Development of new carbon-11 labelled radiotracers for imaging GABA A- and GABAB-benzodiazepine receptors

Article abstract of DOI:10.1016/j.bmc.2012.05.046

Two quinolines identified as positive allosteric modulators of γ-aminobutyric acid (GABA)_A receptors containing the α₂ subunit, 9-amino-2-cyclobutyl-5-(6-methoxy-2-methylpyridin- 3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (4) and 9-amino-2-cyclobutyl- 5-(2-methoxypyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (5), were radiolabelled at the methoxy position with carbon-11 (half-life = 20.4 min). These quinolines represent a new class of potential radiotracers for imaging the benzodiazepine site of GABA_A receptors with positron emission tomography (PET). Both radiotracers were reliably isolated following reaction of their respective pyridinone/pyridinol tautomeric precursors with [ ¹¹C]CH₃I in clinically useful, formulated quantities (2.9% and 2.7% uncorrected radiochemical yield, respectively, relative to [ ¹¹C]CO₂) with high specific activities (>70 GBq μmol^-1; >2 Ci μmol^-1) and high radiochemical purities (>95%). The radiosyntheses reported herein represent rare examples of selectively isolating radiolabelled compounds bearing [¹¹C]2- methoxypyridine moieties. Although both radiotracers demonstrated promising imaging characteristics based on preliminary ex vivo biodistribution studies in conscious rodents, higher brain uptake was observed with [¹¹C]5 and therefore this radiotracer was further evaluated. Carbon-11 labelled 5 readily penetrated the brain (>1 standard uptake value in cortical regions at 15 min post-injection of the radiotracer), had an appropriate regional brain distribution for GABA_A receptors that appeared to be reversible, and did not show any appreciable radiometabolites in rat brain homogenates up to 15 min post-injection. Preadministration of flumazenil (1, 10 mg kg^-1) or 5 (5 mg kg^-1) effectively blocked >50% of [¹¹C]5 binding to the GABA_A receptor-rich regions, thereby suggesting that this radiotracer is worthy of further evaluation for imaging GABA_A receptors. Additionally (R,S)-N-(1-(3-chloro-4-methoxyphenyl)ethyl)-3,3- diphenylpropan-1-amine, 6, an allosteric modulator of GABA_B receptors, was efficiently labelled in one step using [¹¹C]methyl iodide. Ex vivo biodistribution studies in conscious rats showed low brain uptake, therefore, efforts are underway to discover alternative radiotracers to image GABA_B. In conclusion, [¹¹C]5 is worthy of further evaluation in higher species for imaging GABA_A receptors in the central nervous system.

Full text of DOI:10.1016/j.bmc.2012.05.046

Bioorganic & Medicinal Chemistry 20 (2012) 4482–4488
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of new carbon-11 labelled radiotracers for imaging
GABAA- and GABAB-benzodiazepine receptors
Matthew D. Moran a, Alan A. Wilson a, Charles S. Elmore b,c, Jun Parkes a, Alvina Ng a, Oleg Sadovski a,
Ariel Graff a, Zafiris J. Daskalakis a, Sylvain Houle a, Marc J. Chapdelaine b, Neil Vasdev a, ,
⇑
a Centre for Addiction and Mental Health and University of Toronto, 250 College St., Toronto, ON, Canada M5T 1R8
b Department of Chemistry, AstraZeneca Pharmaceuticals, Wilmington, DE 19850, United States
c Department of DMPK, AstraZeneca Pharmaceuticals, Mölndal, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 February 2012
Revised 5 May 2012
Accepted 12 May 2012
Available online 24 May 2012
Two quinolines identified as positive allosteric modulators of
c-aminobutyric acid (GABA)A receptors
containing the a2 subunit, 9-amino-2-cyclobutyl-5-(6-methoxy-2-methylpyridin-3-yl)-2,3-dihydro-1H-
pyrrolo[3,4-b]quinolin-1-one (4) and 9-amino-2-cyclobutyl-5-(2-methoxypyridin-3-yl)-2,3-dihydro-
1H-pyrrolo[3,4-b]quinolin-1-one (5), were radiolabelled at the methoxy position with carbon-11
(half-life = 20.4 min). These quinolines represent a new class of potential radiotracers for imaging the
benzodiazepine site of GABAA receptors with positron emission tomography (PET). Both radiotracers
were reliably isolated following reaction of their respective pyridinone/pyridinol tautomeric precursors
with [11C]CH3I in clinically useful, formulated quantities (2.9% and 2.7% uncorrected radiochemical yield,
Keywords:
Radiopharmaceuticals
Positron emission tomography
GABAA receptors
GABAB receptors
Carbon-11
respectively, relative to [11C]CO2) with high specific activities (>70 GBq molÀ1; >2 Ci mol–1) and high
l l
radiochemical purities (>95%). The radiosyntheses reported herein represent rare examples of selectively
isolating radiolabelled compounds bearing [11C]2-methoxypyridine moieties. Although both radiotracers
demonstrated promising imaging characteristics based on preliminary ex vivo biodistribution studies in
conscious rodents, higher brain uptake was observed with [11C]5 and therefore this radiotracer was
further evaluated. Carbon-11 labelled 5 readily penetrated the brain (>1 standard uptake value in cortical
regions at 15 min post-injection of the radiotracer), had an appropriate regional brain distribution for
GABAA receptors that appeared to be reversible, and did not show any appreciable radiometabolites in
rat brain homogenates up to 15 min post-injection. Preadministration of flumazenil (1, 10 mg kgÀ1) or
5 (5 mg kgÀ1) effectively blocked >50% of [11C]5 binding to the GABAA receptor-rich regions, thereby
suggesting that this radiotracer is worthy of further evaluation for imaging GABAA receptors. Additionally
(R,S)-N-(1-(3-chloro-4-methoxyphenyl)ethyl)-3,3-diphenylpropan-1-amine, 6, an allosteric modulator of
GABAB receptors, was efficiently labelled in one step using [11C]methyl iodide. Ex vivo biodistribution
studies in conscious rats showed low brain uptake, therefore, efforts are underway to discover alternative
radiotracers to image GABAB. In conclusion, [11C]5 is worthy of further evaluation in higher species for
imaging GABAA receptors in the central nervous system.
Published by Elsevier Ltd.
1. Introduction
neurosteroids, etc.), binds to GABAA receptors, conformational
changes increase the permeability of the central pore to chloride
Gamma-aminobutyric acid (GABA) receptors constitute the
largest population of inhibitory neurotransmitter receptors in the
mammalian brain. The GABAA receptor is a gated chloride-ion
channel with a primary GABA binding site as well as multiple allo-
steric modulatory sites. When GABA, in conjunction with positive
allosteric modulatory compounds (benzodiazepines, barbituates,
ions, resulting in chloride flux that hyperpolarizes the neuron.1
Of all the allosteric sites known for GABAA, the benzodiazepine
(BZ) site has been the most studied. The GABAA receptor has a pen-
tameric structure that is composed of seven families of protein
subunits: a1–6, b1–4, c1–3, d, e, p and q1–3. a subunit is of par-
2,3 The
ticular importance in determining the pharmacology of BZ-type
drugs and alcohol.4,5 Benzodiazepines (and related drugs that bind
to the BZ site) are amongst the most widely prescribed drugs in
modern medicine: in addition to their anxiolytic, anticonvulsant
and sedative effects, they are extensively used in the treatment
of several illnesses of high socioeconomic burden including
Alzheimer’s Disease, schizophrenia, chronic alcohol dependency,
⇑
Corresponding author. Tel.: +1 617 643 4736; fax: +1 617 726 6165.
E-mail address: vasdev.neil@mgh.harvard.edu (N. Vasdev).
Present address: Department of Nuclear Medicine and Molecular Imaging,
Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114,
United States.
0968-0896/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.05.046

M. D. Moran et al. / Bioorg. Med. Chem. 20 (2012) 4482–4488
4483
O
O
O
N
N
N
N
N
O
N
N
O
O
F
N
N
CH
*
N3
CH
*
3
3
*
O
I
O
O
[11C]1
[11C]3
[
123I]2
Figure 1. Commonly used radiotracers for imaging the BZ site of GABAA.
mood disorders, post-traumatic stress disorder, etc., and are also
postulated to play a role in cognition, consciousness and sleep.6
Further research has shown that subtype-selective compounds
for the benzodiazepine site can have anti-anxiolytic effects while
avoiding the side effects experienced from earlier BZs:7,8 including
ataxia, sedation, potentiation of alcohol, tolerance and dependence
upon chronic administration.9,10
reported attempts to develop radiolabelled compounds for imaging
GABAB receptors: CGP62349, a GABAB receptor antagonist, was
successfully labelled with carbon-11 (11C; t = 20.4 min) but did
½
not penetrate rat brain,23 while [11C]-bacolofen, a radiotracer
based on a GABAB receptor agonist, has not been evaluated
in vivo.34 Kerr and co-workers have identified a number of
fendiline derivatives that are potent positive allosteric modulators
for the GABAB receptor, with (R,S)-N-(1-(3-chloro-4-methoxy-
phenyl)ethyl)-3,3-diphenylpropan-1-amine (6) displaying high
potency for this target (EC50, 10 nM).35 Herein we also report
the radiosynthesis of carbon-11 labelled (R,S)-N-(1-(3-chloro-4-
methoxyphenyl)ethyl)-3,3-diphenylpropan-1-amine ([11C]6) as a
potential radiotracer for imaging GABAB receptors, and preliminary
ex vivo brain biodistribution studies following tail-vein injection of
this radiotracer in conscious rats.
Positron emission tomography (PET) has been extensively used
to image GABAA receptors, and radiopharmaceutical development
for this target has been extensively reviewed.6 The most well char-
acterized radiopharmaceutical for imaging GABAA receptors is
[
11C]flumazenil ([11C]1, Ro 15-1788, Fig. 1), a potent, non-subtype
selective BZ inverse agonist that binds to the a1,2,3, and 5 subunits on
the BZ site on GABAA receptors in the human brain.8,11,12 Deriva-
tives of flumazenil have been labelled with carbon-11 at the ester
moiety13 and with fluorine-18 at the 4-position of the phenyl
ring14,15 and evaluated as PET radiopharmaceuticals in human sub-
jects. The structurally related radioiodinated analogue, [123I]-
iomazenil ([123I]2, Ro 16-0154, Fig. 1) has been extensively used
for single photon emission computed tomography (SPECT),16 and
has alternately been radiolabelled with carbon-11 at the nitrogen
atom (Fig. 1).17 Carbon-11 labelled [11C]Ro15 4513 ([11C]3, Fig. 1)
is an established radiotracer for GABAA receptors in human sub-
jects,18,19 and has more recently been proposed to be selective
for the a5 subtype.19,20 Other PET radiotracers for imaging GABAA
2. Results
2.1. Chemistry and radiochemistry
Our radiosynthetic strategy for the preparation of [11C]4 and
[
11C]5, involves reaction of their respective desmethyl precursors
with
[
11C]CH3I. Both 9-amino-2-cyclobutyl-5-(2-methyl-6-oxo-
1,6-dihydropyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-
1-one (7) and 9-amino-2-cyclobutyl-5-(2-oxo-1,2-dihydropyridin-
3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (9), were effi-
receptors include [18F]fluoroethylflumazenil,21,22
[
[
[
11C]suriclone,23
11C]fludiazepam,26 and
18F]oxoquazepam,27 but these radiotracers all lack subtype selec-
11C]flunitrazepam,24 11C]diazepam,24,25
[ [
ciently prepared by demethylation of
4 and 5, respectively
(Scheme 1). Because compounds 7 and 9 exist as tautomers
between their pyridinone and pyridinol forms (Schemes 2 and 3),
reactions with [11C]CH3I will most likely favor the methylation
reaction at the secondary amine of this moiety (vide infra).
Therefore, 9-amino-2-cyclobutyl-5-(1,2-dimethyl-6-oxo-1,6-dihy-
dropyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (8)
tivity. There is still a paucity of radiotracers developed for imaging
the GABAA receptor that are not derived from the BZ core.6
We here report the radiosyntheses of [11C]9-amino-2-cyclobu-
tyl-5-(6-methoxy-2-methylpyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,
4-b]quinolin-1-one ([11C]4)28 and [11C]9-amino-2-cyclobutyl-5-(2-
methoxypyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one
([11C]5) and biological evaluations of these non-benzodiazepine
radiotracers in conscious rodent models with the goal of discover-
ing improved PET radiotracers for imaging the BZ site of GABAA.
Compounds 4 and 5 were identified as having excellent potential
for radiopharmaceutical development for the following reasons:
(1) they are amenable for radiolabelling with carbon-11 at the
methoxy position; (2) recently reported structure-activity relation-
ship studies have identified that these quinolines are potent
and
9-amino-2-cyclobutyl-5-(1-methyl-2-oxo-1,2-dihydropyri-
din-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (10), were
prepared by reaction of methyl iodide in the presence of base, with
7 or 9, respectively (Scheme 1), to serve as reference standards
during radiochromatographic separation and analyses.
Preparation of [11C]4 and [11C]5 were successfully accomplished
by reactions of [11C]CH3I with 7 and 9, respectively (Schemes 2 and
3). While previous work has demonstrated that methylation reac-
tions using silver carbonate as the base most favors formation of
the desired methylated oxygen product with similar structural
motifs,36,37 analogous reactions failed to yield the products in our
hands. Instead, Cs2CO3 was found to give the best conversions
(Table S1) and was utilized as the base to achieve reasonable
conversions to [11C]4 and [11C]5 at room temperature. As antici-
pated, the major radiochemical products, [11C]8 and [11C]10, were
identified by coinjection with authentic 8 and 10, respectively, and
were effectively separated post-synthesis by HPLC (ratio of
towards the GABAA a1 and a2-subunits but selective over a5-sub-
units;28 and (3) these functionally selective compounds would
represent a potentially new class of quinoline-based radiotracers
for imaging the BZ site of GABAA receptors.28,29 Both radiotracers
were evaluated ex vivo in rat brain regions and whole blood, and
where appropriate, metabolite analyses were conducted on both
brain homogenates and plasma.
While GABAA receptors have been extensively studied with
PET,6 there are currently no radiotracers for imaging GABAB recep-
tors in the living human brain. The GABAB receptors are considered
an important target in the brain, as they have been implicated in
addiction to alcohol30,31 and cocaine,32 as well as epilepsy.33 Radi-
otracer leads for this elusive target are still sparse, with only two
[
11C]4:[11C]8 ꢀ [11C]5:[11C]10 ꢀ 1:4). The radiosyntheses reported
herein represent rare examples of selectively isolating [11C]2-
methoxypyridine moieties and should prove to be generally appli-
cable within this class of compounds.

4484
M. D. Moran et al. / Bioorg. Med. Chem. 20 (2012) 4482–4488
NH2
O
NH2
N
NH2
N
O
O
N
N
N
BBr3:Me2S
PhCH3
CH3I, NaOH
N
MeOH
HN
N
N
O
O
O
4
7 (72%)
8 (51%)
NH2
O
NH2
NH2
O
O
N
N
N
BBr3:Me2S
PhCH3
CH3I, NaOH
MeOH
N
N
N
O
O
O
N
HN
N
9 (94%)
5
10 (74%)
Scheme 1. Preparation of compounds 7–10.
NH2
O
NH2
O
N
[
11C]CH3I
N
N
N
Cs2CO3
DMF, rt
HN
N
H311C
'LOOP' method
O
O
[11C]8
NH2
O
NH2
N
O
N
N
[
11C]CH3I
N
Cs2CO3
N
N
DMF, rt
'LOOP' method
O
O
H311C
H
[11C]4
7
Scheme 2. Preparation of [11C]4 and the undesired product, [11C]8.
Automated reactions of 7 (DMF, 25 °C, 1 min, Scheme 2) and 9
(DMSO, 25 °C, 1 min, Scheme 3) with [11C]methyl iodide were
accomplished in a clean, dry HPLC loop (the ‘LOOP’ method)38
using a modified radiofluorination module.39 After purification by
HPLC (Figs. S1a and S2a) and formulation, the products, [11C]4
and [11C]5, were obtained in 2.9% (910.6 MBq, 24.6 mCi) and 2.7%
(847.8 MBq, 23.0 mCi) radiochemical yields, respectively (based
on our full scale production of [11C]CO2 (31.4 GBq; 850 mCi) uncor-
rected for decay). The times to end-of-synthesis (EOS) were
30 min, with the formulated products having specific activities of
different conditions (solvents, pH), with different analytical
columns, futher established the identity of the radiotracers. Under
all conditions
[ [
11C]4 and 11C]5 co-chromatographed with
authentic 4 and 5. On storage for 120 min, both compounds did
not show signs of radiolysis as determined by HPLC analysis.
Compound 635 was O-demethylated in moderate yield to
prepare the desmethyl precursor, 11 (Scheme 4). Clinically useful
quantities of [11C]6 (5.0 GBq, 136 mCi) were efficiently achieved
by reactions of 11 with [11C]CH3I (Scheme 4) in DMF in the
presence of tetrabutylammonium hydroxide (1 N TBAOH in MeOH)
via the ‘LOOP’ method38 using a modified GE TRACERlabTM FXFN
radiosynthesis module.39 After purification by preparative HPLC
(Fig. S3) and formulation, [11C]6 was obtained in a radiochemical
yield (RCY) of 16% (uncorrected for decay, based on production of
100 30 GBq
l
molÀ1 (2.7 0.9 Ci
l ; n = 6) and 70 20
molÀ1
GBq
l
molÀ1 (2.0 0.5 Ci
l
mol–1; n = 6), respectively, with radio-
chemical purities >95% (Figs. S1b and S2b). Co-injection of the
radioactive products with authentic standards of 4 and 5 under

M. D. Moran et al. / Bioorg. Med. Chem. 20 (2012) 4482–4488
4485
NH2
N
O
NH2
N
O
N
[
11C]CH3I
N
O
Cs2CO3
O
DMSO, rt
HN
N
H311C
'LOOP' method
[11C]10
NH2
N
NH2
O
O
N
N
[
11C]CH3I
N
HO
O
Cs2CO3
H311C
N
DMSO, rt
N
'LOOP' method
[11C]5
9
Scheme 3. Preparation of [11C]5 and the undesired product, [11C]10.
HO
Cl
O
O
H311C
H
H
N
H
N
HBr, HOAc
[
11C]CH3I
N
Cl
Cl
1N TBAOH/DMF
120 ºC, 24 h
rt, 5 min
'LOOP' method
11C]6 (16% RCY)
11 (52%)
6
[
Scheme 4. Preparation of the desmethyl precursor, 11, and radiosynthesis of [11C]6.
31.4 GBq (850 mCi) of [11C]CO2), in a synthesis time of 30 min.
Analysis of the final formulated product by HPLC (Fig. S4) gave a
Upon tail-vein injection of [11C]5, moderate levels of radioac-
tivity (>0.4% ID/g) were taken up in rat brain at early time points
(Table 1, n = 4 per time point), and were effectively cleared by
60 min post injection. The regional heterogeneity of radioactivity
reflects the known distribution of GABAA in rat brain,44 with the
highest levels of radioactivity in the frontal cortex and rest of
cortex. The fast kinetics of uptake and washout of radioactivity
is also a favorable characterisitic for a potential radiotracer
labelled with carbon-11.
specific activity of 160 40 GBq l l ;
molÀ1 (4.3 1.2 Ci molÀ1
n = 3), and a radiochemical purity >99% at end of synthesis.
2.2. Ex vivo biodistribution studies with [11C]5 in rats
Preliminary biodistribution studies of [11C]4 and [11C]5 (Fig. S5)
and [11C]6 (Table S2) were carried out as previously described by
our group,40 and demonstrated that [11C]5 had higher uptake in
rat brain than [11C]4; as such, this compound was chosen for fur-
ther evaluations for imaging GABAA. Upon tail-vein injection of
2.3. Metabolism studies
[
11C]6, however, only low levels of radioactivity (<0.2% injected
The presence of significant quantities of radioactive metabolites
in the regions of interest during a PET scan may confound mea-
surements (i.e. improper quantification of the images and interpre-
tation of the results).45 In order to quantify the extent of
dose per gram of wet tissue (%ID/g) across all brain regions at all
time points) were taken up in rat brain (Table S2). Because success-
ful radiotracers for imaging the central nervous system (CNS) typ-
ically express a %ID/g of tissue P0.5% in rat brain,41 the relatively
low brain uptake of [11C]6 precluded its further study for imaging
central GABAB in vivo.
Table 1
Ex vivo biodistribution of [11C]5 in rodents (%ID/g (decay-corrected), n = 4).
While physicochemical properties of a potential PET radiotracer
for imaging neuroreceptors has not been well determined, log P
values (lipophilicities) between 2 and 3 are considered ideal.42 At
physiological pH (7.40), using the octanol/buffer shake-flask meth-
od,43 the logD7.4 of [11C]4 was 4.12 0.06 (n = 8), while the logD7.4
of [11C]5 was 3.00 0.06. The logD7.4 of [11C]6 was 3.2 0.1. Our
future efforts are aimed at developing less lipophilic fendiline
derivatives as potential therapeutics and radiotracers for imaging
GABAB receptors.
Time (post injection)
5 min
15 min
30 min
60 min
Cerebellum
Hippocampus
Striatum
Cortex (front)
Cortex (rest)
Rest
0.36 0.02
0.20 0.06
0.26 0.02
0.47 0.07
0.45 0.03
0.25 0.02
0.14 0.1
0.34 0.05
0.29 0.05
0.21 0.04
0.53 0.08
0.46 0.06
0.27 0.04
0.15 0.04
0.26 0.05
0.24 0.04
0.15 0.06
0.44 0.09
0.37 0.06
0.22 0.05
0.11 0.03
0.13 0.02
0.09 0.02
0.06 0.01
0.21 0.05
0.16 0.02
0.11 0.02
0.08 0.01
Blood

4486
M. D. Moran et al. / Bioorg. Med. Chem. 20 (2012) 4482–4488
metabolism of [11C]5, rats (n = 2 per time point) were injected with
the radiotracer (tail-vein), and homogenized brain extracts and
plama were isolated at 5 and 15 min post injection. While the HPLC
analysis of radioactivity in rat plama at 15 min post injection
showed significant metabolism to both a hydrophilic (tR = 1 min)
and a lipophilic (tR = 5.3 min) metabolite (71% parent compound
at 15 min post injection, Fig. 2b; showing the rapid metabolism
at 5 min), no radioactive metabolites were detected in the brain,
with [11C]5 accounting for >99% of the radioactivity present in
the rat brain tissue at 15 min post injection (Fig. 2a). Thus, the
metabolites present in the plama are not reflected in brain tissue
and unlikely to interfere with imaging studies of central nervous
system (CNS) GABAA receptors.
Table 2
Effect of drug pretreatment on the specific binding of [11C]5 in rat brain (%ID/g).
Treatment (dose, mg/kg)
Vehicle
5 (0.1)
5 (5.0)
1 (10)
Cerebellum
Hippocampus
Striatum
Cortex (front)
Cortex (rest)
Rest
0.26 0.02
0.22 0.02
0.13 0.02
0.42 0.04
0.36 0.02
0.20 0.01
0.11 0.01
0.22 0.04
0.17 0.04
0.14 0.01
0.36 0.07
0.27 0.11
0.16 0.07
0.11 0.01
0.12 0.03
0.11 0.02
0.10 0.01
0.16 0.06
0.14 0.05
0.09 0.04
0.12 0.01
0.09 0.03
0.08 0.03
0.08 0.02
0.13 0.06
0.11 0.04
0.09 0.02
0.12 0.01
Blood
displays binding that appeared to be reversible. The ex vivo phar-
macology is indicative of selective binding to GABAA receptors, as
pre-administration of flumazenil or 5 blocks specific uptake of
the radiotracer. Plasma and brain radioactive metabolite measure-
ments are in full accord with the desired properties of a selective
GABAA receptor imaging agent for PET. The above-mentioned prop-
erties indicate that [11C]5 and related quinoline derivatives are
leading-candidate radiotracers worthy of further exploration for
imaging GABAA receptors in higher species.
2.4. Blocking studies
In light of the promising biodistribution and metabolism stud-
ies, blocking studies were carried out with [11C]5. Four groups of
rats were pretreated (i.p.) at 30 min prior to administration of
[
11C]5 with: (1) vehicle (5% DMSO/5% EtOH in saline containing
5% Tween80 w/w), (2) 0.1 mg/kg of 5, (3) 5 mg/kg of 5, and (4)
10 mg/kg of 1 (highly selective for the a1 3, and 5 subunits).
All groups were sacrificed at 15 min post injection of the radio-
tracer (Table 2).
Specificity of binding to GABAA was demonstrated by showing
>70% reduction of radioactivity in brain regions of interest, includ-
ing cerebellum, hippocampus and cortical regions, by treatment
with high doses of 5 (5 mg/kg) and 1 (10 mg/kg; Table 2). These
findings indicate that [11C]5 likely binds selectively to GABAA in
the rat brain.
,
a2
,
a
a
4. Experimental section
4.1. General methods
The compounds 9-amino-2-cyclobutyl-5-(6-methoxy-2-meth-
ylpyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (4)
and 9-amino-2-cyclobutyl-5-(2-methoxypyridin-3-yl)-2,3-dihy-
dro-1H-pyrrolo[3,4-b]quinolin-1-one (5), were prepared as previ-
ously described.28,29 All other chemicals were obtained from
commercial sources and were used as received without further
purification unless indicated. For radiochemical syntheses, THF
was freshly distilled under nitrogen from LiAlH4. All water used
was distilled and deionized (unless otherwise stated). A Scandi-
tronix MC 17 cyclotron was used for radionuclide production.
Purifications and analyses of radioactive mixtures were performed
by HPLC with an in-line UV (254 nm) detector in series with a NaI
crystal radioactivity detector (radiosynthesis and QC). Isolated
radiochemical yields were determined with a dose calibrator
(Capintec CRC-712M).
3. Conclusion
The present work has demonstrated that [11C]4, [11C]5 and
[
11C]6 can be reliably synthesized in quantities and at the specific
activities and radiochemical purities required for human PET stud-
ies, and the radiolabelling of a pyridinone/pyridinol tautomeric
precursor developed herein should be broadly applicable to the
preparation of structurally related [11C]2-methoxypyridine moie-
ties with [11C]CH3I. Furthermore, [11C]6 represents a rare example
of a radiotracer developed for imaging GABAB receptors that has
been evaluated in vivo. While [11C]6 was not readily taken up in
rat brain, the ex vivo biodistribution studies of [11C]5 demonstrate
that this radiotracer readily crosses the blood-brain barrier, has an
appropriate regional brain distribution for GABAA receptors, and
All animal experiments were carried out under humane condi-
tions, with approval from the Animal Care Commitee at the Centre
for Addiction and Mental Health and in accordance with the guide-
lines set forth by the Canadian Council on Animal Care.
4.2. Chemistry
4.2.1. 9-Amino-2-cyclobutyl-5-(2-methyl-6-oxo-1,6-
dihydropyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-
one (7)
A solution of 0.100 g (0.27 mmol) of 9-amino-2-cyclobutyl-5-
(6-methoxy-2-methylpyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]
quinolin-1-one (4) in 2 mL of toluene was stirred under N2 as
0.167 g (0.53 mmol) of BBr3ÁMe2S complex was added. The mixture
was heated at 100 °C for 2 h and was then cooled to room temper-
ature. It was diluted with 4 mL of 1 N HCl and the resulting solid
was removed by filtration. The solid was taken up in 80 mL of
30% MeOH in CH2Cl2 and the resulting slurry was sonicated and
filtered to give 7 as an off-white solid (0.069 g, 72% yield): mp:
257–258 °C; 1H NMR (400 MHz, MeOD/DMSO-d6) d (ppm) = 1.70–
1.76 (m, 2H), 1.91 (s, 3H), 2.13–2.22 (m, 2H), 2.24–2.35 (m, 2H),
4.55–4.78 (m, 3H), 6.36 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 9.2 Hz, 1H),
7.75 (t, J = 7.9 Hz, 1H), 7.83 (d, J = 7.4 Hz, 1H), 8.66 (d, J = 8.6 Hz,
1H). HRMS (ESI, positive mode) m/z, [M+H]+ calcd for
Figure 2. HPLC chromatograms of (a) rat brain extract after injection of [11C]5
(15 min post injection), (b) rat plasma after injection of
[
11C]5 (5 min post
injection), and (c) control plasma containing [
11C]5. The peak at ca. 6.2 min
C
21H21N4O2+: 361.1659, found: 361.1629.
corresponds to unmetabolized [11C]5.

M. D. Moran et al. / Bioorg. Med. Chem. 20 (2012) 4482–4488
4487
4.2.2. 9-Amino-2-cyclobutyl-5-(1,2-dimethyl-6-oxo-1,6-dihydro-
pyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (8)
A suspension of 0.131 g (0.36 mmol) of 9-amino-2-cyclobutyl-
5-(2-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2,3-dihydro-1H-pyr-
rolo[3,4-b]quinolin-1-one (7) in 4 mL of MeOH was stirred as
0.533 mL (0.80 mmol) of 1.5 M NaOH was added. After stirring
the resulting solution for 10 min, 0.150 g (1.06 mmol) of iodometh-
ane was added and the solution was stirred at room temperature
overnight. The reaction mixture was diluted with 10 mL of 10%
NaHCO3 and the resulting solution was extracted three times with
10 mL of CH2Cl2. The combined organics were dried with MgSO4
and then filtered, and the resulting solution concentrated to dry-
ness. Purification by silica gel chromatography afforded 8 as an
off-white solid (0.069 mg, 51%): mp: 235–236 °C; 1H NMR
(400 MHz, CDCl3) d (ppm) = 1.74–1.85 (m, 2H), 2.13 (s, 3H), 2.19–
2.31 (m, 2H), 2.32–2.43 (m, 2H), 3.69 (s, 3H), 4.37–4.54 (m, 2H),
4.73–4.84 (m, 1H), 6.55 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 9.2 Hz, 1H),
7.54 (dd, J = 7.1, 8.3 Hz, 1H), 7.60–7.65 (m, 1H), 8.26 (dd, J = 1.6,
8.4 Hz, 1H). HRMS (ESI, positive mode) m/z, [M+H]+ calcd for
a 1 M NaHCO3 solution. The aqueous phase was back-extracted
with a 1:5 mixture of MeOH/CHCl3 (6 mL) and the combined
organic extracts were dried (MgSO4), filtered and concentrated
under vacuum. The product was purified by preparative TLC
(10:1 CHCl3/MeOH) to give 71 mg of 11 (52%) as an off-white solid:
mp: 62 °C (free base); 1H NMR (400 MHz, CDCl3) d (ppm) = 1.29
(d, J=6.65 Hz, 3H), 2.14–2.36 (m, 2H), 2.39–2.58 (m, 2H), 3.63
(q, J = 6.65 Hz, 1H), 3.96 (t, J = 7.83 Hz, 1H), 6.85 (d, J = 8.22 Hz,
1H), 7.01 (dd, J-1.96, 8.22 Hz, 1H), 7.11–7.33 (m, 11H). HRMS
(ESI, positive mode) m/z, [M+H]+ calcd for C23H25NOCl+:
366.1619, found: 366.1603.
4.3. Radiosynthesis of [11C]4 and [11C]5
The method of Crouzel,46 with some modifications, was used to
produce [11C]CH3I. Preparation of the carbon-11 labelled com-
pounds was accomplished via the ‘LOOP’ method38 using a modi-
fied radiosynthesis module as previously described.39 Precursors
7 (0.25 mg, 0.69
in 80 L of DMF and DMSO, respectively. The solution of 7 (9) was
added to a vial filled with 6 mg (18.4 mol) of Cs2CO3 and vortexed
lmol) and 9 (0.25 mg, 0.72 lmol) were dissolved
C
22H23N4O2+: 375.1816, found: 375.1788.
l
l
for 3 min. The resulting bright yellow solution was then decanted
from the excess solid carbonate via syringe and injected into a
clean, dry loop.38 The [11C]CH3I was then introduced, and the mix-
ture was reacted for 1 minute at ambient temperature. The reac-
tion mixture was then purified via HPLC (LUNA C18(2),
4.2.3. 9-Amino-2-cyclobutyl-5-(2-oxo-1,2-dihydropyridin-3-yl)-
2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (9)
A solution of 0.042 g (0.12 mmol) of 9-amino-2-cyclobutyl-5-
(2-methoxypyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-
1-one (5) in 1 mL of toluene under nitrogen was stirred as
0.073 mg (0.23 mmol) of BBr3ÁMe2S complex was added and the
resulting mixture was heated in at 100 °C for 3 h. After cooling to
room temperature, 2 mL of aq 1 N HCl was added and the resulting
precipitate was collected by filtration to afford of 9 as an off-white
solid (0.040 g, 94%): mp: 242–243 °C; 1H NMR (400 MHz, DMSO-
d6) d (ppm) = 1.65–1.78 (m, 2H), 2.10–2.23 (m, 2H), 2.26–2.40
(m, 2H) 4.62–4.71 (m, 1H), 4.74 (s, 2H), 6.43 (t, J = 6.7 Hz, 1H),
7.65 (d, J = 6.8 Hz, 2H), 7.74 (dd, J = 7.4, 8.4 Hz, 1H), 7.84 (dd,
J = 1.2, 7.2 Hz, 1H), 8.71 (dd, J = 1.4, 8.4 Hz, 1H), 8.8 (s, 1H), 9.95
(s, 1H), 12.09 (s, 1H). HRMS (ESI, positive mode) m/z, [M+H]+ calcd
for C20H19N4O2+: 347.1503, found: 347.1472.
250 Â 10 mm, 10
l
; 60:40 CH3CN/H2O + 0.1 N NH4+HCO2À; 5 mL/
min (3 mL/min), 254 nm; Figs. S1a and S2a). The radiochemical
peak (7.3 min, [11C]4; 9.2 min, [11C]5) was collected and formu-
lated as previously described.47 The formulated product was ana-
lyzed by HPLC (Prodigy C18 ODS3 (250 Â 4.6 mm, 10
l); 60:40
CH3CN/H2O + 0.1 N NH4+HCO2À; 2 mL/min (1.5 mL/min), 254 nm)
with a retention time of 3.6 min (3.9 min; Figs. S1b and S2b). The
radiochemical yield (2.9%, 910.6 MBq, 24.6 mCi,
[
11C]4; 2.7%,
847.8 MBq, 23.0 mCi, [11C]5), uncorrected for decay (based on our
full scale production of [11C]CO2 (31.4 GBq; 850 mCi), had specific
activities of 100 30 GBq
l l
molÀ1 (2.7 0.9 Ci molÀ1; n = 6) and
70 20 GBq l
l
molÀ1 (2.0 0.5 Ci molÀ1; n = 6), respectively, with
radiochemical purities >95%.
4.2.4. 9-Amino-2-cyclobutyl-5-(1-methyl-2-oxo-1,2-dihydro-
pyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (10)
A suspension of 150 mg (0.35 mmol) of 9-amino-2-cyclobutyl-
5-(2-oxo-1,2-dihydropyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]
quinolin-1-one (9) hydrochloride in 3 mL of MeOH was stirred at
room temperature as 0.517 mL (0.78 mmol) of aq 1.5 M NaOH
was added. After 10 min, 150 mg (1.06 mmol) of iodomethane
was added and the suspension was stirred at room temperature
overnight. The solid was removed by filtration and was washed
2 Â 10 mL water, 2 Â 10 mL of ether, and 2 Â 10 mL hexane. It
was then purified by silica gel chromatography to give 10 as a
white solid (0.125 g, 74%): mp: 187–188 °C, 1H NMR (400 MHz,
CDCl3) d (ppm) = 1.69–1.82 (m, 2H), 2.26 (m, 4H), 3.65 (s, 3H),
4.39 (s, 2H), 4.84 (m, 1H), 6.31 (t, J = 6.8 Hz, 1H), 6.68 (s, 2H),
7.17 (dd, J = 7.1, 8.3 Hz, 1H), 7.38 (dd, J = 2.0, 6.7 Hz, 1H), 7.48
(dd, J = 2.0, 6.8 Hz, 1H), 7.60 (dd, J = 1.5, 7.1 Hz, 1H), 7.70 (dd,
J = 1.4, 8.4, 1H). HRMS (ESI, positive mode) m/z, [M+H]+ calcd for
4.4. Radiosynthesis of [11C]6
The desmethyl precursor, 11 (1.4 mg. 3.8
(with vigorous vortexing for ca. 2 min) in a mixture of 72.5
DMF and 3.5 L of 1 N TBAOH (3.5 mol, in MeOH), followed by
loading onto the HPLC loop. After 5-min reaction with
11C]CH3I, the product was purified using a Phenomenex Luna
l
mol), was dissolved
lL of
l
l
a
[
(2) C18 10
l
(250 Â 10 mm) column and 60:40 CH3CN/
H2O + 0.1 N NH4HCO2 as the mobile phase with a flow rate of
5.0 mL min–1 (k = 254 nm; Fig. S3). The [11C]CH3I reacted with
11 to form [11C]6 (81% conversion by HPLC, tR ꢀ 9 min) and was
well separated during purification. Purified [11C]6 was formulated
in 10% EtOH/saline as described previously.39 At the end of syn-
thesis (30 min) the final product contained 4.9 GBq (133 mCi)
starting [11C]CO2: 31.4 GBq, 850 mCi. The radiochemical purity
was determined by analytical HPLC using a Phenomenex Luna
C
21H21N4O2+: 361.1659, found: 361.1628.
C18 10
l
(250 Â 4.6 mm) column using
a
flow rate of
2.0 mL minÀ1 and eluted with 60:40 CH3CN/H2O + 0.1 N NH4HCO2
(k = 280 nm; Fig. S4). Co-injections of [11C]6 with authentic 6 were
performed under several other HPLC conditions (columns, wave-
lengths, mobile phases) to confirm radiochemical purity. The
radiochemical yield of [11C]6 was 16% (5.0 GBq, 136 mCi); uncor-
rected for decay, based on production of 31.4 GBq (850 mCi) of
4.2.5. (R,S)-N-(1-(3-chloro-4-hydroxyphenyl)ethyl)-3,3-diphenyl-
propan-1-amine (11)
To 0.14 g (0.37 mmol) of (R,S)-N-(1-(3-chloro-4-methoxy-
phenyl)ethyl)-3,3-diphenylpropan-1-amine (6) was added 2 mL
of a 48% HBr solution in acetic acid and 4 mL of glacial acetic acid.
The mixture was heated to 120 °C and stirred for 24 h. The solution
was evaporated under vacuum and the crude product was redis-
solved in 7 mL of CHCl3, and the organic phase was washed with
[
11C]CO2), with
(4.3 1.2 Ci
molÀ1; n = 3) and radiochemical purity >99% at end
of synthesis.
a specific activity of 160 40 GBq l
molÀ1
l

4488
M. D. Moran et al. / Bioorg. Med. Chem. 20 (2012) 4482–4488
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11C]6 in conscious male Sprague-Dawley rats were conducted
(n = 1 rat per time point) as previously described.40,48 For the full
biodistribution study, all rats received ca. 37 MBq (1 mCi) of
[
11C]5 in 0.3 mL of buffered saline (containing 10% ethanol) via
the tail vein and were sacrificed by decapitation at either 5, 15,
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and then counted for radioactivity (Table 1).
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4.6. Blocking studies
Rats received intraperitoneal injections of a solution (0.4–
0.6 mL) of 5% DMSO/5% EtOH in saline (containing 5% Tween80
w/w; vehicle), 0.1 mg/kg of 5, 5 mg/kg of 5, or 10 mg/kg of flu-
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4.7. Metabolism studies
Following tail-vein injection of [11C]5, as described above,
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HPLC using the method of Hilton and coworkers,49 with minor
modifications.50 Briefly, rat plasma from either time point was di-
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capture column (4.6 mm  20 m) that was packed in-house with
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OASIS HLB 30 lm (Waters, NJ). The capture column was eluted
with 1% aqueous CH3CN (2 mL/min) for approximately 3 min and
À
then back-flushed with 50:50 CH3CN/H2O + 0.1 N NH4+HCO2 (pH
4); 2.0 mL/min onto
a
Phenomenex Luna C18 Column
(250 Â 4.6 mm, 10
l
). The column effluents from both columns
29. Chang, H.-F.; Chapdelaine, M. J.; Dembofsky, B. T.; Herzog, K. J.; Horchler, C.;
Schmiesing, R. J. 2008. WO Patent 2008/155572 A2.
were monitored through a flow detector (Bioscan Flow-Count)
operated in coincidence mode. Whole brain from a control rat
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was removed and treated with ca. 1.3 MBq (37 l
Ci) of [11C]5, and
the whole brain was removed from rats sacrificed at 5 or 15 min
post-injection. Brains were individually homogenized with ice-
cold 80:20 CH3CN/H2O + 0.01 N HCl and centrifuged as previously
described by our laboratory,50 prior to radio-HPLC analysis of the
supernatant using the aforementioned method.
Acknowledgements
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The authors thank Armando Garcia, Winston Stableford and
Min Wong for assistance with the radiochemisty, and Justin Hicks
for assistance with the logD7.4 determinations. CAMH is gratefully
acknowledged for support in the form of a post-doctoral fellowship
(M.D.M.).
Supplementary data
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Supplementary data associated with this article can be found, in
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