Radiosyntheses and in vivo evaluation of carbon-11 PET tracers for PDE10A in the brain of rodent and nonhuman primate

Article abstract of DOI:10.1016/j.bmc.2014.03.028

The radiosyntheses and in vivo evaluation of four carbon-11 labeled quinoline group-containing radioligands are reported here. Radiolabeling of [¹¹C]1-4 was achieved by alkylation of their corresponding desmethyl precursors with [¹¹C]CH₃I. Preliminary biodistribution evaluation in Sprague-Dawley rats demonstrated that [¹¹C]1 and [ ¹¹C]2 had high striatal accumulation (at peak time) for [ ¹¹C]1 and [¹¹C]2 were 6.0-fold and 4.5-fold at 60 min, respectively. Following MP-10 pretreatment, striatal uptake in rats of [ ¹¹C]1 and [¹¹C]2 was reduced, suggesting that the tracers bind specifically to PDE10A. MicroPET studies of [¹¹C]1 and [ ¹¹C]2 in nonhuman primates (NHP) also showed good tracer retention in the striatum with rapid clearance from non-target brain regions. Striatal uptake (SUV) of [¹¹C]1 reached 1.8 at 30 min with a 3.5-fold striatum:cerebellum ratio. In addition, HPLC analysis of solvent extracts from NHP plasma samples suggested that [¹¹C]1 had a very favorable metabolic stability. Our preclinical investigations suggest that [ ¹¹C]1 is a promising candidate for quantification of PDE10A in vivo using PET.

Full text of DOI:10.1016/j.bmc.2014.03.028

Bioorganic & Medicinal Chemistry 22 (2014) 2648–2654
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Radiosyntheses and in vivo evaluation of carbon-11 PET tracers
for PDE10A in the brain of rodent and nonhuman primate
Jinda Fan a, Xiang Zhang a, Junfeng Li a, Hongjun Jin a, Prashanth K. Padakanti a, Lynne A. Jones a,
Hubert P. Flores b, Yi Su b, Joel S. Perlmutter a,b, Zhude Tu a,
⇑
a Department of Radiology, Washington University School of Medicine, 510 South Kingshighway Blvd., St. Louis, MO 63110, USA
b Department of Neurology, Washington University School of Medicine, 510 South Kingshighway Blvd., St. Louis, MO 63110, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The radiosyntheses and in vivo evaluation of four carbon-11 labeled quinoline group-containing radioli-
gands are reported here. Radiolabeling of [11C]1–4 was achieved by alkylation of their corresponding
desmethyl precursors with [11C]CH3I. Preliminary biodistribution evaluation in Sprague-Dawley rats
demonstrated that [11C]1 and [11C]2 had high striatal accumulation (at peak time) for [11C]1 and
Received 18 January 2014
Revised 6 March 2014
Accepted 17 March 2014
Available online 26 March 2014
[
11C]2 were 6.0-fold and 4.5-fold at 60 min, respectively. Following MP-10 pretreatment, striatal uptake
in rats of [11C]1 and [11C]2 was reduced, suggesting that the tracers bind specifically to PDE10A. MicroPET
studies of [11C]1 and [11C]2 in nonhuman primates (NHP) also showed good tracer retention in the stri-
atum with rapid clearance from non-target brain regions. Striatal uptake (SUV) of [11C]1 reached 1.8 at
30 min with a 3.5-fold striatum:cerebellum ratio. In addition, HPLC analysis of solvent extracts from
NHP plasma samples suggested that [11C]1 had a very favorable metabolic stability. Our preclinical inves-
tigations suggest that [11C]1 is a promising candidate for quantification of PDE10A in vivo using PET.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
PDE10A
Carbon-11
PET imaging
MP-10
CNS
1. Introduction
Positron emission tomography (PET) allows noninvasive mea-
surement of drug disposition, localization, and quantification in
Phosphodiesterase 10A (PDE10A) is an enzyme that hydrolyzes
the phosphodiester bond in both cyclic adenosine monophosphate
(cAMP) and cyclic guanosine monophosphate (cGMP), resulting in
deactivation of intracellular secondary messengers which regulate
a wide variety of biological processes in the brain.1,2 PDE10A has
low expression in peripheral tissues and uniquely limited distribu-
tion in the brain, especially in the striatal medium spiny neurons
(MSN),3–5 which are the principal input sites of the basal ganglia,
and are involved in the regulation of motor, appetitive, and cogni-
tive processes.6–8 Numerous studies have shown that PDE10A
plays a central role in striatal signaling and is implicated in several
neuropsychiatric disorders including schizophrenia, Huntington’s
disease, Parkinson’s disease, obsessive–compulsive disorder, and
addiction.9–12 Since the enzyme structure was first reported in
1999,2,13,14 tremendous effort has been dedicated to developing
PDE10A inhibitors for the treatment of the aforementioned dis-
eases and other disorders characterized by reduced MSN activity.
animal models used for preclinical studies and in humans. PET
methodology is useful for the evaluation of drugs that target the
central nervous system (CNS). The efforts in developing therapeu-
tic agents for PDE10A inhibition has been accompanied by a grow-
ing interest in PET imaging agents for PDE10A. Our group reported
the first 11C-labeled PET tracer for PDE10A: [11C]papaverine.15 De-
spite promising in vitro results, in vivo imaging in NHP showed ra-
pid washout after high initial uptake; low tracer retention was
observed in both rat and NHP striatum. Although papaverine is
behaviorally active, kinetics did not allow PDE10A visualization
with PET. The fast washout from the CNS was attributed to the rel-
ative low affinity of papaverine for PDE10A (IC50 = 36 nM). Subse-
quent efforts in developing a PET imaging agent focused on more
potent inhibitors including MP-10 (IC50 = 1.26 nM).16,17 Several re-
search groups, including ours, independently reported the synthe-
sis and in vivo evaluation
[
11C]MP-10.18,19 Our preliminary
evaluation in rats demonstrated that the tracer bind specifically
to rodent striatum, with washout from non-target brain regions.19
Specific striatal binding of [11C]MP-10 was also observed by Plisson
et al. in pig and baboon using two different blocking strategies.18
Our tissue time–activity curve (TAC) analysis of the NHP imaging
⇑
Corresponding author. Tel.: +1 314 362 8487; fax: +1 314 362 8555.
E-mail address: tuz@mir.wustl.edu (Z. Tu).
http://dx.doi.org/10.1016/j.bmc.2014.03.028
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

J. Fan et al. / Bioorg. Med. Chem. 22 (2014) 2648–2654
2649
studies showed continuously increasing brain accumulation.
Radioactive metabolism studies in NHP plasma extracts and rodent
brain homogenates identified a major radiometabolite generated
by the breakdown of the phenol–ether linkage on MP-10 in both
species. The resulting tricyclic radiometabolite crossed the blood
brain barrier (BBB) and accumulated in CNS tissue. Polar metabo-
lites which accumulate in rodent brain have also been reported
for 18F-labeled PDE10A ligands.20
vessel. The mixture was purified by reversed phase HPLC (Agilent
Zorbax, SB-C18 column, 250 Â 10 mm, flow rate: 4.0 mL/min, UV
detection 254 nm). Inline radioactive detector signals were used
to identify product fractions which were collected in a vial contain-
ing 50 mL sterile water. The diluted eluent was passed through a
C-18 Plus Sep-Pak cartridge to remove the mobile phase; the radio-
active product was then rinsed with 10 mL sterile water; and then,
the product was eluted with 0.6 mL ethanol followed by 5.4 mL
saline, which was efficient to elute 80–95% of the radioactive prod-
uct from the cartridge. The formulated solution was passed
Given these findings, we proposed to introduce the [11C]meth-
oxy group at the quinoline fragment of the structures. Our hypoth-
esis was that by changing the labeling position on MP-10
analogues, we would avoid generation of radiometabolites capable
of crossing the BBB and thus improve quality of the brain imaging.
Structure–activity analysis (SAR) of 28 newly synthesized ana-
logues of MP-10, showed that derivatives with a methoxy group
on 3-, 4- and 6-positions of the quinoline fragment and their corre-
sponding analogues with N-methyl group on the 2-position of the
pyrazole ring exhibited high potency and selectivity over other
PDEs.21 Compounds 1, 2, 3, and 4 have IC50 values of 0.40 0.02,
0.28 0.06, 1.82 0.25, and 0.36 0.03 nM, respectively, (Fig. 1).
In this study we report the radiosynthesis of [11C]1–4, their biodis-
tribution in SD rats, microPET imaging studies in NHP, as well as
the metabolite analysis in NHP plasma.
through a 0.22 lm syringe filter into a sterile glass vial. The total
synthesis time was 50–55 min. Quality control was conducted on
an analytical HPLC system (Agilent Zorbax, SB-C18 column,
250 Â 4.6 mm, UV detection 254 nm).
2.1.1. 3-(Methoxy-11C)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-
pyrazol-3-yl)phenoxy)methyl)quinoline ([11C]1)
The mobile phase used in purification was acetonitrile/0.1 M
ammonium formate buffer pH = 4.5 (45:55, v/v); product fractions
were collected between 14.5 and 16.1 min; precursor (2-((4-(1-
methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)quino-
lin-3-ol) (5) retention time was 6.7 min. The QC mobile phase was
acetonitrile/0.1 M ammonium formate buffer pH 4.5 (59:41, v/v).
For the quality control using the analytical HPLC system, the reten-
tion time for [11C]1 was 4.7 min at a flow rate of 1.5 mL/min. The
sample was authenticated by co-injecting with the cold standard.
Radiochemical purity was >99%, chemical purity was >95%, radio-
chemical yield (RCY) was 55–70% (n > 5, decay corrected to EOB)
2. Experimental
2.1. General procedure of the radiosynthesis
Production of [11C]CH3I was via a [14N(p, 11C] nuclear reaction
a)
and the specific activity (SA) was >425 GBq/lmol (decay corrected
following the reported procedure.22,23 Briefly, [11C]CH3I was pro-
duced on-site from [11C]CO2 using a GE PETtrace CH3I Microlab.
Up to 51.8 GBq of [11C]CO2 was produced from the JSW BC-16/8
to EOB).
2.1.2. 4-(Methoxy-11C)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-
pyrazol-3-yl)phenoxy)methyl)quinoline [11C]2
cyclotron by irradiating
a gas target of 0.5% O2 in N2 for
The mobile phase used in the purification was acetonitrile/
0.1 M ammonium formate buffer pH = 6.5 (50:50, v/v); product
fractions were collected between 14.6 and 16.5 min; precursor
(2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)
quinolin-4-ol) (6) retention time was 5.6 min. The QC mobile phase
was acetonitrile/0.1 M ammonium formate buffer pH 6.5 (60:40, v/
v). The retention time for [11C]2 was 4.7 min at a flow rate of
1.0 mL/min. The sample was authenticated by co-injecting with
the cold standard. Radiochemical purity was >99%, chemical purity
was >95%, RCY 30-35% (n > 5, decay corrected to EOB) and SA
15–30 min with a 40 A beam of 16 MeV protons in the Barnard
l
Cyclotron Facility of Washington University School of Medicine.
After the GE PETtrace CH3I Microlab system converted the [11C]CO2
to [11C]CH4 using a nickel catalyst [Shimalite-Ni (reduced), Shima-
dzu, Japan P.N.221-27719] in the presence of hydrogen gas at
360 °C; the [11C]CH4 was further converted to [11C]CH3I by reaction
with iodine in the gas phase at 690 °C. Approximately 12 min fol-
lowing the end-of-bombardment (EOB), several hundred millicu-
ries of [11C]CH3I were delivered in the gas phase to the hot cell
where the radiosynthesis was accomplished.
[
11C]CH3I was bubbled for a period of 2–3 min into a solution of
the corresponding precursor24 (1–2 mg) in DMSO (0.20 mL) con-
taining aqueous sodium hydroxide (NaOH) solution (5 N, 3 L) at
>477 GBq/lmol (decay corrected to EOB).
2.1.3. 6-(Methoxy-11C)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-
pyrazol-3-yl)phenoxy)methyl)quinoline [11C]3
l
room temperature. When the trapping of radioactivity was com-
plete, the sealed reaction vessel was heated at 85 °C for 5 min.
After the heat source was removed, 1.8 mL high performance liquid
chromatography (HPLC) mobile phase was added to the reaction
The mobile phase used in the purification was acetonitrile/
0.1 M ammonium formate buffer pH = 4.5 (45:55, v/v); product
fractions were collected between 15.6 and 17.5 min; precursor
(2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)
quinolin-6-ol) 7 retention time was 6.1 min. The QC mobile phase
was acetonitrile/0.1 M ammonium formate buffer pH 6.5 (60:40, v/
v). The retention time for [11C]3 was 4.5 min at a flow rate of
1.0 mL/min. The sample was authenticated by co-injecting with
the cold standard. Radiochemical purity was >99%, chemical purity
was >95%, RCY 20-30% (n = 4, decay corrected to EOB) and SA
N
N
N
N
N
CH3
N
N
CH3
1
N
>480 GBq/lmol (decay corrected to EOB).
8
O
2
7
6
O
2.1.4. 4-(Methoxy-11C)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-
pyrazol-5-yl)phenoxy)methyl)quinoline [11C]4
3
R
OCH3
5
4
MP-10, R = H,
IC50 = 1.26 nM
The mobile phase used in the purification was acetonitrile/
0.1 M ammonium formate buffer pH = 6.5 (50:50, v/v); product
fractions were collected between 18.4 and 20.5 min; precursor
(2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-5-yl)phenoxy)methyl)
quinolin-4-ol) 8 retention time was 5.6 min. The QC mobile phase
4, IC50 = 0.36 0.03 nM
1,
2,
3,
R = 3-OCH3, IC50 = 0.40 0.02 nM
R = 4-OCH3, IC50 = 0.28 0.06 nM
R = 6-OCH3, IC50 = 1.82 0.25 nM
Figure 1. Structure of MP-10 and compounds 1, 2, 3, and 4.

2650
J. Fan et al. / Bioorg. Med. Chem. 22 (2014) 2648–2654
was acetonitrile/0.1 M, ammonium formate buffer pH 6.5 (60:40,
v/v). The retention time for [11C]4 was 4.8 min at a flow rate of
1.0 mL/min. The sample was authenticated by co-injecting with
the cold standard. Radiochemical purity was >99%, chemical purity
was >95%, the RCY 20–30% (n = 4, decay corrected to EOB) and SA
check the positioning; once confirmed, a 45 min transmission scan
was obtained for attenuation correction. After that, the animal was
administrated 260–370 MBq of the radiotracer via the venous
catheter. Subsequently, a 120 min dynamic (three 1 min frames,
four 2 min frames, three 3 min frames, and twenty 5 min frames)
PET scan was acquired. During the whole procedure, core temper-
ature was kept constant at 37 °C with a heated water blanket. In
each microPET scanning session, the head was positioned supine
in the adjustable head holder with the brain in the center of the
field of view. For each subject, at least three independent PET stud-
ies were performed (n = 3). All animal experiments were con-
ducted in compliance with the Guidelines for the Care and Use of
Research Animals established by the Animal Studies Committee
at Washington University School of Medicine in St. Louis.
>318 GBq/lmol (decay corrected to EOB).
2.2. In vivo biodistribution and regional brain uptake in rats
All animal experiments were conducted in compliance with the
Guidelines for the Care and Use of Research Animals approved by
Washington University’s Animal Studies Committee. For the bio-
distribution studies, 14.8À18.5 MBq of the 11C-tracer in 200
lL of
10% ethanol/saline solution (v/v) was injected into the tail vein
of mature male Sprague-Dawley rats (250À400 g) under anesthe-
sia (2.5% isoflurane in oxygen). At 5, 30, and 60 min post injection
(n = 4 per study group), rats were anesthetized and euthanized.
The whole brain was quickly harvested and dissected into seg-
ments comprising hippocampus, striatum, cortex, thalamus, brain
stem, and cerebellum. The remainder of the brain was also col-
lected in order to determine total brain uptake. Peripheral tissues
including blood, heart, lung, liver, spleen, pancreas, kidney, muscle,
fat, and tail were also collected. Samples were counted in an auto-
matic gamma counter (Beckman Gamma 8000 well counter) with a
diluted sample of the injectate and the %ID/g was calculated; val-
ues are represented as mean SD unless otherwise stated. When
appropriate, a 2-tailed paired student t test was used. A p value
2.4. Metabolite studies on NHP plasma
Arterial blood samples (1.2–1.5 mL) were collected at 5, 15, 30,
and 60 min post-iv injection of the radiotracers in a heparinized
syringe. Aliquots of whole blood (1 mL) were counted in a well
counter, then centrifuged to separate red blood cells from plasma.
Aliquots of plasma (400 lL) were solvent deproteinated using
0.92 mL ice-cold methanol and separated by centrifugation. The
supernatant was mixed with water, 1:1 (v/v) and the mixture
was injected to a HPLC system for radioactive metabolite determi-
nation. The HPLC system consisted of an Agilent SB C-18 analytic
HPLC column (250 mm  4.6 mm, 5
lA) and an UV detector with
of 0.05 was considered
significance.
a
threshold criterion of statistical
wavelength set at 254 nm. For [11C]1, the mobile phase was aceto-
nitrile/0.1 M, pH 4.5 ammonium formate buffer (52:48, v/v), and
the flow rate was 1.0 mL/min. For [11C]2, the mobile phase was
acetonitrile/0.1 M, pH 4.5 ammonium formate buffer (48:52, v/v),
and the flow rate was 1.1 mL/min. The HPLC fractions were col-
lected at 1 min intervals for 16 min; each fraction was counted
by a well counter to determine the radioactivity of each collection.
The results were corrected for background radiation and physical
decay.
2.3. In vivo microPET brain imaging studies in male cynomolgus
monkeys
A microPET Focus 220 scanner (Concorde/CTI/Siemens Micro-
systems, Knoxville, TN) was used for the imaging studies of
[
11C]1–2 in male cynomolgus monkey (4À6 kg). The animals were
fasted for 12 h before the study. The animals were initially anes-
thetized using an intramuscular injection with ketamine (10 mg/
kg) and glycopyrulate (0.13 mg/kg), and intubated with an endo-
tracheal tube under anesthesia (maintained at 0.75À2.0% isoflu-
rane in oxygen) throughout the PET scanning procedure. After
intubation, a percutaneous venous catheter was placed for radio-
tracer injection. A 10 min transmission scan was performed to
2.5. MicroPET image processing and analysis
PET image reconstructed resolution was <2.0 mm full width half
maximum for all 3 dimensions at the center of the field of view.
Emission scans were corrected using individual attenuation and
model-based scatter correction and reconstructed using filtered
N
N
N
N
CH3
CH3
11C]MeI, 5 N NaOH (aq.)
N
N
[
1
1
8
8
N
2
N
2
7
6
7
6
O
O
DMSO, 85 °C, 5 min
3
3
O11CH3
OH
4
5
4
5
11
11
[
[
[
C] : 3-O11CH3
1
2
5
: 3-OH
C] : 4-O11CH3
6: 4-OH
7
: 6-OH
11C]3: 6-O11CH3
N
N
N
N
[
11C]MeI, 5 N NaOH (aq.)
DMSO, 85 °C, 5 min
N
N
CH3
CH3
N
O
N
O
11
4
C]
[
8
O11CH3
Scheme 1. Radiosynthesis of [11C]1–4 through O-methylation of corresponding precursor 5–8 with [11C]CH3I.
OH

J. Fan et al. / Bioorg. Med. Chem. 22 (2014) 2648–2654
2651
Table 1
Biodistribution of [11C]1–4 in male SD rats (%ID/gram)
Organ
5 min
30 min
60 min
5 min
11C]2
30 min
60 min
[
11C]1
[
Blood
Lung
Liver
Kidney
Muscle
Fat
0.679 0.116
0.321 0.021
0.408 0.083
7.345 1.472
1.647 0.459
0.244 0.073
0.851 0.314
0.296 0.038
0.097 0.016
0.452 0.086
0.133 0.024
0.275 0.028
0.299 0.042
6.398 0.945
1.275 0.141
0.142 0.014
0.500 0.087
0.199 0.013
0.064 0.006
0.380 0.048
0.095 0.008
0.268 0.053
0.890 0.206
7.080 0.435
2.340 0.295
0.258 0.035
0.242 0.065
0.464 0.023
0.215 0.031
0.352 0.052
0.210 0.035
0.192 0.075
0.233 0.045
10.085 1.972
1.294 0.268
0.133 0.035
0.371 0.091
0.187 0.042
0.066 0.023
0.230 0.030
0.087 0.016
0.152 0.030
0.163 0.027
8.941 1.805
0.758 0.112
0.084 0.013
0.278 0.040
0.121 0.025
0.046 0.014
0.201 0.037
0.057 0.008
1.602 0.509
11.005 0.758
4.354 0.473
0.412 0.064
0.935 0.462
0.733 0.039
0.229 0.039
0.488 0.101*,a
0.231 0.038
Heart
Cerebellum
Striatum
Total brain
*,a
*,a
*,a
*,a
*,a
[
11C]3
[
11C]4
Blood
Lung
Liver
Kidney
Muscle
Fat
0.282 0.025
0.744 0.241
5.357 0.989
1.333 0.282
0.151 0.027
0.139 0.038
0.416 0.026
0.251 0.031
0.293 0.075*,b
0.249 0.037
0.275 0.041
0.389 0.082
3.172 0.606
0.883 0.123
0.175 0.009
0.221 0.060
0.264 0.032
0.090 0.018
0.160 0.023
0.105 0.011
0.210 0.023
0.249 0.015
1.724 0.510
0.637 0.052
0.129 0.006
0.135 0.026
0.182 0.020
0.066 0.008
0.099 0.015
0.643 0.196
3.312 0.494
1.744 0.316
0.146 0.027
0.066 0.016
0.335 0.026
0.094 0.017
0.067 0.006
0.156 0.020
4.430 0.481
0.645 0.088
0.093 0.010
0.139 0.027
0.132 0.019
0.024 0.007
0.067 0.011
0.107 0.016
4.588 0.577
0.575 0.075
0.052 0.009
0.139 0.058
0.103 0.012
0.015 0.003
Heart
Cerebellum
Striatum
Total brain
*,a
0.089 0.014*,a
0.074 0.005
0.151 0.033*,a
0.091 0.017
0.083 0.010*,a
0.025 0.004
0.037 0.004*,a
0.015 0.002
*
p Value was determined by using 2-tailed paired student t test for the uptake of striatum versus that of cerebellum.
a
p < 0.05.
b
p > 0.05.
Figure 2. striatum:cerebellum ratios of [11C]1–4 in rat brain. (A) [11C]1 and [11C]2 displayed higher ratio than [11C]3 and [11C]4. For [11C]1, the ratio reached 6-fold at 60 min
p.i., that of [11C]2 reached 4.5-fold. (B) Blocking studies were performed for [11C]1 and [11C]2 by pretreating rats with MP-10 (2 mg/kg, 5 min prior to tracer injection).
Significant reduction in striatal binding at 30 min p.i. was obtained for both [11C]1 and [11C]2; striatum:cerebellum ratio was decreased for 50% on [11C]1, and 60% decreased
for [11C]2. ⁄p < 0.05.
back projection as described previously.25 The first baseline PET
image for each animal acted as the target image with the MPRAGE
and subsequent PETs coregistered to it using automated image reg-
istration program AIR.26,27 All MPRAGE-based volume of interest
(VOI) analyses were accomplished by investigators blinded to the
clinical status of the monkeys. For quantitative analyses, three-
dimensional regions of interest (ROI) (cerebellum, frontal occipital,
striatum, temporal, white matter, midbrain and hippocampus)
were transformed to the baseline PET space and then overlaid on
all reconstructed PET images to obtain time–activity curves. Activ-
ity measures were standardized to body weight and dose of radio-
activity injected to yield standardized uptake value.
of the NaOH as outlined in Scheme 1 to give [11C]1–4 with 20–70%
RCY after HPLC purification. The radiochemical purity of [11C]1–4
was >99% and chemical purity was >95% . [11C]1–4 were identified
by co-eluting with the standard. The entire synthetic procedure
Table 2
The distribution of [11C]1 and [11C]2 in control rats and rats pretreated using MP-10
(2.0 mg/kg, 5 min prior to tracer injection) at 30 min p.i. (%ID/gram)
[
11C]1
MP-10 block
[
11C]2
MP-10 block
Organ
Control
Control
Blood
Lung
0.321 0.021 0.407 0.026
0.408 0.083 0.404 0.024
0.192 0.075 0.343 0.007
0.233 0.045 0.362 0.021
Liver
7.345 1.472 5.781 0.696 10.085 1.972 4.723 0.489
3. Results and discussion
3.1. Radiochemistry
Kidney
Muscle
Fat
1.647 0.459 1.523 0.162
0.244 0.073 0.257 0.026
0.851 0.314 1.353 0.202
0.296 0.038 0.340 0.036
1.294 0.268 1.072 0.069
0.133 0.035 0.224 0.012
0.371 0.091 0.697 0.219
0.187 0.042 0.310 0.040
0.066 0.023 0.112 0.011
0.230 0.030* 0.175 0.022*
0.087 0.016 0.114 0.007
Heart
Cerebellum 0.097 0.016 0.119 0.006
Striatum
0.452 0.086* 0.294 0.013*
Total brain 0.133 0.024 0.128 0.003
The radiolabeling of [11C]1–4 was accomplished by employing
standard conditions for methylation of an O-desmethyl precursor
with [11C]CH3I. Reaction of the corresponding desmethyl precursor
5–824 with [11C]CH3I was performed in DMSO and in the presence
*
p Value was determined by using 2-tailed paired student t test for the striatum
uptake in control rats versus rats pretreated using MP-10. p < 0.05.

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J. Fan et al. / Bioorg. Med. Chem. 22 (2014) 2648–2654
Figure 3. MicroPET studies of [11C]1 (A) and [11C]2 (B) in an adult male cynomolgus monkey. Upper panel: MicroPET images (left), co-registered images (middle) and MRI
images (right). High uptake of [11C]1 and [11C]2 in striatum produced clear visualization of tracer in putamen and caudate; low panel: tissue time activity curves for
cerebellum (black), frontal cortex (blue), occipital cortex (magenta), striatum (red), temporal cortex (olive), white matter (navy), middle brain (violet), and hippocampus
(brown).
including the production of [11C]CH3I, HPLC purification and for-
mulation of the radiotracer for in vivo studies, was completed
3.2. Biodistribution
within 50–55 min.
(>318 GBq/
mol, n ꢀ 4, decay corrected to EOB), which was suffi-
cient for in vivo validation.
[
11C]1–4 were obtained with high SA
The rat biodistribution data of [11C]1–4 are summarized in
Table 1. The brain uptake of [11C]1–4 at 5 min ranged from 0.09
to 0.25 (I.D./g), all four of radioligands displayed heterogeneous
l

J. Fan et al. / Bioorg. Med. Chem. 22 (2014) 2648–2654
2653
Figure 4. Radiometabolite analysis of NHP arterial plasma samples post injection of [11C]1 (A) or [11C]2 (B). This black histogram represents the percentage radioactivity from
the intact parent compound, while the gray histogram represents the percentage radioactivity from the metabolite at 2, 15, 30 and 60 min post-injection. The stability of
radioligand [11C]1(A) are better than that of radioligand [11C]2 (B) in vivo.
distribution throughout the brain with the highest accumulation of
radioactivity in striatal tissue and gradual washout. [11C]1–2
showed the slowest washout from the striatal region between 5
and 60 min post injection (p.i.). The target to non-target ratio
was calculated using cerebellum as the non-target reference re-
gion. The striatum to cerebellum ratio of radioactivity accumula-
tion and washout is shown in Figure 2. A blocking dose of 2 mg/kg
MP-10 was injected iv 5 min prior to injection of [11C]1 or [11C]2
(Table 2), and resulted in a significant reduction in striatal binding
(p <0.05). These observations are consistent with the reported that
striatal region has the highest expression of PDE10A compared to
other non-target brain regions such as cerebellum and cortex.1,28
5, 15, 30, and 60 min. After centrifugation of arterial blood sam-
ples, 65–75% of the radioactivity was in the plasma and 35–25%
of the radioactivity was in the red blood cell (RBC). Following sol-
vent deproteination, 88–95% of the radioactivity in plasma was in
the supernatant while 5–12% of the radioactivity in plasma was
in the protein pellet. These results suggested that the supernatant
contains the majority of the radioactivity. As shown in Figure 4,
HPLC analysis of plasma samples revealed only two radioactive
peaks for both [11C]1 and [11C]2, a hydrophilic metabolite with
retention time at 3–5 min; the retention time for parent
compound [11C]1 was 8–10 min, whereas that of [11C]2 was
9–12 min. For [11C]1, a stable metabolism profile was obtained:
at 2 min p.i. 97% of the extracted activity represented the parent
compound, at 15 min 87% parent was measured, and by 30 min
82% of the recovered activity was parent, while 60 min p.i., 65%
of the recovered radioactivity in the arterial blood sample repre-
sented intact [11C]1 while only 30% of the recovered radioactivity
was the radiometabolite. For [11C]2, 85% of the extracted activity
was parent, which decreased to 75% at 15 min p.i. and 60% parent
at 30 min p.i.; by 60 min p.i. equal percentages (42%) of parent
[
11C]1 displayed a 4.6-fold ratio at 30 min p.i. and a 6.0-fold ratio
at 60 min p.i. The ratio for [11C]2 was 3.8-fold at 30 min and 4.5-
fold at 60 min p.i. [11C]4 displayed a 3.6-fold ratio at 30 min p.i.;
however the absolute brain uptake of [11C]4 was significantly low-
er than that of the other tracers (Table 1). Based on this promising
preliminary rodent evaluation, both [11C]1 and [11C]2 showed po-
tential and were worth for further evaluation.
3.3. MicroPET studies in nonhuman primate (NHP)
[
11C]2 and metabolite were observed. Due to the both the excel-
lent image quality and the superior in vivo metabolic stability, we
concluded that [11C]1 is the more promising probe for imaging
PDE10A.
To confirm the feasibility of using [11C]1 or [11C]2 to measure
the levels of PDE10A in vivo, PET imaging studies were conducted
in an adult male NHP on a microPET Focus 220 scanner. The repre-
sentative summed images from 0 to 120 min were co-registered
with MRI images to accurately identify the regions of interest
(Fig. 3). The summed images revealed high uptake of both [11C]1
and [11C]2 in striatum (Fig. 3A and B, top panels), which are regions
that have high expression of PDE10A. The TACs (Fig. 3A and B, low-
er panels) also indicated the highest uptake in striatum and wash-
out trend in other brain regions for both [11C]1 and [11C]2.
Specifically, for [11C]1, striatum uptake (SUV) reached 1.8 at
30 min p.i. with a 3.5-fold striatum to cerebellum uptake ratio.
For [11C]2, the striatum equilibrium was observed at 25 min p.i.
with 2.7-fold striatum to cerebellum uptake ratio. These data indi-
cate: (a) both [11C]1 and [11C]2 can readily penetrate the blood–
brain-barrier and enter into the NHP brain; (b) the distribution of
both tracers is consistent with the distribution of the PDE10A in
brain; (c) the data supports our hypothesis that by labeling on
the quinoline fragment of the molecule, striatum uptake were
equilibrated for both [11C]1 and [11C]2; (d) TACs showed higher
PDE10A binding for [11C]1 than [11C]2, which is further supported
by the microPET images, in that better signal to background ratio
was found for [11C]1.
4. Conclusions
Previous studies have suggested that [11C]MP-10 has limited
utility as a PDE10A PET tracer for human brain imaging due to a
brain penetrable radiometabolite. Similar problems were observed
in the preclinical evaluation of 18F-labeled PDE10A tracers.20 In a
PET tracer recently approved for human studies of PDE10A,
although reproducible brain imaging was obtained, significant lev-
els of radiometabolite were also reported: >50% was metabolized
with a half-life of 18.7 min.29 To address the challenges involved
with radiometabolites, we introduced the [11C]methoxy group
onto the quinoline fragment of structures that belongs to this ser-
ies, unlike [11C]MP-10 which was 11C-labeled on the pyrazole frag-
ment. Here we reported the radiosyntheses and the preclinical
evaluation of [11C]1–4 in rats and the subsequent evaluation of
the two promising radioligands, [11C]1 and [11C]2, in cynomolgus
macaques. Rat biodistribution studies indicated that [11C]1 had
the highest brain uptake, good striatal retention and the highest
striatum to cerebellum ratio. MicroPET studies in NHP also re-
vealed high accumulation of [11C]1 in the striatum, which platea-
ued at 80 min p.i. with a 4.5-fold target to non-target ratio.
Although target to non-target ratios were acceptable for [11C]2,
3.4. Metabolite studies in NHP plasma
Because MicroPET studies in NHP identified both [11C]1 and
[
11C]1 had favorable metabolic stability. We observed no signifi-
cant interference from radiometabolites, suggesting the strategy
of radiolabeling the quinoline fragment of the structure rather than
[
11C]2 as potential candidates for imaging PDE10A in the brain,
we performed radioactive metabolite analysis of both tracers at

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This work was supported by the National Institute of Mental
Health (NIMH) of the National Institutes of Health under award
no. MH092797 and National Institute of Neurological Disorders
and Stroke (NINDS) under award no. NS075527. The authors grate-
fully thank Christina M. Zukas, John Hood, and Darryl Craig for
their excellent technical assistance for the PET studies in nonhu-
man primate.
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