Radiosynthesis and in vivo evaluation of [11C]MP-10 as a PET probe for imaging PDE10A in rodent and non-human primate brain

Article abstract of DOI:10.1016/j.bmc.2011.01.032

2-((4-(1-[¹¹C]Methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy) methyl)-quinoline (MP-10), a specific PDE10A inhibitor (IC₅₀ = 0.18 nM with 100-fold selectivity over other PDEs), was radiosynthesized by alkylation of the desmethyl precursor with [¹¹C]CH₃I, ～45% yield, >92% radiochemical purity, >370 GBq/μmol specific activity at end of bombardment (EOB). Evaluation in Sprague-Dawley rats revealed that [¹¹C]MP-10 had highest brain accumulation in the PDE10A enriched-striatum, the 30 min striatum: cerebellum ratio reached 6.55. MicroPET studies of [¹¹C]MP-10 in monkeys displayed selective uptake in striatum. However, a radiolabeled metabolite capable of penetrating the blood-brain-barrier may limit the clinical utility of [¹¹C]MP-10 as a PDE10A PET tracer.

Full text of DOI:10.1016/j.bmc.2011.01.032

Bioorganic & Medicinal Chemistry 19 (2011) 1666–1673
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Radiosynthesis and in vivo evaluation of [11C]MP-10 as a PET probe for imaging
PDE10A in rodent and non-human primate brain
Zhude Tu a, , Jinda Fan a, Shihong Li a, Lynne A. Jones a, Jinquan Cui a, Prashanth K. Padakanti a, Jinbin Xu a,
⇑
Dexing Zeng a, Kooresh I. Shoghi a, Joel S. Perlmutter a,b, Robert H. Mach a
a Department of Radiology, Washington University School of Medicine, Mallinckrodt Institute of Radiology, 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
2-((4-(1-[11C]Methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)-quinoline (MP-10),
a specific
Article history:
Received 14 December 2010
Revised 13 January 2011
Accepted 16 January 2011
Available online 22 January 2011
PDE10A inhibitor (IC50 = 0.18 nM with 100-fold selectivity over other PDEs), was radiosynthesized by
alkylation of the desmethyl precursor with
>370 GBq/
[
11C]CH3I, ꢀ45% yield, >92% radiochemical purity,
l
mol specific activity at end of bombardment (EOB). Evaluation in Sprague–Dawley rats
revealed that [11C]MP-10 had highest brain accumulation in the PDE10A enriched-striatum, the 30 min
striatum: cerebellum ratio reached 6.55. MicroPET studies of [11C]MP-10 in monkeys displayed selective
uptake in striatum. However, a radiolabeled metabolite capable of penetrating the blood–brain-barrier
may limit the clinical utility of [11C]MP-10 as a PDE10A PET tracer.
Keywords:
PDE10A
PET imaging
Carbon-11
MP-10
Ó 2011 Elsevier Ltd. All rights reserved.
Huntington’s disease
1. Introduction
(HD), Parkinson disease (PD), Tourette syndrome, and drug
abuse.6–9 PDE10A plays a major role in the regulation of cyclic
Phosphodiesterase 10A (PDE10A) is a unique dual specificity
phosphodiesterase that converts both cyclic adenosine monophos-
phate (cAMP) to adenosine monophosphate (AMP) and cyclic gua-
nosine monophosphate (cGMP) to guanosine monophosphate
(GMP).1,2 Unlike other phosphodiesterase (PDE) families, PDE10A
is uniquely expressed in the brain where it plays a critical role in
dopaminergic neurotransmission.3,4 The expression of PDE10A is
highest in the medium spiny neurons of the striatum (caudate
and putamen), nucleus accumbens, and olfactory tubercle of mice,
dogs, cynomolgus and humans.5 Abnormal striatal levels of
PDE10A impair striatal output that may contribute substantially
to the pathophysiology of schizophrenia, Huntington’s disease
nucleotide signaling cascades; inhibition of the enzyme causes
cAMP response element-binding (CREB) phosphorylation and acti-
vation.10 For Huntington’s disease, western analysis of 1
lg protein
extracts from the striatum of R6/1 and R6/2 Huntington’s disease
mice prior to motor manifestation development displayed lower
PDE10A proteins levels than in wild type mice, and may be an early
marker of neuronal dysfunction. Post mortem analysis of human
brain tissue has shown similar results: western analysis of 5 lg
of total protein samples from the caudate, nucleus accumbens
and putamen from three patients with grade 3 HD demonstrated
reduced PDE10A protein levels compared with age-matched
neurologically normal control subjects.10–12 Inhibition studies
with 2-{4-[pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-
phenoxymethyl}-quinoline (TP-10), an analogue of 2-((4-(1-methyl-
4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)-methyl)quinoline (MP-10,
7) in a rodent quinolinic acid model of Huntington’s disease sug-
gest that PDE10A inhibition may be a novel neuroprotective ap-
proach to the treatment of HD. In addition to the potential
application of PDE10A inhibitors in HD, the observation that
PDE10A expression parallels D2-receptor distribution suggests that
PDE10A inhibition might yield therapeutic effects similar to D2
antagonism for schizophrenia patients, without accompanying
untoward effects. Inhibition of PDE10A in rodents significantly in-
creased extra-cellular levels of cAMP in the striatum and also
Abbreviations: AMP, adenosine monophosphate; cAMP, cyclic adenosine mono-
phosphate; CNS, central nervous system; cGMP, cyclic guanosine monophosphate;
DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; GMP, guanosine mono-
phosphate; HD, Huntington disease; LDA, lithium diisopropylamide; MP-10, 2-[4-
(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline;
MSNs,
medium-sized spiny neurons; PCP, phenylcyclohexylpiperidine; PDE10A, phospho-
diesterase 10A; PDE3A, phosphodiesterase 3A; PDE3B, phosphodiesterase 3B; PET,
positron emission tomography; THF, tetrahydrofuran; TLC, thin layer chromatography;
TP-10, 2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-
quinoline; MP-10, 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-
quinoline; EOB, end of bombardment.
⇑
Corresponding author. Tel.: +1 314 362 8487; fax: +1 314 362 8555.
E-mail address: tuz@mir.wustl.edu (Z. Tu).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.032

Z. Tu et al. / Bioorg. Med. Chem. 19 (2011) 1666–1673
1667
mediated certain clinical antipsychotic effects13,14 with significant
differences from D2-receptor antagonists in preclinical behavioral
models of schizophrenia including the induction of catalepsy and
prepulse inhibition of startle.14,15
of ꢀ370 GBq/
lmol (decay corrected to EOB n = 15), which is suffi-
cient for in vivo validation. However, while labeling [11C]MP-10,
we found that 1.5–4 equiv of NaH was necessary for the N-
alkylation to obtain satisfactory yield. When aqueous NaOH solu-
tion was used, no product was obtained. In addition, adding the
solution of NaH in DMF to the reaction vial 2–3 min prior to the
Over the past 10 years, tremendous efforts have been made to de-
velop PDE10A inhibitors for treatment of schizophrenia and associ-
ated mental disorders.16–19 MP-10 has been identified as
a
[
11C] CH3I delivery is necessary to obtain satisfactory yield. Either
promising therapeutic inhibitor of PDE10A and is in clinical evalua-
tion.16,17 However, most evidence supporting the role of PDE10A in
CNS disorders comes from behavioral studies and post mortem tissue
analysis. Positron emission tomography (PET) is a non-invasive imag-
ing modality that can provide functional information about molecu-
lar and cellular processes in living subjects. Therefore, a PET
radioligand having high affinity and selectivity for PDE10A would
provide a unique tool to study changes in PDE10A levels of living sub-
jects and investigate the physiological function of PDE10A in the CNS.
Our group reported initial results for imaging PDE10A in vivo.20,21
Recently, an initial validation of an MP-10 analogue, 2-((4-(1-
adding NaH in DMF too early or large excess of NaH in DMF will
reduce radiolabeling yield. Solid phase extraction to process the
HPLC collection fraction of the product prevents the final
[
11C]MP-10 production from decomposition. When rotary evapora-
tor was used to concentrate the [11C]MP-10 product, the product
11C]MP-10 decomposed easily.
[
2.3. Biodistribution
The radioactivity organ distribution after injection of [11C]MP-10
into rats is summarized in Table 1. From 5 min to 60 min post-
injection, [11C]MP-10 displayed heterogenous distributions in the
brain: the striatum displayed highest uptake compared to other
brain regions. The percent injected doses per gram (%I.D./g) of the
radioactivity post-injection of [11C]MP-10 at 5, 30, and 60 min were
0.300 0.094, 0.236 0.035, and 0.164 0.036 for striatum,
0.133 0.025, 0.044 0.009, and 0.044 0.014 for cortex,
0.169 0.049, 0.050 0.010, and 0.051 0.009 for hippocampus,
0.133 0.023, 0.036 0.003, and 0.035 0.003 for cerebellum.
These observations are consistent with the observation that stria-
tum has the highest density of PDE10A whereas the density of
PDE10A is relatively low in cortex.2,16 The ratios of the striatum to
non-target regions including blood, hippocampus, cortex and cere-
bellum reached 2.54, 1.88, 2.35, and 2.23 respectively, at 5 min; at
30 min, the ratios were 3.82, 4.80, 5.44, and 6.55 for striatum versus
blood, hippocampus, cortex and cerebellum respectively. As shown
in Figure 1, from 5 min to 30 min, the striatum: non-target ratios
displayed an increase trend in rat brain, which suggested that radio-
activity [11C]MP-10 in the non PDE10A specific region was rapidly
cleared. The higher striatum/organ ratios of [11C]MP-10 and the
increasing trend of striatum/organ ratios in rat brain indicated that
[
18F]ethyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)-methyl)quin-
oline (18F-JNJ41510417) was reported.22 To confirm if MP-10 could
serve as a PET probe for quantifying PDE10A in vivo, we radiosynthe-
sized [11C]MP-10 and conducted in vivo validation studies in rodent
and non-human primate models. In this paper, we will describe the
radiosynthesis of [11C]MP-10, its biodistribution and validation
in vivo in Sprague–Dawley rats, MicroPET imaging studies of
[
11C]MP-10 in rhesus macaque, as well as the metabolite analysis
of the rat brain tissue, plasma and monkey plasma post-injection of
11C]MP-10 in animals.
[
2. Results and discussion
2.1. Chemistry
The target compound MP-10 is a pyrazole derivative, that pos-
sesses a methyl group on the nitrogen, which gives easy access
to 11C-labeling by N-methylation of the corresponding desmethyl
precursor (6) with [11C]methyl iodide ([11C]CH3I). The synthesis
of the N-desmethyl precursor 6 and the standard compound 7
was accomplished by starting with methyl 4-hydroxybenzoate
(1) as shown in Scheme 1 following the reported procedure.16,23
However, reference compound 7 and its isomer 8 were synthesized
from the key intermediate 5 by reacting it with methylhydrazine
instead of following the literature procedure, in which 4-(1-
methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenol was treated with
quinoxaline-2-yl-methanol.18 Compound 4-(1-methyl-4-pyridin-
4-yl-1H-pyrazol-3-yl)-phenol that matched the major radiolabeled
metabolite (9) was synthesized following the literature
procedure.18
[
11C]MP-10 has specific binding with PDE10A enriched-striatum.
Among the peripheral tissues, the liver showed a relative high initial
uptake of [11C]MP-10, which reached 2.766 0.350 (%I.D./g) in
5 min.; at 30 min the uptake of [11C]MP-10 in liver still retained as
3.364 0.613 (% I.D./g). There was a rapid clearance of the radioac-
tivity from blood, lung, muscle, heart, kidney, and testes.
2.4. MicroPET studies
The results of the rodent brain distribution studies suggest that
[
11C]MP-10 can be a suitable ligand for in vivo imaging of PDE10A.
To confirm the feasibility of using [11C]MP-10 to measure the levels
of PDE10A in vivo, PET imaging studies of [11C]MP-10 were con-
ducted in three male rhesus monkeys (Fig. 2) on a microPET Focus
2.2. Radiochemistry
The radiolabeling of [11C]MP-10 was accomplished by employ-
ing convenient conditions for methylation of N-desmethyl precur-
sor with [11C]MeI. Reaction of the desmethyl precursor 6 with
220 scanner. The representative summed images from
0 to
120 min were co-registered with MRI images to accurately identify
the regions of interest. The summed images of MicroPET studies in
male rhesus macaque post-injection of [11C]MP-10 revealed high
uptake of [11C]MP-10 in caudate and putamen (Fig. 2 upper left)
and give clear visualization of striatum, which is a region that ex-
presses high level of the PDE10A. These data indicate: (a) [11C]MP-
10 can readily penetrate the blood–brain-barrier and enter into the
non-human primate brain and, (b) the distribution of [11C]MP-10 is
consistent with the distribution of the PDE10A in brain. The tissue
time–activity curves (Fig. 2 lower panel) indicate an increasing
trend of radioactivity accumulation in caudate, putamen and cere-
bellum post-injection of [11C]MP-10; Striatum: cerebellum ratio
reaches maximum at 30 min post-injection of [11C]MP-10 with
[
11C]MeI was performed in DMF and in the presence of the NaH
in DMF as outlined in Scheme 2 to give corresponding [11C]MP-
10 with approximately 45% yield and [11C]8 with approximately
36% yield after HPLC purification. The radiochemical purity of
[
11C]MP-10 was greater than 92% and chemical purity was great
than 95%. [11C]MP-10 was identified by co-eluting with the solu-
tion of standard MP-10. The retention time of [11C]MP-10 on the
analytical HPLC system was 10.0–10.5 min. The entire synthetic
procedure including the production of [11C]MeI, HPLC purification
and formulation of the radiotracer for vivo studies, was complete
within 50–55 min. [11C]MP-10 was obtained in a specific activity

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Z. Tu et al. / Bioorg. Med. Chem. 19 (2011) 1666–1673
O
O
OCH3
O
OCH3
N
N
a
O
OCH3
b, c, d
N
CH3
O
HO
2
1
3
O
N
O
N
N
N
N
f
N
e
O
N
N
O
5
4
N
H
g
5
N
O
6
N
N
N
CH3
N
N
CH3
h
N
+
N
5
O
O
7 (MP-10)
8
Reagent and Reaction condition:
(a) 2-(chloromethyl)quinoline, K2CO3/Acetone, Reflux; (b) NaOH, CH3OH; (c) SOCl2; (d) CH3NHOCH3/acetonitrile;
(e) 4-methylpyridine, Lithium diisopropylamide (LDA); (f) 1,1-dimethoxy-N,N-dimethylmethanamine,reflux;
(g) NH2NH2; (h) NH2NHCH3.
Scheme 1. Synthesis of MP-10.
a
2.0-fold ratio for caudate-to-cerebellum and 1.5-fold for
compound [11C]MP-10 in plasma decreased from 98.35 to 49.28.
The percentage of metabolite #1 in plasma increased to 40.72 at
60 min (Fig. 3). The percentage of the metabolite #2 reached the
highest of 12.19 at 40 min and then began to decrease.
putamen-to-cerebellum ratio. The increasing trend of radioactivity
in the regions of interest may reflect a radiolabeled lipophilicity
metabolite entering into the brain and accumulating in the brain
regions.
For the rat brain samples, the percentages for [11C]MP-10 were
98.71, 94.85, 84.74, and 78.62 at 2, 20, 40, and 60 min (Table 2). For
metabolite #1, the percentages were 1.03, 3.45, 11.47, and 15.87
for 2, 20, 40, and 60 min brain tissue samples. The radioactive
metabolite #1 was the major metabolite component that crossed
the blood–brain-barrier and entered the brain. For metabolite #2,
the percentages were 0.27, 0.93, 3.82, and 1.24 for 2, 20, 40, and
60 min brain tissue samples. Although metabolite #2 was found
in the brain samples, it is negligible relative to metabolite #1
and to the parent compound. When comparing rat brain tissue
HPLC data and rat plasma data at 60 min (Fig. 3), radioactive
metabolite #1 was the major metabolite which had the capability
of crossing the blood–brain-barrier and enter the brain. The accu-
mulation of metabolite #1 in the brain resulted in increasing non-
specific binding in the cerebellum.
2.5. Metabolite studies in monkey plasma, rat plasma as well as
rat brain
2.5.1. In vivo metabolite analysis in rat blood and rat brain
To confirm whether or not radiolabeled metabolites enter the
brain, the metabolite analysis of rat brain and rat plasma samples
were performed post-injection of [11C]MP-10 into Sprague–Dawley
rats. The plasma radioactivity at 2 min post-injection displayed
three radioactivity peaks. The percentage (%) of the three peaks is
1.23 for peak #1 (metabolite #1), a small amount (0.43) for peak
#2 (metabolite #2) and a very large amount (98.35) for peak #3
which is the parent compound, [11C]MP-10 (Table 2). At 20, 40,
and 60 min post-injection of [11C]MP-10, the percentages of the
parent compound remained 74.20, 52.61, and 49.28; the percent-
ages for metabolite #1 were 19.13, 35.19, and 40.72; the percent-
ages for metabolite #2 are 5.06, 12.19, and 5.58. Overall, the parent
To identify the structure of metabolite #1, the rats brain sample
was co-injected into the LC–MS with cold compound 4-(1-methyl-
4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenol, the m/z+ value of

Z. Tu et al. / Bioorg. Med. Chem. 19 (2011) 1666–1673
1669
N
NH
N
N
O
6
[
11C]CH3I/DMF, NaH
5 min, 85°C
N
N
N
N11
CH3
N
N
11CH3
N
O
N
O
+
[
11C]MP-10
[
11C]8
Scheme 2. Synthesis of [11C]MP-10.
suggested that radioactive 4-(1-[11C]methyl-4-(pyridin-4-yl)-1H-
Table 1
Biodistribution of [11C]MP-10 in male Sprague–Dawley rats (ID%/gram)
pyrazol-3-yl)phenol was generated from the O-dealkylation
[
11C]MP-10 post-injection, as outlined in Scheme 3.
Organ
5 min
30 min
60 min
Blood
Lung
Liver
Muscle
Fat
Heart
Kidney
Testes
Total brain
Brain regions
Cortexa,b
Hippocampusa,b
Striatum
Cerebelluma,b
0.123 0.033
0.317 0.037
2.766 0.350
0.093 0.021
0.124 0.059
0.264 0.080
1.229 0.215
0.160 0.048
0.162 0.025
0.065 0.016
0.155 0.053
3.395 0.448
0.059 0.010
0.075 0.020
0.102 0.041
0.366 0.075
0.098 0.006
0.066 0.008
0.047 0.012
0.090 0.012
3.364 0.613
0.039 0.011
0.100 0.013
0.068 0.023
0.314 0.060
0.068 0.021
0.056 0.006
2.5.2. The non-human primate plasma metabolite analysis
To check the metabolic stability of [11C]MP-10 post-injection
into non-human primates, radiolabeled metabolite analysis of
monkey plasma sample was performed. HPLC analysis of rhesus
macaque plasma samples at 5, 15, 30, and 60 min revealed only
two radioactive peaks, a potential lipophilic metabolite (retention
time ꢀ3 min) and the parent compound; [11C]MP-10 (retention
time is ꢀ10.5 min) were observed post-injection of [11C]MP-10 as
shown in Figure 4. The first radioactive peak in the HPLC chromato-
gram of rhesus plasma has the same retention time as the radioac-
tive metabolite #1 from rat brain tissue and rat plasma samples.
However, in rhesus monkey plasma, only one radiolabeled peak,
metabolite #1 were observed in the HPLC chromatograms. This
observation is consistent with the metabolite analysis result from
the fluorine version of MP-10 analogue, 18F-JNJ41510417.22 The
metabolite #2 in the rat plasma sample as a minor lipophilic
metabolite was not observed in the arterial blood samples of rhe-
sus monkeys. The difference in metabolism results from rat and
monkey plasma may reflect species differences. Similar results also
were observed in previous studies.24–26 Nonetheless, metabolism
of the radiotracer in rat is still a valuable predicator for metabolism
in non-human primate, which will be used as a selection criteria to
determine if a PET tracer has potential to serve as a PDE10A PET
probes for further evaluations in non-human primate and human
being.
0.133 0.025
0.169 0.049
0.300 0.094
0.133 0.023
0.044 0.009
0.050 0.010
0.236 0.035
0.036 0.003
0.044 0.014
0.051 0.009
0.164 0.036
0.035 0.003
a
p-Value <0.05 for striatum versus non-target tissue, 5 min.
p-Value <0.005 for striatum versus non-target tissue, at 30 and 60 min.
b
3. Materials and methods
3.1. General
Figure 1. The striatum versus non-target tissue ratios of [
11C]MP-10 in were
calculated by using the %ID/g data.
All analytical grade chemicals and reagents were purchased
from Sigma–Aldrich (Milwaukee, WI) and were used without
further purification unless otherwise specified. Flash column
metabolite #1 was 251.2384, which matched with that of
4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenol (9), which

1670
Z. Tu et al. / Bioorg. Med. Chem. 19 (2011) 1666–1673
Figure 2. Upper panel: MicroPET images (left), co-registered images (middle) and MRI images (right), High uptake of [11C]MP-10 in striatum was observed which gave clear
visualization of tracer in putamen and caudate; lower panel: tissue time–activity curves for caudate, putamen and cerebellum (left); target: non-target ratios (right).
Table 2
Metabolite analysis of [11C]MP-10 in rat brain and plasma
Time (min)
Brain tissue
% Peak #2
Plasma
% Peak #1
% [11C]MP-10
% Peak #1
% Peak #2
% [11C]MP-10
2
20
40
60
1.03
3.45
11.47
15.87
0.27
0.93
3.82
1.24
98.71
94.85
84.74
78.62
1.23
19.13
35.19
40.72
0.43
5.06
12.19
5.58
98.35
74.20
52.61
49.28
3.2. Chemistry
The synthesis of MP-10 precursor (6) and reference compound
(7) was accomplished by following procedure reported in the liter-
ature with slight modifications.18 The synthetic approach is out-
lined in Scheme 1. The experimental details for the synthesis of
the intermediates (2–5, 9), precursor (6), the reference compound
(7) and its isomer 8 were provided in Supplementary data.
3.3. Radiochemistry
3.3.1. Production of [11C] CH3I
Briefly, [11C]CH3I was produced from [11C]CO2 using a GE PET-
trace MeI Microlab.27 Up to 51.8 GBq of [11C]CO2 was produced from
Washington University’s JSW BC-16/8 cyclotron by irradiating a gas
Figure 3. HPLC results of rat plasma and brain tissue at 60 min post-iv injection of
11C]MP-10 into rats. Metabolite #1 that is the major metabolite in both plasma and
brain tissue has the capability of crossing the brain–blood-barrier and enter into the
brain.
[
target of 0.2% O2 in N2 for 15–30 min with a 40
protons. The GE PETtrace MeI microlab coverts the [11C]CO2 to
11C]CH4 using a nickel catalyst (Shimalite-Ni,28 Shimadzu, Japan
lA beam of 16 MeV
[
P.N.221-27719) in the presenceof hydrogen gas at 360 °C; it was fur-
ther converted to [11C]CH3I by reaction withiodine that was held in a
column in the gas phase at 690 °C. Approximately 12 min after EOB,
several hundred millicuries of [11C]CH3I were delivered as a gas to
the hot cell where the radiosynthesis was accomplished.
chromatography was conducted using Scientific Adsorbents, Inc.
silica gel, 60 Å, ‘40 Micron Flash’ (32–63 l). Melting points were
determined using MEL-TEMP 3.0 apparatus and uncorrected. 1H
NMR spectra were recorded at 300 MHz on a Varian Mercury-VX
spectrometer with CDCl3 or DMSO-d6 as solvent and tetramethyl-
silane (TMS) as the internal standard. All chemical shift values
are reported in ppm (d). Peak multiplicities were recorded as sin-
glet, S; double, d; triplet, t, multiplet, m. Elemental analyses (C,
H) were determined by Atlantic Microlab, Inc. (Norcross, GA).
3.3.2. Radiosynthesis of [11C]MP-10
A streamof [11C]CH3I in heliumwas bubbled for 5 min into a solu-
tion of 6 (1.5–2.0 mg) in DMF (0.18 mL) and 30
in DMF solution (3 mg of NaH (95%) in 250
water bath). The sealed reaction vessel was heated at 85 °C (oil bath)
lL of sodium hydride
lL DMF) at 0 °C (ice-

Z. Tu et al. / Bioorg. Med. Chem. 19 (2011) 1666–1673
1671
N
N
N
11CH3
metabolism
N
N
11CH3
N
N
O
HO
11C]MP-10
Log P*a = 3.52
Radiolabed metabolite #1 (9)
Log P = 1.4
[
*a: Calculated using the program ACD/log D when the pH = 7.4.
Scheme 3. O-Dealkylation of [11C]MP-10.
Washington University’s Animal Studies Committee. For the bio-
distribution studies, 3.7 Â 10À3–5.55 Â 10À3 GBq of [11C]MP-10 in
100–150 lL of saline containing 10% ethanol was injected via the
tail vein into mature male Sprague–Dawley rats (425–490 g) under
2–3% isoflurane/oxygen anesthesia. Groups of at least four rats
were used for each time point. At 5 min, 30 min and 60 min pi,
the groups of rats were again anesthetized and euthanized. The
whole brain was quickly removed and dissected into segments
consisting of striatum, hippocampus, cortex, and cerebellum. The
remainder of the brain was also collected to determine total brain
uptake. At the same time, samples of blood, lung, liver, muscle, fat,
heart, brain and testes were removed and counted in a Beckman
Gamma 8000 well counter with a standard dilution of the injectate.
Tissues were weighed, and the %ID/g was calculated. The striatum/
organ ratios were calculated by dividing the %ID/g of the striatum
by the %ID/g of the non-target tissue.
Figure 4. HPLC results of monkey arterial blood plasma at 5, 15, 30, and 60 min
post-iv injection of [11C]MP-10 into monkeys. Only one metabolite with a retention
time of 3 min was observed and it matched the metabolite #1 in the rat plasma and
brain tissue post-iv injection of [11C]MP-10. This metabolite has the capability of
crossing the brain–blood-barrier and entering into the brain.
3.5. Imaging studies in male rhesus monkeys
MicroPET studies of [11C]MP-10 were performed on three adult
male rhesus monkeys (ꢀ7–12 kg) with a microPET Focus 220 scan-
ner. The animals were fasted for 12 h before PET study. The animals
were initially anesthetized using intramuscular ketamine
(10–20 mg/kg) and glycopyrollate (0.13–17 mg/kg) and then trans-
ported to the PET scanner suit. Upon arrival, the animal was intu-
bated with an endotracheal tube and anesthesia was maintained at
0.75–2.0% isoflurane/oxygen throughout the PET scanning proce-
dure. After intubation, a percutaneous venous catheter was placed
for radiotracer injection. The temperature was kept constant at
37 °C with a heated water blanket. In each microPET scanning ses-
sion, the head was positioned supine in the adjustable head holder
with the brain in the center of the field of view. A 10 min transmis-
sion scan was performed to check the position; once confirmed, a
45 min transmission scan was obtained for attenuation correction.
Subsequently, a 2 h dynamic emission scan was acquired after
administration of 0.22–0.37 GBq of [11C]MP-10 via the venous
catheter.
for 5 min. The reaction was quenched by adding HPLC solvent
(1.8 mL), and the diluted solution was injected to
a high-
performance liquid chromatography (HPLC) Agilent Zorbax SB-C18
reverse phase column (9.4 Â 250 mm); the product was eluted by
using acetonitrile/0.1 M ammonium formate buffer (40:60, v/v) at
pH ꢀ4.5 as the mobile phase, at a flow rate of 4.0 mL/min and the
UV detection was at 254 nM. Under these conditions, the retention
time of the precursor 6 was ꢀ12.1 min; the retention time of the
[
[
11C]MP-10 was ꢀ19.90 min; the retention time of the other isomer
11C]8 was ꢀ22.53 min. The [11C]MP-10 product was collected into a
vial containing 50 mL milli-Q water and passed through a Sep-Pak
Plus C-18 cartridge (Waters, Milford, MA, USA), in which the product
was trapped. The trapped product was eluted with ethanol (0.6 mL)
followed by 5.4 mL of 0.9% saline. After sterile filtration into a dose
vial, the final product was ready for quality control (QC) analysis
and animal studies. The HPLC was performed on Agilent SB C-18 re-
verse phaseanalyticHPLC column(250 mm  4.6 mm, 5
lA) and UV
detection at 254 nm wavelength. The mobile phase was acetonitrile/
0.1 M ammonium formate buffer (45:55, v/v) using 1.2 mL/min flow
rate. Under these conditions the retention time of [11C]MP-10 was
10.4 min. The radioactive dose sample was authenticated by co-
injection with the cold reference compound MP-10. The radiochem-
ical purity was >92%, the chemical purity was >95%, the labeling
yield was ꢀ45% (n = 15, decay corrected to EOB) and the specific
3.5.1. MicroPET image processing and analysis
Acquired list mode data were histogrammed into a 3D set of
sinograms and binned to the following time frames: 3 Â 1 min,
4 Â 2 min, 3 Â 3 min, and 20 Â 5 min. Sinogram data were cor-
rected for attenuation and scatter. Maximum a posteriori (MAP)
reconstructions were done with 18 iterations and a b value of
0.004. A 1.5 mm Gaussian filter was applied to smooth each MAP
reconstructed image. These images were then co-registered with
MRI images to accurately identify the regions of interest with
Amira software (Visage Imaging, Inc., Carlsbad, CA). The 3D regions
of interest were manually drawn through all planes of co-
registered MRI images for the caudate, putamen, and cerebellum
activity was >370 GBq/lmol (decay corrected to EOB).
3.4. Biodistribution studies
All animal experiments were conducted in compliance with the
Guidelines for the Care and Use of Research Animals established by

1672
Z. Tu et al. / Bioorg. Med. Chem. 19 (2011) 1666–1673
(Fig. 2 upper left). The regions of interest then were overlaid on all
reconstructed PET images to obtain time–activity curves. Activity
measurements were standardized to body weight and dose of
radioactivity injected to yield standardized uptake value (SUV)
(Fig. 2 lower left).
methylating the corresponding desmethyl precursor with
11C]CH3I in DMF in the presence of sodium hydride as the base.
[
Biodistribution study of [11C]MP-10 was conducted in male Spra-
gue–Dawley rats and found that the highest uptake in the brain
was in the striatum, the PDE10A enriched region. The highest tar-
get to non-target (striatum to cerebellum) ratio was found to be
6.55 at 30 min post-injection. The microPET studies performed in
rhesus monkeys revealed that [11C]MP-10 readily enters the brain
of non-human primates and its accumulation was more specific to
PDE10A enriched-striatum. However, tissue time–activity curve
displayed an increase in the radioactivity accumulation in striatum
and cerebellum, post-injection of [11C]MP-10. Analysis of the
metabolites from rat brain tissue, plasma and monkey plasma
post-injection of [11C]MP-10 revealed that a lipophilic radiolabeled
metabolite was formed. This metabolite has the capability to pen-
etrate blood–brain-barrier and enter into the brain. Overall,
3.6. Metabolite studies in monkey plasma, rat plasma as well as
rat brain
3.6.1. In vivo metabolite analysis in rat blood and rat brain
Adult male Sprague–Dawley rats (250–300 g) were anesthe-
tized with 2–3% isoflurane/oxygen and [11C]MP-10 (ꢀ0.04 GBq
for the 2 min, ꢀ0.19 GBq for the 20 min, ꢀ0.24 GBq for 40 min;
ꢀ0.28 GBq for 60 min) was administered via intravenous tail vein
injection. Whole blood samples were collected via cardiac punc-
ture under anesthesia into heparinized syringes immediately be-
fore rats were euthanized. Rats were euthanized under
anesthesia at 2, 20, 40, and 60 min post-injection. The whole brain
was removed from the rat, blotted to remove excess blood, and
homogenized on ice with 2 mL of ice-cold acetonitrile. 1 mL ali-
quots of whole blood were counted in a well counter and then sep-
arated by centrifugation for 2 min into packed red cells (PRC) and
plasma, which were separated and counted. An aliquot of serum
was deproteinated by mixing with 2 parts of ice-cold acetonitrile;
plasma proteins were separated by centrifugation, and both por-
[
11C]MP-10 was found to bind specifically to PDE10A in the rat
and monkey brain and displayed a clear image of striatum, to iden-
tify a clinical PET probe to image PDE10A in human beings. Further
optimization of the structure of [11C]MP-10 is necessary to resolve
the issue of radioactive metabolite.
Acknowledgment
Financial support for these studies was provided by the Na-
tional Institute of Health under 5R33MH081281-04 (RHM),
NS058714, 1R21NS061025-01A2 and Intramural grant MIR-11-
009 of Mallinckrodt Institute of Radiology in Washington Univer-
sity School of Medicine. The authors gratefully thank Christina M.
Zukas, John Hood, Darryl Craig, Ruike Wang, Aixiao Li and Junfeng
Li for their excellent technical assistance for the microPET studies
in non-human primate.
tions were counted. 200
an Agilent SB C-18 analytic HPLC column (250 mm  4.6 mm,
A) and peaks detected at 254 nm UV wavelength. The mobile
lL of the supernatant was injected on
5
l
phase was acetonitrile/0.1 M ammonium formate buffer (48:52,
v/v) and the flow rate was 1.0 mL/min. The HPLC fractions were
collected at 1 min intervals for 16 min; the sample radioactivity
was counted and decay corrected to the time the first sample of
the series was counted. Similarly, a 1 mL aliquot of the brain
homogenate was transferred to a separate tube and counted in a
well counter. The acetonitrile extract was separated from the tis-
sue pellet by centrifugation. The supernatant was used for HPLC
chromatography as described above. The percent of unchanged
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.01.032.
[
11C]MP-10 (parent compound) and radiolabeled metabolites was
calculated by dividing the amount of recovered radioactivity in
the peak by the sum of the total recovered radioactivity in all sam-
ples and multiplying by 100. The parent compound [11C]MP-10
was authenticated by co-injection with cold standard MP-10 sam-
ple; the retention time for [11C]MP-10 was ꢀ10.5 min. It was ob-
served that even using the same brand of analytic column for the
metabolite analysis, in order to keep the similar retention time of
References and notes
1. Loughney, K.; Snyder, P. B.; Uher, L.; Rosman, G. J.; Ferguson, K.; Florio, V. A.
Gene 1999, 234, 109.
2. Bora, R. S.; Gupta, D.; Malik, R.; Chachra, S.; Sharma, P.; Saini, K. S. Biotechnol.
Appl. Biochem. 2008, 49, 129.
3. Hebb, A. L.; Robertson, H. A. Curr. Opin. Pharmacol. 2007, 7, 86.
4. Nishi, A.; Kuroiwa, M.; Miller, D. B.; O’Callaghan, J. P.; Bateup, H. S.; Shuto, T.;
Sotogaku, N.; Fukuda, T.; Heintz, N.; Greengard, P.; Snyder, G. L. J. Neurosci.
2008, 28, 10460.
5. Coskran, T. M.; Morton, D.; Menniti, F. S.; Adamowicz, W. O.; Kleiman, R. J.;
Ryan, A. M.; Strick, C. A.; Schmidt, C. J.; Stephenson, D. T. J. Histochem. Cytochem.
2006, 54, 1205.
6. Groenewegen, H. J.; van den Heuvel, O. A.; Cath, D. C.; Voorn, P.; Veltman, D. J.
Brain Dev. 2003, 25, S3.
7. Winterer, G.; Carver, F. W.; Musso, F.; Mattay, V.; Weinberger, D. R.; Coppola, R.
Hum. Brain Mapp. 2007, 28, 805.
8. Winterer, G.; Weinberger, D. R. Trends Neurosci. 2004, 27, 683.
9. DeLong, M. R.; Wichmann, T. Arch. Neurol. 2007, 64, 20.
10. Menniti, F. S.; Chappie, T. A.; Humphrey, J. M.; Schmidt, C. J. Curr. Opin. Investig.
Drugs 2007, 8, 54.
11. Hebb, A. L.; Robertson, H. A.; Denovan-Wright, E. M. Neuroscience 2004, 123, 967.
12. Hu, H.; McCaw, E. A.; Hebb, A. L.; Gomez, G. T.; Denovan-Wright, E. M. Eur. J.
Neurosci. 2004, 20, 3351.
13. Siuciak, J. A.; Chapin, D. S.; Harms, J. F.; Lebel, L. A.; McCarthy, S. A.; Chambers,
L.; Shrikhande, A.; Wong, S.; Menniti, F. S.; Schmidt, C. J. Neuropharmacology
2006, 51, 386.
14. Schmidt, C. J.; Chapin, D. S.; Cianfrogna, J.; Corman, M. L.; Hajos, M.; Harms, J.
F.; Hoffman, W. E.; Lebel, L. A.; McCarthy, S. A.; Nelson, F. R.; Proulx-LaFrance,
C.; Majchrzak, M. J.; Ramirez, A. D.; Schmidt, K.; Seymour, P. A.; Siuciak, J. A.;
Tingley 3rd, F. D.; Williams, R. D.; Verhoest, P. R.; Menniti, F. S. J. Pharmacol. Exp.
Ther. 2008, 325, 681.
15. Threlfell, S.; Sammut, S.; Menniti, F. S.; Schmidt, C. J.; West, A. R. J. Pharmacol.
Exp. Ther. 2009, 328, 785.
16. Chappie, T.; Humphrey, J.; Menniti, F.; Schmidt, C. Curr. Opin. Drug Discov. Dev.
2009, 12, 458.
[
11C]MP-10 as ꢀ10 min, the mobile phase and the flow rate needs
to be adjusted. To further determine the structure of metabolite #1
which is the first radioactivity peak in the HPLC chromatograph,
60 min of brain tissue supernatant sample (180
injected with 4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenol
acetonitrile solution (10 L, 100 ppm) into a LC–MS, in which the
lL) was co-
l
liquid chromatography condition was the same as HPLC condition
described above. From the LC–MS analysis, the metabolite peak #1
was detected to have m/z+ as 251.2384, which matched with 4-(1-
methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenol.
3.6.2. In vivo metabolite analysis in macaque plasma
Arterial blood samples (1.2–1.5 mL) were collected at 5, 15, 30,
and 60 min post-iv injection of [11C]MP-10. The HPLC injection
sample preparation and data collection were conducted by follow-
ing the similar procedure as described for rat plasma samples.
4. Conclusion
In summary, [11C]MP-10, a high affinity and high selectivity li-
gand for enzyme PDE10A was successfully radiosynthesized by

Z. Tu et al. / Bioorg. Med. Chem. 19 (2011) 1666–1673
1673
17. Walker, M. A. Drug Discovery Today 2010, 15, 79.
24. Honer, M.; Hengerer, B.; Blagoev, M.; Hintermann, S.; Waldmeier, P.;
Schubiger, P. A.; Ametamey, S. M. Nucl. Med. Biol. 2006, 33, 607.
25. Osman, S.; Lundkvist, C.; Pike, V. W.; Halldin, C.; McCarron, J. A.; Swahn, C. G.;
Ginovart, N.; Luthra, S. K.; Bench, C. J.; Grasby, P. M.; Wikstrom, H.; Barf, T.;
Cliffe, I. A.; Fletcher, A.; Farde, L. Nucl. Med. Biol. 1996, 23, 627.
26. Pike, V. W.; McCarron, J. A.; Hume, S. P.; Ashworth, S.; Opacka-Juffry, J.; Osman,
S.; Lammertsma, A. A.; Poole, K. G.; Fletcher, A.; White, A. C.; Cliffe, I. A. Med.
Chem. Res. 1994, 5, 208.
18. Verhoest, P. R.; Chapin, D. S.; Corman, M.; Fonseca, K.; Harms, J. F.; Hou, X.;
Marr, E. S.; Menniti, F. S.; Nelson, F.; O’Connor, R.; Pandit, J.; Proulx-LaFrance,
C.; Schmidt, A. W.; Schmidt, C. J.; Suiciak, J. A.; Liras, S. J. Med. Chem. 2009, 52,
5188.
19. Kehler, J.; Kilburn, J. P. Expert Opin. Ther. Pat. 2009, 19, 1715.
20. Tu, Z.; Xu, J.; Jones, L. A.; Li, S.; Mach, R. H. Nucl. Med. Biol. 2010, 37, 509.
21. Tu, Z.; Xu, J.; Li, S.; Jones, L. A.; Mach, R. H. J. Nucl. Med. 2009, 50, 618.
22. Celen, S.; Koole, M.; De Angelis, M.; Sannen, I.; Chitneni, S. K.; Alcazar, J.;
Dedeurwaerdere, S.; Moechars, D.; Schmidt, M.; Verbruggen, A.; Langlois, X.;
Van Laere, K.; Andres, J. I.; Bormans, G. J. Nucl. Med. 2010, 51, 1584.
23. Moerlein, S. M.; Perlmutter, J. S.; Cutler, P. D.; Welch, M. J. Nucl. Med. Biol. 1997,
24, 311.
27. Tu, Z.; Chu, W.; Zhang, J.; Dence, C. S.; Welch, M. J.; Mach, R. H. Nucl. Med. Biol.
2005, 32, 437.
28. Andrews, T. C.; Brooks, D. J. Mol. Med. Today 1998, 4, 532.