Synthesis and biological characterization of a promising F-18 PET tracer for vesicular acetylcholine transporter

Article abstract of DOI:10.1016/j.bmc.2015.05.058

Nine fluorine-containing vesicular acetylcholine transporter (VAChT) inhibitors were synthesized and screened as potential PET tracers for imaging the VAChT. Compound 18a was one of the most promising carbonyl-containing benzovesamicol analogs; the minus

Full text of DOI:10.1016/j.bmc.2015.05.058

Accepted Manuscript
Synthesis and Biological Characterization of a Promising F-18 PET Tracer for
Vesicular Acetylcholine Transporter
Zhude Tu, Xiang Zhang, Hongjun Jin, Xuyi Yue, Prashanth K. Padakanti, Lihai
Yu, Hui Liu, Hubert P. Flores, Kota Kaneshige, Stanley M. Parsons, Joel S.
Perlmutter
PII:
S0968-0896(15)00482-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.05.058
BMC 12354
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 April 2015
22 May 2015
28 May 2015
Please cite this article as: Tu, Z., Zhang, X., Jin, H., Yue, X., Padakanti, P.K., Yu, L., Liu, H., Flores, H.P., Kaneshige,
K., Parsons, S.M., Perlmutter, J.S., Synthesis and Biological Characterization of a Promising F-18 PET Tracer for
Vesicular Acetylcholine Transporter, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/
j.bmc.2015.05.058
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Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Synthesis and Biological Characterization of a Promising F-18 PET Tracer for
Vesicular Acetylcholine Transporter
Zhude Tua,*, Xiang Zhanga, Hongjun Jina, Xuyi Yuea, Prashanth K. Padakantia, Lihai Yua, Hui Liua, Hubert
P. Floresb, Kota Kaneshigec, Stanley M. Parsons c, and Joel S. Perlmuttera,b
aDepartment of Radiology, Washington University School of Medicine, 510 South Kingshighway Blvd., St. Louis, MO 63110, USA
bDepartment of Neurology, Washington University School of Medicine, 510 South Kingshighway Blvd., St. Louis, MO 63110, USA
cDepartment of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Nine fluorine-containing vesicular acetylcholine transporter (VAChT) inhibitors were
synthesized and screened as potential PET tracers for imaging the VAChT. Compound 18a was
one of the most promising carbonyl-containing benzovesamicol analogues; the minus
enantiomer, (-)-18a displayed high potency (VAChT Ki = 0.59 0.06 nM) and high selectivity
for VAChT versus σ receptors (> 10,000-fold). The radiosynthesis of (-)-[18F]18a was
accomplished by a two-step procedure with 30 – 40% radiochemical yield. Preliminary
biodistribution studies of (-)-[18F]18a in adult male Sprague–Dawley rats at 5, 30, 60 and 120
min post-injection (p.i.) were promising. The total brain uptake of (-)-[18F]18a was 0.684 ID%/g
at 5 min p.i. and by 120 min p.i. slowly washed out to 0.409 %ID/g.; evaluation of regional
brain uptake showed stable levels of ~0.800 %ID/g from 5 to 120 min p.i in the VAChT-
enriched striatal tissue of rats, indicating the tracer had crossed the blood brain barrier and was
retained in the striatum. Subsequent microPET brain imaging studies of (-)-[18F]18a in
nonhuman primates (NHPs) showed high striatal accumulation in the NHP brain; the
standardized uptake value (SUV) for striatum reached a maximum value of 5.1 at 15 min p.i.
The time-activity curve for the target striatal region displayed a slow and gradual decreasing
trend 15 min after injection, while clearance of the radioactivity from the cerebellar reference
region was much more rapid. Pretreatment of NHPs with 0.25 mg/kg of the VAChT inhibitor (-
)-vesamicol resulted in a ~90% decrease of striatal uptake compared to baseline studies. HPLC
metabolite analysis of NHP plasma revealed that (-)-[18F]18a had a good in vivo stability.
Together, these preliminary results suggest (-)-[18F]18a is a promising PET tracer candidate for
imaging VAChT in the brain of living subjects.
Received in revised form
Accepted
Available online
Keywords:
VAChT
PET tracer
Vesamicol
F-18
Striatum
2015 Elsevier Ltd. All rights reserved.
———
Abbreviations: Ac2O, acetic anhydride; AD, Alzheimer disease; BVM, benzovesamicol; calcd, calculated; CH2Cl2, dichloromethane; CNS, central nervous
system; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; [3H]DTG, [3H]ditolylguanidine; EOB, end of bombardment; EtOH, ethanol; [18F]FEOBV,
[18F]fluoroethoxybenzovesamicol; HPLC, high performance liquid chromatography; HRMS, high resolution mass spectrometry; [123I]IBVM, (-)-5-[123I]iodo-
benzovesamicol; IC50, half maximum inhibitory constant; MABV, 5-(N-methyl-amino)benzo-vesamicol; NEFA, N-ethyl-fluoroacetamidobenzo-vesamicol;
NHPs, nonhuman primates; PD, Parkinson Disease; PET, positron emission tomography; p.i., post-injection; PSL, photo-stimulated luminescence; QC, quality
control; SD, Sprague-Dawley; SPECT, single-photon emission computerized tomography; SUV, standardized uptake value; THF, tetrahydrofuran; TLC, thin
layer chromatography; VAChT, Vesicular Acetylcholine Transporter.
*
Corresponding author. Tel.: +1-314-362-8487; fax: +1-314-362-8555.
E-mail address: tuz@mir.wustl.edu (Z. Tu)
1

We previously reported a new class of VAChT analogues in
which a carbonyl group is interposed between the phenyl and
piperidine ring of the benzovesamicol structure; several lead
candidates displayed high in vitro binding affinity and high
selectivity for VAChT versus other CNS targets.[35-40]
Evaluation of these 11C- and 18F-labeled radiotracers in rodents
and microPET imaging studies in NHPs suggested that the
radiolabeled version of several ligands (7-10, Figure 1) bound in
vivo to the VAChT enriched striatal regions.[38-41] The half-life
of 18F (T1/2 = 109.8 min) permits longer scan sessions that
generate higher target-to-reference ratios; 18F PET tracers also
place fewer time constraints on tracer production. Here we
further explore carbonyl-containing benzovesamicol analogues
using the following strategies: 1) Incorporating PEGylated
groups on the phenyl ring linked to the piperidinyl ring by the
carbonyl group (Figure 1), which can improve clearance kinetics
by decreasing lipophilicity;[42-44] 2) Introducing a fluoroethoxy
group on the tetralin moiety to further improve affinity and
selectivity for VAChT versus the σ receptors; 3) Incorporating a
fluorine atom to provide position(s) for 18F radiolabeling. In this
manuscript, we report the synthesis and in vitro and in vivo
biological evaluation of the new fluorinated carbonyl-containing
analogues to determine their suitability as 18F labeled ligands for
imaging VAChT in vivo.
1. Introduction
The loss or decline in memory and other cognitive abilities
is a clinical syndrome characteristic of dementia which is
common seen in Alzheimer disease (AD), Parkinson Disease
(PD), and other neurodegenerative diseases.[1-6] The severity of
dementia is linked to loss of cholinergic neurons and synapses in
the central nervous system (CNS). Identifying a reliable
biomarker that is able to assess the loss of the cholinergic neuron
is imperative. The vesicular acetylcholine transporter (VAChT) is
an important transporter protein in the neuronal cholinergic
system and has been accepted as a reliable cholinergic marker for
more than 25 years.[7-10] Positron emission tomography (PET)
is a non-invasive imaging modality and able to provide
functional information for molecular and cellular processes in
living subjects. A PET radioligand having high affinity and
selectivity for VAChT, and suitable radiopharmaceutical
properties would provide a unique tool to measure the loss of
cholinergic neurons in the brain of subjects with dementia by
assessing changes in VAChT level. The identification of a potent
and selective PET ligand with minimal pharmacological effects
has posed significant difficulties. Vesamicol (1, initially named
AH 5183, Figure 1) was first reported in 1969 [11, 12] as a
potential local anesthetic. Bahr and Parsons reported that L-
isomer and D-isomer of vesamicol had a different binding
activity in Torpedo synaptic vesicles in 1986; vesamicol was
later discovered to be a stereoselective inhibitor of VAChT.[13-
15] Although tritiated vesamicol has been widely used for in
vitro studies of cholinergic loss, vesamicol and many analogues
have significant binding affinity for σ receptors that limits their
use as VAChT imaging agents because σ receptors are highly
expressed throughout the brain.[16-18] To overcome this
challenge, investigators have put tremendous efforts into
exploring vesamicol derivatives to improve the imaging of the
VAChT binding and to reduce the σ receptor binding affinity.
Identification of benzovesamicol (2, BVM, Figure 1) was a
significant achievement, as this structural modification not only
retained the high VAChT affinity, but also offered position(s) on
the aromatic ring in the tetralin fragment for introducing
substitution group(s).[19-22] Among the benzovesamicol
analogues shown in Figure 1, 5-(N-methyl-amino)benzo-
vesamicol (3, MABV) and N-ethyl-fluoroacetamidobenzo-
vesamicol (4, NEFA) are more potent inhibitors and more
selective for VAChT versus σ receptors than vesamicol.[23]
Furthermore, the single-photon emission computerized
tomography (SPECT) radiotracer (-)-5-[123I]iodo-benzovesamicol
([123I]5, [123I]IBVM) was used for mapping cholinergic terminals
in the human brain [7-9, 24-27] although quantification of peak
uptake in the target regions required at least 6 hr post-injection
(p.i.). Although SPECT is a useful tool for studies of ligands with
slow kinetic processes,[28] PET offers higher spatial resolution
and sensitivity for clinical studies of ligands with more rapid
pharmacokinetics. [18F]fluoroethoxy-benzovesamicol ([18F]6,
[18F]FEOBV) was recently approved for the clinical studies in
human subjects as a PET ligand for assessing the VAChT levels
in the brain as a biomarker of cholinergic function.[29-32]
Although equilibrium kinetics in the brain of both NHP and
human subjects show delayed equilibrium of > 360 min p.i.,[32]
[18F]FEOBV offers advantages over SPECT ligands for both
preclinical and clinical imaging of cholinergic loss. Studies of
FEOBV in a rat model of cholinergic dysfunction showed a good
correlation between PET measures of [18F]FEOBV uptake and ex
vivo autoradiography of the same animals post PET; ex vivo brain
autoradiography measures of [18F]FEOBV binding in human AD
cases and age-matched controls also showed differences
consistent with cholinergic loss.[33, 34]
2. Results and Discussion
2.1 Chemistry
The syntheses of the PEGylated compounds 12 – 14 are
shown in Scheme 1. Intermediate 11 was synthesized as
previously reported.[40] Fluoroethoxy-containing 12 was
obtained by O-alkylation of 11 with 2-bromo-1-fluoroethane.
PEGylated analogues 13 and 14 were obtained by treating 11
with the corresponding fluorinated PEGylated tosylates. The
syntheses of benzovesamicol analogues containing
a 5-
substituted carbonyl group are shown in Scheme 2. The epoxide
compound 15 was synthesized as previously described.[36, 38]
Commercially
available
(4-fluorophenyl)(piperidin-4-
yl)methanone (16) was reacted with 15 to afford the phenol
regioisomers 17a and 17b. The desired ligands 18a and 18b were
obtained via O-alkylation of 17a and 17b respectively with -2-
fluoroethyl tosylate.
Since VAChT inhibitors demonstrate stereoselective
binding, we initially attempted to resolve the potent racemic
compound ( )-18a directly using a Chiralcel OD column without
success. To overcome this challenge, racemic compound ( )-18a
was first converted to the acetate ester ( )-19a, which was easily
resolved on Chiralcel OD column using a mobile phase of 3% 2-
propanol in hexanes, as shown in Scheme 3. Using a semi-
preparative Chiralcel OD column, the minus isomer (-)-19a
eluted at 25-29 min and the plus isomer (+)-19a eluted at 30-38
min. Hydrolysis of the enantiomers (-)-19a and (+)-19a using
saturated sodium carbonate aqueous solution afforded the desired
enantiomers (-)-18a and (+)-18a.
2.2 In vitro binding affinity studies
The newly synthesized analogues were screened using our
standard in vitro binding assays [36, 37, 40, 45] to measure their
affinities to VAChT and σ receptors (Table 1). [3H]vesamicol
was used as the radioligand for the VAChT competitive binding
assay; [3H]pentazocine and [3H]ditolylguanidine([3H]DTG) in the
presence of 1 µM (+)-pentazocine were used for σ1 and σ2
receptor binding assays respectively. PEGylated compounds 12-
2

14 displayed similar binding affinities for VAChT (0.87 to 1.74
nM), while the selectivity for VAChT over both σ1 and σ2
receptors was greatly increased by extending the PEG chain: Ki-σ1
values were 13.23 0.20, 1,580 46, and > 10,000 nM for
compounds 12, 13, and 14 respectively. In comparison of
compounds 17a to 17b, and 18a to 18b, the hydroxyl or
fluoroethoxy substitution on the 8-position resulted in > 10-fold
decrease of VAChT affinity with the Ki values of 4.64 0.32,
56.20 4.08 nM for 17a and 17b, 1.55 0.18, 92.80 22.20 nM
for 18a and 18b respectively; the observed stereoselectivity for
VAChT is consistent with previous reports.[46, 47] Enantiopure
(-)-18a was more potent than (+)-18a with Ki values of 0.59
0.06, 13.0 2.20 nM for (-)-18a and (+)-18a respectively. The
results of these in vitro binding studies suggested that: 1)
PEGylation not only reduces the lipophilicity, but also improves
selectivity for VAChT versus σ receptors; 2) the 5-substitution of
the tetralin moiety provides higher binding affinity for VAChT
than the non-substituted compound 10 or the 8-substituted
counterpart; the observation that 5-position substituted
compounds demonstrated high potency is consistent with
literature reports;[46, 47] 3) VAChT binding has
stereoselectivity. For racemic compound 18a, the minus isomer (-
)-18a is more potent than its plus isomer (+)-18a. Enantiopure (-
)-18a displayed high VAChT binding affinity (0.59 0.06 nM)
and high selectivity versus σ receptors (>10,000-fold), and
acceptable lipophilicity, with calculated LogP value of 3.45.
Compound (-)-18a was chosen for radiolabeling with 18F and for
further evaluation in rodents and nonhuman primates.
Biodistribution and regional brain uptake studies of (-)-
[18F]18a were performed in adult male SD rats at 5, 30, 60, and
120 min p.i.; the results are shown in Table 2. The initial uptake
(%ID/g) at 5 min p.i. in blood, heart, lung, muscle, fat, pancreas,
spleen, kidney, liver, and brain was 0.209, 0.706, 2.255, 0.171,
0.144, 1.202, 1.265, 2.363, 1.065 and 0.684 respectively. The
clearance of the radioactivity was rapid from the heart, lung,
spleen, kidney, and liver; the uptake (ID%/g) decreased to 0.390,
0.973, 0.685, 0.883, and 0.523, respectively, at 30 min and
further decreased at 60 and 120 min p.i. The high initial kidney
uptake suggests the radiotracer may be initially excreted through
the urinary system. From 5 to 120 min, the bone uptake of the
radioactivity remained stable, suggesting that there is no
significant metabolic defluorination in vivo. For brain regions of
interest, the uptake (ID%/g) of the cerebellum, brain stem, cortex,
striatum, thalamus, and hippocampus was 0.593, 0.683, 0.777,
0.782, 0.691, and 0.645 respectively at 5 min post injection of (-
)-[18F]18a; the initial uptake of the striatal region was a little
higher than other brain regions but no significant difference was
observed, as shown in Table 2 and Figure 3. However, the
striatal region retained the highest levels (%ID/g) at 0.886, 0.819,
and 0.761 at 30, 60 and 120 min, respectively, in contrast with
other brain regions. The uptake ratios for target to non-target
brain regions (striatum-to-cerebellum) increased from 1.3-fold at
5 min to 3.0-fold at 30 min and remained steady up to 120 min
p.i. as shown in Figure 3.
2.6 MicroPET imaging studies in NHPs
2.3 Radiochemistry
Following the promising data from rat ex vivo
autoradiography and biodistribution studies, microPET imaging
of (-)-[18F]18a in the brains of male cynomolgus monkeys was
used to determine its potential as a PET tracer for imaging the
VAChT in vivo. As shown in Figure 4, the microPET imaging
results suggested that (-)-[18F]18a was able to enter the NHP
brain and had the highest accumulation in the striatal region
including both caudate and putamen. This high accumulation of
radioactivity in the striatal region further indicates that (-)-
[18F]18a binds to VAChT in vivo. The time-activity curves
showed that the radioactivity accumulation in striatum reached
maximum with a standardized uptake value (SUV) of 5.1 at 15
min p.i. and decreased slowly over 120 min p.i. Compared with
[18F]FEOBV which displayed slow striatal kinetics, (-)-[18F]18a
demonstrated a much faster equilibrium for striatal binding in
NHP. The uptake ratio of the target (striatum) versus non-target
(cerebellum) increased gradually and reached ~3.0 at 80 min p.i.
To test the specificity of (-)-[18F]18a binding to VAChT in the
brain, microPET studies were performed in a monkey pretreated
with 0.25 mg/kg (-)-vesamicol, a known VAChT inhibitor, 5 min
before (-)-[18F]18a injection. Striatal uptake in this blocking
study was reduced about ~90% compared to the striatal uptake in
the baseline condition (Figure 4), while the change in the
cerebellar region was negligible (Figure 4); this further
suggested that (-)-[18F]18a binds to VAChT specifically in the
monkey brain.
The radiosynthesis of (-)-[18F]18a was accomplished by a
two-step 18F labeling strategy starting with (-)-17a as shown in
Scheme 4. Ethylene ditosylate was first reacted with
[18F]KF/Kryptofix 2.2.2 in acetonitrile and then purified on a
reversed phase HPLC system to afford [18F]fluoroethyl tosylate
([18F]20) with a 60-75% radiochemical yield. Nucleophilic
substitution of (-)-17a with [18F]20 in dimethyl sulfoxide
(DMSO) followed by HPLC purification afforded (-)-[18F]18a in
50-60% radiochemical yield (decay corrected to end of synthesis,
n > 20). The radioactive (-)-[18F]18a was authenticated by co-
injection with the nonradiolabeled standard (-)-18a on an
analytical HPLC system. The two-step radiosynthesis of (-)-
[18F]18a took ~ 2.0 hr with overall radiochemical yield of 30 to
40%, specific activity > 74 GBq/µmol (decay corrected to EOS)
and radiochemical purity > 98% (n > 20).
2.4 Ex vivo autoradiography study in rats
To confirm the target binding selectivity of (-)-[18F]18a in
the rat brain, ex vivo autoradiography was performed. An adult
male Sprague-Dawley (SD) rat was euthanized 60 min p.i. The
brain was quickly removed and snap-frozen then sectioned at 100
µm and mounted on glass slides. The brain sections were exposed
to the film in an imaging cassette for 12 hr; the distribution of
radioactivity is shown in Figure 2 with the outlined regions of
interest based on standard anatomical guides.[48] The results
show (-)-[18F]18a has the highest accumulation in the VAChT
enriched striatum, which is consistent with the literature
reports.[49] The uptake ratio of radioactivity in striatum versus
cerebellum reached 4.19 0.37 at 60 min p.i.
2.7 Metabolite studies in NHP plasma
Because the microPET imaging data suggested that (-)-
[18F]18a was a promising candidate VAChT-specific PET
radiotracer, radioactive metabolite analysis was performed in
NHP plasma samples post injection of (-)-[18F]18a. 70 - 82% of
the radioactivity was in the plasma after centrifugation to
separate the red blood cells. Following solvent extraction and
deproteination, 76 - 87% of the radioactivity in plasma was in the
supernatant. The solvent extract was then injected onto HPLC to
2.5 Biodistribution studies in rats
3

dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The crude product was purified by silica gel
column chromatography using ethyl acetate/hexane(1/4, v/v) to
give compound 12 (26.1 mg, 77%) as a pale yellow solid.
1HNMR (400 MHz, CDCl3) δ 1.76–1.95 (m, 4 H), 3.88 (dt, J =
2.7, 12.4 Hz, 1 H), 2.75–3.02 (m, 7 H), 3.24-3.35 (m, 2 H), 3.88
(td, J = 5.0, 13.8 Hz, 1 H), 4.05 (t, J = 4.0 Hz, 1 H). 4.33 (t, J =
4.2 Hz, 1 H). 4.71 (t, J = 4.0 Hz, 1 H), 4.87 (t, J = 4.2 Hz, 1 H),
6.99 (d, J = 8.4 Hz, 2 H), 7.09–7.16 (m, 4 H), 7.95 (d, J = 9.0 Hz,
2 H). 13C NMR (75.5 MHz, CD3OD) δ 201.4, 165.9, 162.4,
136.0, 135.0, 131.0, 129.1, 126.2, 116.6, 115.0, 108.6, 82.5 (J =
167.2 Hz), 68.0, 66.3 (d, J = 23.2 Hz), 50.3, 47.0, 43.3, 38.4,
31.2, 29.7, 28.0, 24.3; HRMS (ESI) Calcd for C24H29FNO3
(M+H)+ 398.2131, found: 398.2129. To 26 mg (0.065 mmol) of
12 in a glass vial was added in 2.5 mL of dichloromethane, and
2.94 mg (0.0325 mmol) of oxalic acid in methanol was added.
After recrystallization, 31.4 mg of the oxalate salt was obtained,
Mp: 196.2 – 197.0 °C.
identify the percentage of radioactive parent compound versus
any radiolabeled metabolites. The HPLC metabolite analysis of
the plasma samples showed three radioactive peaks: the parent
compound (-)-[18F]18a, radioactive metabolite 1 and radioactive
metabolite 2; the retention times were 9 – 11 min for (-)-[18F]18a,
2 – 4 min for the major hydrophilic radioactive metabolite 1 and
6 – 7 min for the radioactive metabolite 2 which represented <
1.5% of the total radioactivity collected from the HPLC and is
considered negligible. The percentage of the remaining parent
compound (-)-[18F]18a was 66, 41, 21, and 16% at 15, 30, 60 and
90 min p.i. respectively. The level of radioactive metabolite 1
was 3.0, 30, 59, 78 and 82% at 5, 15, 30, 60 and 90 min
respectively as shown in Figure 5. To test if the hydrophilic
radioactive metabolite 1 has capability in penetrating the blood
brain barrier and thus confound PET measurement, we evaluated
the stability of the tracer in rat blood and brain. The
radiometabolite analysis of the rat plasma samples revealed that
(-)-[18F]18a in rat metabolized much faster than in monkeys,
indicating the metabolism of (-)-[18F]18a has
a species
3.1.2 (4-(2-(2-Fluoroethoxy)ethoxy)phenyl)(1-((2R,3R)-3-hy-
droxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)-
methanone (13)
difference. This limits the utility of rodent data in predicting the
metabolism of (-)-[18F]18a in nonhuman primates or human
subjects. Future metabolite analysis of the human plasma
samples, kinetic modeling of tracer uptake, and clearance in
human subjects are necessary to determine if the major
radioactive metabolite can enter into the human brain.
To a solution of 11 (40 mg, 0.11 mmol) in 2.0 mL DMF was
added 2-(2-fluoroethoxy)ethyl tosylate (60 mg, 0.22 mmol) and
K2CO3 (35 mg, 0.25 mmol). The mixture was stirred at room
temperature overnight. Ethyl acetate (35 mL) was added into the
reaction mixture, and then sequentially washed with saturated
NaHCO3 aqueous solution and brine. The combined organic
solution was dried over anhydrous sodium sulfate, concentrated
under reduced pressure. The crude product was purified on a
silica gel column to give compound 13 (38 mg, 78%) as colorless
syrup. 1HNMR (400 MHz, CDCl3) δ 7.94 (d, J=8.7 Hz, 2H), 7.12
(s br, 4H), 6.98 (d, J=8.4 Hz, 2H), 4.70-4.65 (m, 1H), 4.55-4.49
(m, 1H), 4.25-4.20 (m, 2H), 4.00-3.75 (m, 5H), 3.40-3.10 (m,
2H), 3.10-2.70 (m, 7H), 2.5-2.30 (m, 1H), 2.00-1.70 (m, 4H).
The oxalate salt was obtained as described above. Mp: 203 – 206
°C. Anal. Calcd of C28H34FNO8: C, 63.27; H, 6.45; N, 2.63.
Found: C, 63.29; H, 6.49; N, 2.79. HRMS (ESI) Calcd for
C26H33FNO4 (M+H)+ 442.2394, found: 442.2392.
3. Experimental
3.1 General
All the solvents and reagents were obtained commercially
and used as received, unless otherwise stated. All anhydrous
reactions were carried out in oven-dried or flame-dried, and
nitrogen purged glassware. Anhydrous tetrahydrofuran was dried
over sodium and fresh distilled prior to use. Anhydrous
dichloromethane was distilled over calcium hydride prior to use.
Reactions were monitored by thin layer chromatography (TLC)
using silica gel 60 F254 glass plates (EMD Chemicals Inc.). Flash
column chromatography was performed over silica gel (32-63
1
µm), HPLC grade solvents were used for chromatography. H
NMR were recorded on a Varian Mercury-VX 400 or 300 MHz
spectrometer. The chemical shifts are reported as δ values (ppm)
relative to tetramethylsilane (TMS) as an internal reference. The
SpectraSystem was used for both analytical and semi-preparative
HPLC. A Chiralcel OD normal phase HPLC column was used to
resolve enantiomers. The specific optical rotation was determined
on an automatic polarimeter (Autopol 111, Rudolph Research,
Flanders, NJ). Elemental analyses were determined by Atlantic
Microlab, Inc. (Norcross, GA).
3.1.3 (4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)phenyl)(1-((2R,-
3R)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-
4-yl)methanone (14)
Following the similar procedure for preparing compound 13,
compound 14 was afforded as colorless oil (41.6 mg, 41%).
1HNMR (400 MHz, CDCl3) δ 7.93 (d, J=9.0 Hz, 2H), 7.11 (s br,
4H), 6.97 (d, J=9.0 Hz, 2H), 4.70-4.63 (m, 1H), 4.55-4.45 (m,
1H), 4.25-4.18 (m, 3H), 4.00-3.80 (m, 3H), 3.80-3.70 (m, 6H),
3.40-3.20 (m, 2H), 3.10-2.70 (m, 7H), 2.5-2.30 (m, 1H), 2.00-
1.70 (m, 4H). The oxalate salt was obtained as described above.
Mp: 189.8 – 191.8 °C. Anal. Calcd for C30H38FNO9: C, 62.60; H,
6.65; N, 2.43. Found: C, 62.63; H, 6.62; N, 2.50. HRMS (ESI)
Calcd for C26H37FNO5 (M+H)+ 486.2656, found: 486.2655.
[18F]Fluoride was produced from a RDS111 cyclotron
(Siemens/CTI Molecular Imaging, Knoxville, TN) by 18O(p,
n)18F reaction through proton irradiation of enriched 18O water
(95%). [18F]Fluoride was firstly passed through an ion-exchange
resin and then eluted using 0.02 M potassium carbonate (K2CO3)
solution.
3.1.4 (1-(3,5-Dihydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-
piperidin-4-yl)(4-fluorophenyl)methanone (17a) and (1-(3,8-
dihydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-
yl)(4-fluorophenyl)methanone (17b)
3.1.1 (4-(2-Fluoroethoxy)phenyl)(1-(3-hydroxy-1,2,3,4-tetra-
hydronaphthalen-2-yl)piperidin-4-yl)methanone (12)
To a solution of 11 (30 mg, 0.08 mmol) in acetone (10 mL)
was added K2CO3 (12 mg, 0.08 mmol) and 2-bromo-1-
fluoroethane (54 mg, 0.2 mmol). The reaction mixture was
refluxed overnight until the reaction was completed as
determined by TLC. After the solvent was removed, 20 mL of
water was added to the flask, and the mixture was extracted with
ethyl acetate (10 mL × 3). The combined organic layers were
Compound
15
(200
mg,
1.2
mmol),
(4-
fluorophenyl)(piperidin-4-yl)methanone 16 (585 mg, 2.4 mmol)
and triethylamine (1 mL) were mixed in ethanol (5 mL) and
heated at 60 °C for 2 d. Ethyl acetate (50 mL) was used to dilute
the solution and then washed with brine (2 x 10 mL). After
concentration, the residue was loaded onto silica gel column and
4

eluted using CH2Cl2/methanol (20/1, v/v). Compound 17a was
obtained as the first component, a white solid (109 mg, 25%). Mp
229 – 230 oC. 1H NMR (300 MHz, CDCl3) δ 8.00 – 7.94 (m, 2H),
7.15 – 7.11 (m, 2H), 7.02 (t, J = 7.8 Hz, 1H), 6.66 (dd, J = 25.2
Hz, 7.8 Hz, 2H), 5.01 (br, 1H), 3.92 – 3.83 (m, 1H), 3.32 – 3.23
(m, 2H), 3.07 – 2.97 (m, 2H), 2.89 – 2.77 (m, 4H), 2.64 – 2.45
(m, 2H), 1.97 – 1.89 (m, 3H), 1.88 – 1.76 (m, 1H). 13C NMR
(75.5 MHz, CDCl3) δ 201.1, 166.2 (d, J = 301.0 Hz), 153.6,
135.8, 130.9, 130.8, 126.9, 121.8, 121.4, 115.8 (d, J = 21.7 Hz),
112.1, 66.5, 65.3, 51.9, 43.8, 37.8, 29.5, 29.2, 28.5, 20.0. HRMS
(ESI) Calcd for C22H25FNO3 (M+H)+ 370.1818, found: 370.1817.
Racemic compound ( )-18a (140 mg, 0.337 mmol), acetic
anhydride (200 µL) and pyridine (200 µL) were dissolved in
CH2Cl2 (10 mL) and stirred at ambient temperature overnight.
The product was extracted using ethyl acetate, and then dried
over anhydrous Na2SO4. After solvent was removed under
vacuum, the residue was purified on silica gel column by ethyl
acetate/hexane (1/5 to 1/3, v/v) to afford ( )-19a as a white solid
(148 mg, 96%). Mp 70 - 73 C. H NMR (400 MHz, CDCl3) δ
1.58-1.86 (m, 4H), 2.10 (s, 3H), 2.45-2.76 (m, 3H), 2.80-3.20 (m,
7H), 4.02-4.25 (m, 3H), 4.65 (s, 1H), 4.80 (s, 1H), 6.57-6.74 (m,
2H), 7.01-7.15 (m, 3H), 7.95 (br s, 2H). 13C NMR (75.5 MHz,
CD3OD) δ 202.0, 171.0, 156.2, 131.0, 130.9, 126.6, 121.1, 120.8,
115.4 (d, J = 21.9 Hz), 115.2, 108.6, 108.4, 81.9 (d, J = 169.3
Hz), 70.0, 66.2 (d, J = 25.3 Hz), 63.3, 49.5, 43.5, 37.9, 34.7, 29.0,
22.2, 21.1, 20.0. HRMS (ESI) Calcd for C26H30F2NO4 (M+H)+
458.2143, found: 458.2152.
o
1
Compound 17b was obtained as the second component, a
o
white solid (120 mg, 27%). Mp 208 – 210 C. 1H NMR (400
MHz, CDCl3) δ 1.76-1.93 (m, 4H), 2.46-2.64 (m, 2H), 2.76-2.89
(m, 4H), 2.97-3.07 (m, 2H), 3.25-3.32 (m, 2H), 3.88 (s, 1H), 6.61
(d, J = 8.4Hz, 2H), 6.72 (d, J = 7.5Hz, 1H), 7.15 (t, J = 8.1Hz,
2H), 7.98 (t, J = 8.4Hz, 2H). 13C NMR (75.5 MHz, CD3SOCD3)
δ 201.5, 165.4 (d, J = 251.6 Hz), 155.2, 137.1, 132.8, 131.6 (d, J
= 9.3 Hz), 126.6, 121.6, 119.7, 116.2 (d, J = 21.7 Hz), 112.1,
66.5, 65.7, 50.3, 46.6, 43.6, 32.9, 29.6, 29.4, 27.8. HRMS (ESI)
Calcd for C22H25FNO3 (M+H)+ 370.1818, found: 370.1833.
3.1.8 (-)-(1-(8-(2-Fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahy-
dronaphthalen-2-yl)piperidin-4-yl)(4-fluorophen-yl)-
methanone ((-)-18a)
Enantiomers of ( )-19a (132 mg, 0.29 mmol) were separated
by HPLC using a Chiralcel OD column (250 mm × 10 mm), 2-
propanol/hexane (3/97, v/v) as mobile phase, flow rate of 4.0
mL/min, and UV wavelength at 254 nm) to give (-)-19a (53 mg,
40%) and (+)-19a (61 mg, 46%) respectively. Each enantiopure
compound was mixed with saturated Na2CO3 aqueous solution (3
mL) in EtOH (3 mL) separately and stirred for 72h at room
temperature. Water (20 mL) was used to dilute the reaction
mixture followed by extraction using CH2Cl2 (20 mL × 3). The
combined organic phase was dried over anhydrous Na2SO4, and
the solvent was removed under reduced pressure. The crude
product was purified on silica gel column using ethyl
acetate/hexane (1/1, v/v) to afford (-)-18a as a white solid (40
3.1.5 (1-(8-(2-Fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydro-
naphthalen-2-yl)piperidin-4-yl)(4-fluorophenyl)methanone
(18a)
Compound 17a (74 mg, 0.2 mmol), 2-fluoroethyl tosylate
(52 mg, 0.24 mmol) and Cs2CO3 (98 mg, 0.3 mmol) were mixed
in THF (5 mL) and refluxed overnight. The reaction mixture was
partitioned between ethyl acetate and water. After drying over
anhydrous Na2SO4, the organic phase was concentrated and
purified on silica gel column by CH2Cl2/ethyl acetate (1/1, v/v) to
o
1
get a white solid 18a (65 mg, 78%). Mp 149 – 152 C. H NMR
(400 MHz, CDCl3) δ 1.79 -1.95 (m, 4H), 2.47-2.61 (m, 2H),
2.72-2.99 (m, 5H), 3.10 (d, J = 16.5Hz, 1H), 3.24-3.35 (m, 2H),
3.84 (m, 1H), 4.18 (s, 1H), 4.27 (s, 1H), 4.72 (s, 1H), 4.88 (s,
1H), 6.65 (d, J = 8.4Hz, 1H), 6.76 (d, J = 8.4Hz, 1H), 7.08-7.19
(m, 3H), 8.00 (t, J = 8.7Hz, 2H). 13C NMR (75.5 MHz,
CD3COCD3) δ 200.5, 165.4 (d, J = 254.2 Hz), 156.3, 135.7,
132.8, 131.2 (d, J = 9.2 Hz), 126.6, 124.0, 121.5, 115.5 (d, J =
22.0 Hz), 108.3, 82.1 (d, J = 168.4 Hz), 67.4 (d, J = 19.6 Hz),
66.5, 65.2, 51.6, 44.6, 43.5, 38.2, 20.1; HRMS (ESI) Calcd for
C24H28F2NO3 (M+H)+ 416.2037, found: 416.2041. The free base
was converted to oxalate salt as described above.
1
mg, 88% yield). H NMR (400 MHz, CDCl3) δ 8.00 (t, J = 8.0
Hz, 2H), 7.17 – 7.09 (m, 3H), 6.76 (d, J = 8.0 Hz, 1H), 6.65 (d, J
= 8.0 Hz, 1H), 4.87 - 4.85 (m, 1H), 4.75 - 4.73 (m, 1H), 4.27 –
4.15 (m, 3H), 3.85 – 3.75 (m, 1H), 3.30 – 3.20 (m, 2H), 3.13 –
3.02 (m, 1H), 2.99 – 2.70 (m, 5H), 2.60 – 2.43 (m, 2H), 1.98 –
1.73 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 200.9, 165.6 (d, J =
253.5 Hz), 156.1, 135.6, 132.3, 130.8, 126.7, 124.0, 122.0, 115.8
(d, J = 21.7 Hz), 108.1, 82.0 (d, J = 169.8 Hz), 67.2 (d, J = 20.2
Hz), 66.4, 65.1, 52.1, 44.3, 43.8, 37.9, 29.6, 29.2, 20.0.The free
base of (-)-18a was converted to oxalate salt, Mp: 185.0-185.3
20
°C. The optical rotation of the oxalate salt was [α]D = -37.1°
(0.7 mg/mL in 1/1 acetonitrile/H2O). HRMS (ESI) Calcd for
C24H28F2NO3 (M+H)+ 416.2032, found: 416.2022.
3.1.6 (1-(5-(2-Fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydrona-
phthalen-2-yl)piperidin-4-yl)(4-fluorophenyl)methanone
(18b)
3.1.9 (+)-(1-(8-(2-Fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahy-
dronaphthalen-2-yl)pipe-ridin-4-yl)(4-fluorophenyl)-
methanone ((+)-18a)
Compound 18b was obtained by using 17b as the starting
material, following the procedure for making 18a. Mp 137 – 138
1
oC. H NMR (400 MHz, CDCl3) δ 1.79 -1.92 (m, 4H), 2.36-2.54
Starting with (+)-19a (58 mg), the procedure of making (-)-
18a afforded compound (+)-18a as a yellow oil (44.0 mg, 83%
(m, 2H), 2.75-3.02 (m, 6H), 3.27 (s, 1H), 3.50(d, J = 16.8Hz,
2H), 3.84 (m, 1H), 4.11 (s, 1H), 4.24 (s, 1H), 4.67 (s, 1H), 4.83
(s, 1H), 6.66 (d, J = 8.1Hz, 1H), 6.74 (d, J = 7.5Hz, 1H), 7.07-
7.17(m, 3H), 7.98 (t, J = 8.7Hz, 2H). 13C NMR (75.5 MHz,
CD3COCD3) δ 200.4, 165.4 (d, J = 256.4 Hz), 156.3, 136.8,
132.8 (d, J = 3.0 Hz), 131.1 (d, J = 9.2 Hz), 130.4, 123.2, 121.5,
115.6 (d, J = 22.0 Hz), 108.4, 82.1 (d, J = 168.4 Hz), 67.4 (d, J =
19.8 Hz), 66.1, 65.7, 51.4, 44.9, 43.6, 32.3, 26.3. HRMS (ESI)
Calcd for C24H28F2NO3 (M+H)+ 416.2032, found: 416.2029.
1
yield). H NMR (400 MHz, CDCl3) δ 8.00 (t, J = 8.0 Hz, 2H),
7.17 – 7.09 (m, 3H), 6.76 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 8.0 Hz,
1H), 4.87 - 4.85 (m, 1H), 4.75 - 4.73 (m, 1H), 4.27 – 4.15 (m,
3H), 3.85 – 3.75 (m, 1H), 3.30 – 3.20 (m, 2H), 3.13 – 3.02 (m,
1H), 2.99 – 2.70 (m, 5H), 2.60 – 2.43 (m, 2H), 1.98 – 1.73 (m,
4H). 13C NMR (100 MHz, CDCl3) δ 200.9, 165.6 (d, J = 253.4
Hz), 156.1, 135.6, 130.8, 126.7, 124.0, 122.0, 115.8 (d, J = 21.7
Hz), 108.2, 82.0 (d, J = 169.9 Hz), 67.2 (d, J = 20.2 Hz), 66.5,
65.1, 52.1, 44.3, 43.8, 37.9, 29.6, 29.2, 20.0. The free base was
converted to oxalic salt, Mp: 191.0 – 192.2 °C. The optical
3.1.7 3-(4-(4-Fluorobenzoyl)piperidin-1-yl)-5-(2-fluoro-
ethoxy)-1,2,3,4-tetrahydronaphthalen-2-yl acetate (19a)
20
rotation of (+)-18a was [α]D = 38.4o (0.65 mg/mL in 1/1
acetonitrile/H2O). HRMS (ESI) Calcd for C24H28F2NO3 (M+H)+
416.2032, found: 416.2021.
5

alumina Neutral Sep-Pak Plus cartridge. The crude product was
then loaded onto an Agilent SB-C18 semi-preparative HPLC
column (250 mm × 10 mm) with a UV detector set at 254 nm.
The HPLC system was equipped with a 5 mL injection loop. At
the flow rate of 4.0 mL/min, the retention time of the product was
9.5-10 min. The retention time of the precursor was 23-24 min.
The product eluted from the HPLC was diluted using ~50 mL
sterile water and then passed through a C-18 Sep-Pak Plus
cartridge to trap [18F]20 on the Sep-Pak. Using ether (2.5 mL) to
elute the radioactivity afforded [18F]20 (2.60-3.0 MBq, 60-75%
radiochemistry yield, decay corrected to end of synthesis, EOS).
The synthesis of [18F]20 took about 40 min.
3.2 In vitro biological evaluation
3.2.1 VAChT binding affinity studies
Binding affinities of the new compounds were determined
by competing them against 5 nM [3H]vesamicol for binding to
human VAChT present in synaptic-like microvesicles in
postnuclear supernatant prepared from PC12A123.7 cells.[50]
Nonspecific binding was determined from samples that contained
1 µM of nonradioactive ( )-vesamicol. The test compounds were
assayed in increments of 10-fold from 0.1 to 10,000 nM
concentration. The surfaces of containers were pre-coated with
Sigmacote (Sigma-Aldrich, MO). Samples containing 200 µg
postnuclear supernatant in 200 µL of 110 mM potassium tartrate,
20 mM HEPES (pH 7.4 with KOH), 1 mM dithiothreitol and
0.02 % sodium azide were incubated at 22 °C for 24 h. A volume
of 90 µL was filtered in duplicate through GF/F glass fiber filters
coated with polyethylenimine and washed. Filter-bound
radioactivity was determined by liquid scintillation spectrometry
for 10 min per sample. Averaged data were fitted by regression
with a rectangular hyperbola to estimate Ki. All compounds were
independently assayed at least two times.
3.3.2 Radiosynthesis of (-)-[18F]18a
The upper ether layer of the eluted solution of [18F]20 was
transferred into a vial first, and the bottom aqueous phase was
extracted with an additional 1 mL of ether. The combined ether
solution was passed through a set of two Sep-Pak Plus dry
cartridges into a reaction vessel. After the ether was evaporated
with a nitrogen stream at 25 °C, the solution of precursor (-)-17a
(1.0 - 1.4 mg) in DMSO (200 µL), and Cs2CO3 (1.0 - 1.5 mg)
were added to the abovementioned reaction vessel containing
[18F]20. The vessel was capped, vortexed, and then heated at 100
°C for 15 min. Subsequently, the residual was diluted with 3 mL
HPLC mobile phase (35:65 acetonitrile/0.1 M ammonium
formate buffer (pH ~4.5)) and loaded onto a semi-preparative
HPLC system for purification. The HPLC system contains a 5
mL injection loop, an Agilent SB-C18 column, a UV detector at
254 nm and a radioactivity detector. With 35:65 acetonitrile/0.1
M ammonium formate buffer (pH ~4.5) as eluent, at 4.0 mL/min
flow rate, the retention time of the product was 24 - 26 min,
whereas the retention time of (-)-17a was 9 - 10 min. The product
collection was diluted using sterile water (~50 mL) and then
passed through a C18 Sep-Pak Plus cartridge. The trapped
product was eluted using ethanol (0.6 mL), followed by 0.9%
saline (5.4 mL). After sterile filtration into a glass vial, the (-)-
[18F]18a was ready for quality control (QC) analysis and animal
studies. To check the quality of the -(-)-[18F]18a, an aliquot of
sample was co-injected with nonradiolabeled standard (-)-18a
sample solution onto an analytical HPLC system equipped with
an Agilent Zorbax SB-C18 column (250 × 4.6 mm) and UV
absorbance at 254 nm; the mobile phase consisted of
acetonitrile/0.1 M ammonium formate buffer (52/48, v/v). Under
these conditions, the retention time of (-)-[18F]18a was ~ 5.0 min
at a flow rate of 1.5 mL/min. For this step, the radiochemical
purity was > 98%, the radiochemical yield was 50 - 60% (n > 20,
decay corrected to EOS) and the specific activity was > 74
GBq/µmol (decay corrected to EOS). The synthesis of (-)-
[18F]18a starting with [18F]20 took about 80 min. The entire two-
step procedure of making (-)-[18F]18a starting with K[18F]F took
~2.0 h and the radiochemical yield was 30 – 40%.
3.2.2 Sigma receptors binding affinity studies
The σ receptor binding affinity studies were performed
following our previously reported procedure. [51, 52] The σ1
receptor binding assays were performed in 96-well plates by
incubating test compounds with approximately 300 µg protein of
guinea pig brain membrane homogenates and 5 nM (+)-
[3H]pentazocine (1.30 GBq/µmol, Perkin Elmer, Boston, MA).
Non-specific binding was determined from samples containing
10 µM cold haloperidol. The σ2 receptor binding assays were
similarly performed by incubating test compound(s) with rat liver
membrane homogenates (∼300 µg protein) and ∼5 nM [3H]DTG
(2.15 GBq/µmol, Perkin Elmer, Boston, MA) in the presence of 1
µM (+)-pentazocine to block σ1 sites. Non-specific binding was
determined from samples that contained 10 µM of cold
haloperidol. Data from the competitive inhibition experiments
were modeled using nonlinear regression analysis to determine
the concentration that inhibits 50% of the specific binding of the
radioligand (IC50 value). Competitive binding curves were best fit
to a one-site fit and gave pseudo-Hill coefficients of 0.6 – 1.0. Ki
values were calculated using the method of Cheng and Prusoff 36
and are presented as the mean SEM. For these calculations, we
used a Kd value of 7.89 nM for (+)-[3H]pentazocine in guinea pig
brains and a Kd value of 30.73 nM for [3H]ditolylguanidine in rat
livers.
3.3 Radiochemistry
3.3.1 Radiosynthesis of 1-[18F]fluoro-2-tosyloxyethane
([18F]20)
A sample of ~5.50 GBq [18F]/fluoride was added to a
reaction vessel containing Kryptofix 2.2.2 (6.5 - 7.0 mg). To the
mixture, acetonitrile (3 × 1.0 mL) was added to remove
azeotropically water using nitrogen gas to bubble though the
mixture at ~110 °C. After all the water was removed, 1, 2-
ethylene ditosylate (5.0 – 5.5 mg) was dissolved into acetonitrile
(200 µL) under vortex, and then the solution was transferred to
the reaction vessel containing [18F]fluoride/Kryptofix /K2CO3.
The reaction vessel was capped and the reaction mixture was
briefly mixed, then heated about 10 min in an oil bath that was
preheated to 110 °C. After reaction, the vessel was lifted up from
the oil bath, the reaction mixture was diluted using 3.0 mL of
HPLC mobile phase composed of 50/50 ratio of acetonitrile/0.1
M aqueous ammonium formate (pH ~6.5), and passed through an
3.4 Biodistribution studies in rats
All animal experiments were conducted in compliance with
the Guidelines for the Care and Use of Research Animals under
protocols approved by Washington University's Animal Studies
Committee. For the biodistribution studies, the solution of (-)-
[18F]18a (~2.2 MBq/120 µL) in 10% ethanol in saline was
injected via the tail vein into adult male SD rats under 2-3%
isoflurane/oxygen anesthesia. At 5, 30, 60, and 120 min p.i. (n =
4 for each time point), the rats were euthanized under anesthesia.
The whole brain was quickly harvested and dissected into regions
of cerebellum, brain stem, thalamus, striatum, cortex and
hippocampus; the remainder of the brain was also collected to
determine the total brain uptake. Samples of blood, heart, lung,
6

muscle, fat, pancreas, spleen, kidney, liver, and bone were also
collected, and all the samples were counted in an automated
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 using the
striatal uptake (%ID/g) divided by the %ID/g of the different
brain regions.
to body weight and dose of radioactivity injected to yield
standardized uptake value (SUV).
3.7 Metabolite studies on NHP plasma
Arterial blood samples (1.2 - 1.5 mL) were collected at 5, 15
30, 60, 90, and 120 min post-injection of the radiotracer in a
heparinized syringe.[41, 53] 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 µL) 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 determination. The HPLC system
consisted of an Agilent SB C-18 analytic HPLC column (250 mm
× 4.6 mm, 5 µA) and an UV detector with wavelength set at 254
nm. The mobile phase was acetonitrile/0.1 M ammonium formate
buffer (pH, ~ 4.5, 52/48, v/v), and the flow rate was 1.5 mL/min.
The HPLC fractions were collected 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.
For the ex vivo autoradiography study, an adult male SD rat
was injected with (-)-[18F]18a (~63.0 MBq) via the tail vein
under anesthesia and euthanized at 60 min p.i. The brain was
quickly removed, snap-frozen, and horizontal sections (100 µm)
were sliced using a chrome brain matrix (Zivic Instruments Inc.,
Pittsburg, PA). Frozen slides were directly exposed to film in an
imaging cassette (Fuji Photo Film Co., Tokyo, Japan) for 12 hrs
at -80 ˚C in the dark. The distribution of radioactivity was
visualized by a Fuji Bio-Imaging Analyzer FLA-7000 (Fuji
Photo Film Co., Tokyo, Japan). Photo-stimulated luminescence
(PSL) from the striatum and cerebellum was quantified using
Multi Gauge v3.0 software (Fuji Photo Film Co., Tokyo, Japan).
Data were background-corrected, and expressed as photo-
stimulated luminescence signals per square millimeter
(PSL/mm2).
To confirm if the major radioactive metabolite is able to
enter into the brain, metabolite analysis was performed for the rat
plasma samples post injection of (-)-[18F] 18a. 15 – 106 MBq of
(-)-[18F] 18a was injected into male SD rats which were
euthanized 5, 15, 30 and 60 min p.i. The rat plasma samples were
prepared as described above. The rat brain tissue homogenate
samples were prepared by following our published
procedure,[41, 48] briefly, the whole brain was harvested and the
excess blood was blotted; the brain tissue was homogenized on
ice; acetonitrile (1.2 mL) was then added to extract the
radioactivity and homogenize further. A 1.0 mL aliquot of this
brain homogenate was centrifuged to remove the debris; 0.2 mL
of the clear supernatant was diluted with 0.2 mL of distilled
water to form the HPLC injection sample. The rat plasma and rat
brain tissue samples were used for HPLC analysis following the
procedure described above.
3.5 In vivo microPET imaging
Three PET studies were performed on adult male
cynomolgus monkeys (6-8 Kg) with a microPET Focus 220
scanner (Concorde/CTI/Siemens Microsystems, Knoxville, TN).
The animals were fasted for 12 h prior to each PET study. The
animals were initially anesthetized using an intramuscular
injection with ketamine (10 - 20 mg/kg) and glycopyrrolate
(0.013 - 0.017 mg/kg) and then transported to the PET scanner
suit. Upon arrival, the animal was intubated with an endotracheal
tube and anesthesia was maintained at 0.75
- 2.0%
isoflurane/oxygen throughout the procedure. After intubation, a
percutaneous venous catheter was placed for radiotracer
injection. Core temperature 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. A 10-min transmission
scan was performed to check positioning; once confirmed, a 45
min transmission scan was obtained for attenuation correction.
Subsequently, a 2 hr dynamic emission scan was acquired after
administration of 296 - 410 MBq of (-)-[18F]18a via the venous
catheter. In the blocking studies, (-)-vesamicol (0.25 mg/kg) was
administrated to the animal 5 min prior to the (-)-[18F]18a
injection via the venous catheter.
4. Conclusions
In this study, we successfully synthesized nine new
carbonyl-containing fluorinated VAChT ligands. In vitro binding
studies suggested that (-)-18a is a potent and selective candidate
PET ligand. Ex vivo autoradiography studies and biodistribution
studies in rodents and microPET studies in nonhuman primates
suggest that (-)-[18F]18a specifically accumulates in the striatum,
the VAChT enriched region of the brain. Striatal uptake of (-)-
[18F]18a measured in monkeys using PET reached maximum
uptake (SUV = 5.1) at 15 min p.i. More importantly, the striatal
activity levels gradually decreased over time. Washout of the
radioactivity from the nonspecific binding reference region,
cerebellum, was much faster than in the target striatal regions. A
blocking study using vesamicol strongly suggested that (-)-
[18F]18a specifically binds to VAChT in the striatal regions. Over
all, (-)-[18F]18a is a promising PET radiotracer candidate for
quantifying VAChT levels in vivo. Currently an exploratory
Investigational New Drug (IND) application has been approved
by the Food and Drug Administration. The translational clinical
investigation of (-)-[18F]18a in normal control subjects and
patients is in progress.
3.6 MicroPET image processing and analysis
PET scans were collected from 0 – 120 min with the
following time frames: 3×1 min, 4×2 min, 3×3 min and 20×5
min. 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 data were corrected using individual
attenuation and model-based scatter correction and reconstructed
using filtered back projection. The first baseline PET image for
each animal acted as the target image with the MPRAGE and
subsequent PETs co-registered using automated image
registration program AIR. All MPRAGE-based volume of
interest (VOI) analyses were accomplished by experienced
investigators. For quantitative analyses, three-dimensional ROIs
(cerebellum and striatum) were transformed to the baseline PET
space and then overlaid on all reconstructed PET images to
obtain time-activity curves. Activity measures were standardized
Acknowledgements
This work was supported by NIH grants NS061025,
NS075527 and MH092797, grants from the National Institute of
General Medical Sciences (8 P41 GM103422-35). The authors
7

of derivatives and analogues of trans-2-(4-phenylpiperidino)cyclohexanol
(vesamicol), J Med Chem, 32 (1989) 1217-1230.
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thank William H. Margenau, and Robert Dennett in the
Cyclotron Facility for 18F radioisotope production. Optical
rotation was determined in the laboratory of Dr. Douglas F.
Covey in the Department of Molecular Biology and
Pharmacology of Washington University. The authors thank John
Hood, Christina Zukas and Darryl Craig for their assistance with
the nonhuman primate microPET studies. The authors
acknowledge NMR core facility and microPET facility of
Washington University St. Louis for research assistance.
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9

Figure 1. Vesamicol, BVM and their derivatives.
10

Figure 2. Autoradiography of (-)-[18F]18a in the male Sprague-Dawley (SD) rat brain. A). The
representative horizontal slice (100 µm thick) of the SD rat brain shows the highest accumulation of
radioactivity in the VAChT enriched striatal regions. B). Quantification of autoradiography from entire 30
brain slices indicates a striatum-to-cerebellum ratio of 4.19 0.37 at 60 min post-injection. Histograms are
the average of relative radioactivity of 30 brain slices. Error bars are the standard derivations.
11

Figure 3. Regional brain uptake and striatum/organ ratio of (-)-[18F]18a in male SD rats. Both figures
showed the radioactivity retention was the highest in the striatum at 30, 60 and 120 min p.i., while the
radioactivity was quickly washed out in other brain regions. CBL: cerebellum; STR, striatum; BSTM, brain
stem; CTX, cortex; THA, thalamus.
12

Figure 4. Representative microPET images of (-)-[18F]18a in cynomolgus monkey brain. Upper panel:
PET image (left) at baseline condition and PET image, pretreated the animals using 0.25 mg/kg (-)-
vesamicol 5 min prior to injection of the radiotracer (right). High uptake of (-)-[18F]18a in striatum
produced clear visualization of striatum (putamen and caudate) in the baseline condition. The pretreatment
using 0.25 mg/kg (-)-vesamicol significantly reduced the uptake of (-)-[18F]18a in the striatum (right).
Lower panel: time tissue-activity curves for cerebellum (red open circles), striatum (red open triangles) at
baseline condition, cerebellum (blue filled circles), and striatum (blue filled triangles) at the condition that
animal was pretreated using 0.25 mg/kg (-)-vesamicol. SUV: standardized uptake value.
13

Figure 5. Radiometabolite analysis of NHP arterial plasma samples post injection of (-)-[18F]18a. The
black diamonds represent the percentage intact radioactive parent compound at 5, 15, 30, 60, 90, and 120
min p.i.
14

Scheme 1. Syntheses of compounds 12 - 14
Reagents and conditions: a) BrCH2CH2F, K2CO3, acetone, reflux, 12 h, 77%; b) Ts(OCH2CH2)nF, K2CO3, DMF, rt,
12 h, 78% for ( )-13 and 41% for ( )-14.
15

Scheme 2. Syntheses of compounds 18a and 18b
OH
O
15
a
O
O
OH
N
+
N
F
F
OH
OH
OH
17a
17b
b
b
O
F
O
N
O
F
N
F
OH
O
OH
F
18b
18a
Reagents and conditions: a) (4-fluorophenyl)(piperidin-4-yl)methanone (16), Et3N, EtOH, 60 °C, 2d; b)
TsOCH2CH2F, Cs2CO3, THF, reflux, overnight.
16

Scheme 3. Chiral separation of ( )-18a
Reagents and conditions: a) Ac2O, pyridine, CH2Cl2, rt, overnight; b) separation of enantiomers of ( )-19a on chiral
HPLC: Chiralcel OD semi-preparative column, mobile phase: 3% 2-propanol in hexanes; flow rate: 4.0 mL/min; c)
sat. Na2CO3: EtOH 1:1, rt, 3 d.
17

Scheme 4. Radiosynthesis of (-)-[18F]18a
18

Table 1. Binding affinities of the fluoro-containing VAChT inhibitors
Ligands
R1
R2
Ki-VAChT
Ki-σ1
Ki-σ2
VAChT/ σ1 VAChT/ σ2 Log P*a
Vesamicol*b
FBBV*c
12
20.00
73.80
346.0
3.69
71
17.30
93
1.40
2.99
3.15
2.79
2.43
2.63
2.59
3.45
3.45
3.45
3.47
H
H
H
2.70 0.40
1.74 0.32
0.87 0.09
1.23 0.09
4.64 0.32
191.1 57.7 251.3 39.2
13.23 0.20 1,885 239
FCH2CH2O
7.60
1083
13
F(CH2CH2O)2
H
1,580 46
>10,000
8,200 97
>10,000
1800
>8,000
1,862
0.51
9300
14
F(CH2CH2O)3
H
>9,000
398.0
46.30
>6000
600.0
>10,000
77.00
17a
F
F
F
F
F
F
5-OH
8-OH
8,640 974 1,851 408
17b
56.20 4.08 28.70 4.10 2,600 178
18a
5-OCH2CH2F 1.55 0.18
5-OCH2CH2F 13.00 2.19
5-OCH2CH2F 0.59 0.06
8-OCH2CH2F 92.80 22.20
821 34
>10,000
>10,000
>10,000
>10,000
7,800 882
>10,000
529
(+)-18a
(-)-18a
18b
1022
>100,00
667.0
7,145 549
*a Calculated value at pH = 7.4 by ACD/I-Lab ver. 7.0 (Advanced Chemistry Development, Inc., Canada).
b. Reference 37. c. Reference 36.
19

Table 2. Biodistribution of (-)-[18F]18a in male Sprague-Dawley ratsa
Organs
Blood
5 min
30 min
60 min
120 min
0.209 0.029
0.706 0.170
2.255 0.415
0.171 0.055
0.144 0.054
1.202 0.323
1.265 0.452
2.363 0.520
1.065 0.121
0.426 0.086
0.684 0.079
0.270 0.033
0.390 0.031
0.973 0.107
0.258 0.025
0.324 0.071
1.200 0.347
0.685 0.082
0.883 0.068
0.523 0.044
0.369 0.020
0.482 0.037
0.338 0.088
0.357 0.068
0.738 0.087
0.253 0.044
0.548 0.354
1.324 0.346
0.505 0.069
0.604 0.092
0.456 0.122
0.403 0.095
0.425 0.075
0.356 0.019
0.336 0.016
0.551 0.070
0.235 0.010
0.560 0.135
0.819 0.367
0.376 0.034
0.489 0.052
0.382 0.029
0.537 0.035
0.409 0.016
Heart
Lung
Muscle
Fat
Pancreas
Spleen
Kidney
Liver
Bone
Total brain
a %ID/g values (mean SD) with 4 rats per group
20

Graphical
Synthesis and Biological
Leave this area blank for abstract info.
Characterization of a Promising F-18
Tracer of Vesicular Acetylcholine
Transporter
Zhude Tua*, Xiang Zhanga, Hongjun Jina, Xuyi Yuea, Prashanth K. Padakantia, Lihai Yua, Hui
Liua, Hubert P. Floresb, Kota Kaneshigec, Stanley M. Parsonsc, and Joel S. Perlmuttera,b
aDepartment of Radiology, Washington University School of Medicine, 510 South Kingshighway Blvd., St. Louis, MO 63110, USA
bDepartment of Neurology, Washington University School of Medicine, 510 South Kingshighway Blvd., St. Louis, MO 63110, USA
cDepartment of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA