cannabinoid CB1 receptors in nonhuman
primate brain. Neuropsychopharmacology 2008, 33, 259–269.
(30) Laruelle, M.; Slifstein, M.; Huang, Y. Relationships between ra-
diotracer properties and image quality in molecular imaging of the
JM800329Z