Synthesis and in vitro and in vivo evaluation of18F-labeled positron emission tomography (PET) ligands for imaging the vesicular acetylcholine transporter

Article abstract of DOI:10.1021/jm8012344

A new class of vesicular acetylcholine transporter inhibitor that incorporates a carbonyl group into the benzovesamicol structure was synthesized, and analogues were evaluated in vitro. (±)-trans-2-Hydroxy-3- (4-(4-[¹⁸F]fluorobenzoyl)piperidino)tetralin (9e) has K_i values of 2.70 nM for VAChT, 191 nM for σ₁ and 251 nM for σ₂. The racemic precursor (9d) was resolved via chiral HPLC, and (±)-[¹⁸F]9e,(-)-[¹⁸F]9e, and (+)-[ ¹⁸F]9e were respectively radiolabeled via microwave irradiation of the appropriate precursors with [¹⁸F]/F- and Kryptofix/K ₂CO₃ in DMSO with radiochemical yields of ～50-60% and specific activities of >2000 mCi/μmol. (-)-[¹⁸F]9e uptake in rat brain was consistent with in vivo selectivity for the VAChT with an initial uptake of 0.911 %ID/g in rat striatum and a striatum/cerebellum ratio of 1.88 at 30 min postinjection (p.i.). MicroPET imaging of macaques demonstrated a 2.1 ratio of (-)-[¹⁸F]9e in putamen versus cerebellum at 2 h p.i. (-)-[¹⁸F]9e has potential to be a PET tracer for clinical imaging of the VAChT.

Full text of DOI:10.1021/jm8012344

1358
J. Med. Chem. 2009, 52, 1358–1369
18
Synthesis and in Vitro and in Vivo Evaluation of F-Labeled Positron Emission Tomography
(PET) Ligands for Imaging the Vesicular Acetylcholine Transporter
Zhude Tu,*,† Simon M. N. Efange,‡ Jinbin Xu,† Shihong Li,† Lynne A. Jones,† Stanley M. Parsons,§ and Robert H. Mach†
DiVision of Radiological Sciences, Washington UniVersity School of Medicine, 510 South Kingshighway BouleVard, St. Louis, Missouri 63110,
Department of Radiology, UniVersity of Minnesota, Minneapolis, Minnesota 55455, and Department of Chemistry and Biochemistry, UniVersity
of California, Santa Barbara, California 93106
ReceiVed September 29, 2008
A new class of vesicular acetylcholine transporter inhibitor that incorporates a carbonyl group into the
benzovesamicol structure was synthesized, and analogues were evaluated in vitro. (()-trans-2-Hydroxy-3-
(4-(4-[18F]fluorobenzoyl)piperidino)tetralin (9e) has Ki values of 2.70 nM for VAChT, 191 nM for σ1, and
251 nM for σ2. The racemic precursor (9d) was resolved via chiral HPLC, and (()-[18F]9e, (-)-[18F]9e,
and (+)-[18F]9e were respectively radiolabeled via microwave irradiation of the appropriate precursors with
[18F]/F- and Kryptofix/K2CO3 in DMSO with radiochemical yields of ∼50-60% and specific activities of
>2000 mCi/µmol. (-)-[18F]9e uptake in rat brain was consistent with in vivo selectivity for the VAChT
with an initial uptake of 0.911 %ID/g in rat striatum and a striatum/cerebellum ratio of 1.88 at 30 min
postinjection (p.i.). MicroPET imaging of macaques demonstrated a 2.1 ratio of (-)-[18F]9e in putamen
versus cerebellum at 2 h p.i. (-)-[18F]9e has potential to be a PET tracer for clinical imaging of the VAChT.
Introduction
mine, and galantamine are four of the most widely used
acetylcholinesterase inhibitors.29-31
Neurodegenerative diseases such as Alzheimer’s disease,
Down’s syndrome, Parkinson’s disease, and schizophrenia are
generally characterized by progressive diminution in cognitive
function associated with the loss of cholinergic neurons and
synapses in the brain. For Alzheimer’s disease, the severity of
cognitive dysfunction is correlated with loss of cholinergic
synaptic elements in the cortex and subcortical brain areas.1-7
For Down’s syndrome, presynaptic cholinergic deficits also
contribute to loss of cognitive function.8-11 For Parkinson’s
disease, patients exhibit subtle but early cognitive disturbances
that correlate with cholinergic deficits in cortical areas.12,13 For
schizophrenia, cognitive dysfunction occurs in the early phases
of the disease and remains throughout its course.14 Although
these disorders are all characterized by cognitive deficits, clinical
and preclinical evidence demonstrates that the cholinergic
deficits are localized to different components of the cholinergic
system15-18 and cholinergic innervations.19,20 To date, the use
of drugs that inhibit acetylcholinesterase to increase the avail-
ability of acetylcholine (ACha) in the central cholinergic system
is a major therapeutic approach to treatment of the cognitive
dysfunctions.12,13,21-28 For example, tacrine, donepezil, rivastig-
In the central nervous system, ACh is the primary signaling
molecule of cholinergic neurons. The vesicular acetylcholine
transporter (VAChT) is responsible for transporting newly
synthesized ACh into synaptic vesicles.32-34 VAChT has been
cloned from 12 species that include C. elegans, Torpedo, rat,
and human.34-39 It is a reliable molecular marker of the
cholinergic system. Prado and colleagues40 reported a strong
relationship between the levels of VAChT expression and ACh
release in both the peripheral and central nervous systems. A
marked reduction of VAChT expression can affect neurotrans-
mission at the neuromuscular junction. Even a modest deficiency
of VAChT is sufficient to interfere with release of ACh in the
brain and to alter cognitive behavior in object and social
recognition and memory function.40 Changes in VAChT expres-
sion levels have also been implicated in drug addiction,
including addiction to nicotine, ethanol, and neurostimulants
such as cocaine and amphetamine and opiates.41,42 VAChT level
in the brain is affected with drug abuse in humans; the levels
of VAChT are subject to both up- and down-regulation as part
of compensatory processes that attempt to maintain homeostasis
of neuronal cholinergic activity.41 If PET tracers could measure
VAChT levels in vivo, they could be used as a biomarker for
studying changes in cholinergic function in living animal or
human brains in response to therapy.
The lead compound for all radiotracer development in this
field is the aminoalcohol derivative vesamicol [(-)-trans-2-(4-
phenylpiperidinyl)cyclohexanol]. However, since (-)-[3H]ve-
samicol was found to have a high affinity for σ1 and σ2 receptors
in addition to the VAChT and to have marginal selectivity for
VAChT versus σ1 and σ2 receptors,43 there has been a vigorous
effort to develop more selective VAChT ligands. Examples of
the more prominent compounds are shown in Figure 1.
The class of compounds receiving the most attention for in
vivo imaging is the benzovesamicol analogues, which consist
of five compounds: aminobenzovesamicol (ABV), 4-fluorom-
ethylvesamicol (FMV), (-)-5-(N-methylamino)benzovesamicol
(MABV), (2)-N-ethyl-N-fluoroacetamidobenzovesamicol (NEFA),
* To whom correspondence should be addressed. Telephone: 314-362-
8487. Fax: 314-262-8555. E-mail: tuz@mir.wustl.edu.
†
Washington University School of Medicine.
University of Minnesota.
University of California.
‡
§
a Abbreviations: VAChT, vesicular acetylcholine transporter; ACh,
acetylcholine; HPLC, high performance liquid chromatography; TLC, thin
layer chromatography; %ID/g, percent injected dose per gram; p.i.,
postinjection; PET, positron emission tomography; SPECT, single photon
emission computed tomography; MRI, magnetic resonance imaging; ABV,
aminobenzovesamicol; FMV, 4-fluoromethylvesamicol; MABV, (-)-5-(N-
methylamino)benzovesamicol; NEFA, (2)-N-ethyl-N-fluoroacetamidoben-
zovesamicol; FEOBV, (-)-5-fluoroethoxybenzovesamicol; FBOV, 4-flu-
orobenzyloxyvesamicol;
p-fluorobenzyltrozamicol; IBVM, (-)-5-iodobenzovesamicol; DTG, 1,3-
ditolyguanidine; Kryptofix, 4,7,13,16,21,24-hexaoxa-1,10-
MIBT,
m-iodobenzyltrozamicol;
FBT,
diazabicyclo[8.8.8]hexacosane; DMSO, dimethyl sulfoxide; DMF, N,N-
dimethylformamide; NBS, N-bromosuccinimide; ND, not determined.
10.1021/jm8012344 CCC: $40.75
2009 American Chemical Society
Published on Web 02/09/2009

18F-Labeled PET Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1359
Figure 1. Structures and in vitro biological properties of ligands for the vesicular acetylcholine transporter: (*a) ND, not determined. The new
benzoylbenzovesamicol analogues interpose a carbonyl group between regions B and C.
and (-)-5-fluoroethoxybenzovesamicol (FEOBV) (Figure 1).
Another analogue, (-)-5-iodobenzovesamicol (IBVM), has been
radiolabeled with 123I and used as a SPECT radiotracer to
measure the cholinergic terminal density in human AD
patients.44,45 However, the high lipophilicity of [123I]IBVM, slow
clearance from noncholinergic regions of brain, and irreversible
kinetics from the regions with high density of cholinergic
terminals in brain required that SPECT imaging studies be
conducted 22 h following iv injection of the radiotracer. The
long time interval required to achieve equilibrium is not
compatible with the radioisotopes used in PET, particularly
carbon-11 (t1/2 ) 20.4 min) and fluorine-18 (t1/2 ) 109.8 min).
Recently, two 4-fluorobenzyloxy analogues of vesamicol, 4-(4-
fluorobenzyloxy)vesamicol (4-FBOV) and 5-(4-fluorobenzy-
loxy)vesamicol (5-FBOV), were reported to have moderate or
poor affinity for the VAChT.46 These compounds also displayed
a high affinity for σ1 receptors (Figure 1). In addition, a number
of benzovesamicol analogues displaying moderate to high
affinity (Kd ) 0.45-8.8 nM) for the VAChT have been recently
reported (Figure 1) as well as a series of methylvesamicol
analogues.47,48 It is not clear if these compounds will be useful
for imaging the VAChT with PET or SPECT.
BT) and[125I]-m-iodobenzyltrozamicol ([125I]MIBT). The tracer
(+)-[18F]FBT displayed good properties in animal imaging
studies of rodents49 and rhesus monkeys,50-52 and has appropri-
ate in vivo kinetics for PET studies of the VAChT in
anesthetized nonhuman primates with a low test-retest vari-
ability. However, (+)-[18F]FBT has a relatively high affinity
for σ receptors, which resulted in unacceptably high nonspecific
binding in human subjects (Mach unpublished data). Therefore,
(+)-[18F]FBT is not clinically useful for imaging the VAChT.
PET studies with VAChT radiotracers have shown that most
vesamicol analogues also display high affinity for σ receptors.
This observation has been attributed to the high degree of
structural similarity between pharmacophores for VAChT and
σ receptors. It has also been reported that in the rat brain, σ1
receptor density is highest in Purkinje cells of the cerebellum,
the pons-medulla, midbrain, and hippocampus.56 Moderate
densities of σ receptor are found in the hypothalamus and
cerebral cortex. Consequently, imaging data generated from a
PET tracer for VAChT that also labels σ receptors could lead
to an inaccurate estimation of the amount of the VAChT within
these brain regions. Even though [123I]IBVM has been used as
a SPECT imaging radiotracer to measure the VAChT level in
brain,45 no PET radiotracers have been used in human imaging
studies of the VAChT. The higher sensitivity and quantitative
Another class of compounds are the azavesamicol analogues
represented by 4-[18F]-(+)-fluorobenzyltrozamicol ((+)-[18F]F-

1360 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Tu et al.
to generate 3a and 3b. The bromophenyl analogue 3b was
treated with potassium phthalimide to give 4, which was then
hydrolyzed to generate the 4-amino intermediate 3c. The 4-nitro
compound, 3d was obtained by oxidation of 3c with m-
chloroperoxybenzoic acid. Hydrolytic removal of the benzoyl
group in 3a-d generates the corresponding 4-benzoylpiperidine
analogues 5a-d. The fluorine-substituted analogue, 4-fluo-
robenzoylpiperidine 5e, is commercially available.
Synthesis of 8 began with 1,4-dihydronaphthalene 6 as shown
in Scheme 2. Compound 7 was generated by reacting 6 with
N-bromosuccinimide. Then 8 was obtained by refluxing 7 in
chloroform in the presence of aqueous sodium hydroxide.
Reaction of 5a-e with compound 8 in ethanol in the presence
of Na2CO3 afforded the corresponding products 9a-e in
moderate yields (Scheme 2). Compounds 9a-e were converted
into the corresponding hydrochloride salts for in vitro binding
studies to determine the affinities for the VAChT and σ1 and
σ2 receptors.
In vitro binding studies revealed that the racemic compound
9e displayed a high affinity for the VAChT and moderate
affinities for σ1 and σ2 receptors and was identified as a potential
candidate for radiolabeling with 18F. Compound 9e was further
resolved from the racemic mixture via chiral HPLC as described
below to obtain the enantiomerically pure isomers.
Figure 2. Chemical structures of 9d and 9e and the crystal structure
of (-)-9e.
capability of PET versus SPECT indicate that there is a need
to develop PET tracers for imaging the VAChT.
The structure of vesamicol, the prototypical VAChT ligand,
may be divided into three identifiable regions of fragments
labeled A, B, and C (Figure 1).43 In this paper, we report the
synthesis and in vitro characterization of a new class of
vesamicol analogues that interpose a carbonyl group between
regions B and C. We also described the optical resolution,
radiolabeling, and in vitro and in vivo characterization of (()-
trans-2-hydroxy-3-(4-(4-[18F]fluorobenzoyl)piperidino)tetralin
(9e), the most promising compound that emerged from this new
group of vesamicol analogues. The crystal structure of 9e is
displayed in Figure 2.
Because the fluorine-containing analogue, 9e, displayed high
affinity for VAChT (Ki ) 2.7 nM) and good selectivity for the
VAChT compared to σ1 and σ2 receptors (Ki ) 191 nM for σ1
and Ki ) 251 nM for σ2), the racemic mixture (()-9e was
resolved using chiral HPLC to obtain the pure enantiomers
which were characterized in vitro. The minus isomer (-)-9e
(Ki ) 4.1 nM) was more potent than the plus isomer (+)-9e
(Ki ) 108 nM). In order to compare the enantiomers in vivo,
the racemic precursor for radiolabeling (9d) was also resolved
using chiral HPLC. The racemic mixture (()-[18F]9e, plus
isomer (+)-[18F]9e, and minus isomer (-)-[18F]9e were radio-
synthesized successfully by microwave irradiation of the cor-
responding nitro substituted precursors with [18F]/fluoride and
Kryptofix/K2CO3inDMSOandevaluatedinmaleSprague-Dawley
rats to confirm that the distribution in the brain of (-)-[18F]9e,
the most potent isomer, was consistent with the expression of
the VAChT. (-)-[18F]9e was then evaluated in male rhesus
monkeys to determine its potential as a PET radioligand for
clinical imaging of the VAChT receptor.
Resolution of (()-9e was performed by HPLC on a Chiralcel
OD HPLC column, 250 mm × 10 mm, with a mobile phase of
isopropanol/hexane (10:90) eluting at a flow rate of 5.0 mL/
min. A UV wavelength of 254 nm was used for chiral resolution.
Under these conditions, the retention time of (+)-9e is 14.38
min while the retention time of (-)-9e is 25.18 min.
Compound 9d serves as the precursor for radiosynthesis of
[18F]9e. In order to prepare the pure enantiomers (+)-[18F]9e
and (-)-[18F]9e, the corresponding enantiomerically pure iso-
mers (+)-9d and (-)-9d were also obtained by performing
HPLC on a Chiralcel OD HPLC column (250 mm × 10 mm)
using isopropanol/hexane (18:82) at a flow rate of 5.0 mL/min.
A UV wavelength of 254 nm was used for chiral resolution.
Under these conditions the retention time of (+)-9d is 22.13
min while the retention time of (-)-9d is 38.13 min.
The crystal structure of (-)-9e was unequivocally determined
by X-ray diffraction of the corresponding (-)-9e hydrochloride.
The colorless crystals of (-)-9e were grown by slow evaporation
of ethanol/methylene chloride solutions of the compound at 23
°C. The crystal structure of (-)-9e is presented in Figure 2.
The radiosynthesis of (+)-[18F]9e, (-)-[18F]9e, and (()-
[18F]9e was accomplished by treating the corresponding precur-
sors (+)-9d, (-)-9d, and (()-9d with [18F]fluoride/K2CO3 and
Kryptofix in DMSO (Scheme 3). The reaction mixture was
irradiated for 30-40 s in a microwave oven, and the crude
product was separated from the unreacted [18F]fluoride by
passing the mixture through a Sep-Pak light alumina N cartridge
with 3.0 mL of mobile phase (THF/0.1 M ammonium formate
buffer (23:77)). The crude product was then purified by HPLC
on an Alltech Econosil C-18 reversed-phase column (250 mm
× 10 mm). At 3.5 mL/min flow rate using the mobile phase
described above, the retention time of the production is ∼12.6
min. The entire procedure required ∼2 h, and the radiochemical
yield was 50-60% (decay-corrected to the start of synthesis).
The specific activity was >2000 Ci/mmol.
Results and Discussion
Chemistry. Vesamicol analogues are routinely synthesized
by reacting a 4-susbstituted piperidine with an epoxide. Con-
sequently, the synthesis of the target compounds began with
the preparation of the corresponding 4-benzoylpiperidines 5a-d
(Scheme 1). Conversion of 1 into ethyl 1-benzoylpiperidine-4-
carboxylate and then hydrolysis of the ester with sodium
hydroxide gave the N-benzoyl protected isonipecotic acid 2 that
provided a versatile intermediate for Friedel-Crafts acylation
In Vitro Binding Studies. In vitro binding studies were
conducted to measure the affinities of the target compounds for
the VAChT and for σ1 and σ2 receptors.
The binding assays used [3H]vesamicol for VAChT, (+)-
[3H]pentazocine for the σ1 receptors, and [3H]DTG in the

18F-Labeled PET Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1361
Scheme 1a
a Reagents and conditions: (a) (i) benzoyl chloride, Et3N; (ii) NaOH, aq EtOH; (b) (i) oxalyl chloride, CH2Cl2; (ii) arene, AlCl3, CS2, reflux; (c) phthalimide-
K, CuI, DMA, heat; (d) 32% HBr-HOAc, 45 °C; (e) mCPBA, CHCl3, heat; (f) 6 N HCl, heat.
Scheme 2a
analogues, 9b and 9e, affinities of σ1 and σ2 receptors were at
least 10-fold lower than those reported for (+)-FBT. In terms
of VAChT/σ selectivity, the bromine analogue 9b was superior
to the fluorine compound 9e, with a calculated selectivity ratio
of 297 versus 71.
By comparison of the two halogenated compounds, it was
observed that the bromine analogue 9b displays higher affinity
for VAChT than the fluorine compound 9e. Additionally, 9b
exhibits lower affinity for both σ1 and σ2 receptors than 9e. As
a result, the bromine compound displays considerably higher
VAChT/σ selectivity than 9e. Both compounds are moderately
lipophilic, as revealed by their calculated log P values (Table
1); consequently, they are expected to cross the blood-brain
barrier. However, the radionuclidic properties of 76Br make it
less than ideal for PET imaging when compared with 18F.
Radiotracers containing 18F give higher quality PET images than
those containing 76Br because of both the prompt γ emissions
and the long positron range of 76Br. Although correction factors
have been proposed to remove the cascade coincidences, because
positron range is a limiting factor for spatial resolution on PET
scanners, radionuclides like 76Br with a longer mean positron
range (3.47 mm in water) than the mean positron range of 18F
(0.51 mm in water) create inherently “blurred” images.53-55
Because of these considerations, we chose to initially radiolabel
9e rather than 9b. Optical resolution of (()-9e and characteriza-
tion of the enantiomers showed that the (-)-9e displayed
improved VAChT/σ selectivity over the racemic mixture in
vitro, thereby providing further support for radiolabeling the
ligand for in vivo studies.
a Reagents and conditions: (a) NBS, aqueous THF; (b) aqueous NaOH,
CHCl3, reflux; (c) EtOH, Et3N, reflux.
presence of 1 µM (+)-pentazocine for the σ2 receptor. The Ki
values were determined from competition plots. The results of
the binding assays for compounds 9a-d, (()-9e, (+)-9e, (-)-
9e are shown in Table 1. Substituting the 4-postion of the phenyl
group of 9a with Br, NH2, NO2, or F generated 9b, 9c, 9d, or
9e. The affinities for VAChT are Br > NO2 > NH2 > F > H,
corresponding to the following IC50 values: 0.25 ( 0.02, 0.48
( 0.03, 1.68 ( 0.04, 2.70 ( 0.40, 4.30 ( 1.00 nM. The bromine
compound 9b has the highest affinity for the VAChT. The
fluorine substituted compound 9e also has a moderate affinity
of 2.70 ( 0.40 nM, which is comparable to the Ki value of
vesamicol. The bromine and fluorine substituted compounds 9b
and 9e are potential PET radiotracers when labeled respectively
with 76Br and 18F. Three of the five benzoylpiperidine-containing
compounds, 9a, 9b, and 9e, displayed significantly lower affinity
for VAChT than (+)-FBT (Table 1). For the two halogenated
In Vivo Rat Biodistribution Studies. The results of the tissue
biodistribution studies of (-)-[18F]9e, (+)-[18F]9e, and (()-
[18F]9e in adult male Sprague-Dawley rats are reported as
percent injected dose per gram (%ID/g) and summarized in
Table 2. The initial brain uptake values were 0.823, 0.350, 0.606
%ID/g for (-)-[18F]9e, (+)-[18F]9e, (()-[18F]9e, respectively.
At all time points postinjection (p.i.), the minus isomer (-)-
[18F]9e displayed a higher brain uptake and lower initial blood

1362 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Tu et al.
Scheme 3. Radiosynthesis of (()-[18F]9e, (+)-[18F]9e, and (-)-[18F]9
Table 1. Affinites (Ki) of the Benzoylbenzovesamicol Analogues 9a-e, (+)-9e, (-)-9e for VAChT and σ1 and σ2 Receptors Assayed in Vitroa
Ki (nM)
VAChT/σ ratio
VAChTb
σ1
σ2
VAChT/σ1
VAChT/σ2
log Pe
c
d
ligands
vesamicolf
2.0 ( 1.0
0.055 ( 0.01
0.22 ( 0.05
4.30 ( 1.00
0.25 ( 0.02
1.68 + 0.14
0.48 + 0.03
2.70 ( 0.40
108 ( 15
25.8 ( 8.0
ND
21.6 ( 3.9
219.6 ( 17.3
297.7 ( 18.7
ND
34.5 ( 2.0
ND
35.9 ( 6.3
320.0 ( 6.6
592.6 ( 95.2
ND
25.8
ND
98
34.5
ND
163
74
2368
ND
ND
93
1.40
2.44
1.99
2.78
3.73
1.56
2.16
2.99
2.99
2.99
benzovesamicolg
(+)-FBTh
9a
9b
9c
9d
(()-9e
(+)-9e
(-)-9e
51
1188
ND
ND
71
1.7
160
ND
ND
191.1 ( 57.7
185.97 ( 38.89
658.60 ( 118.40
251.3 ( 39.2
277.93 ( 26.66
319.23 ( 36.64
2.8
78
4.1 ( 0.6
a Ki values (mean ( SEM) were determined from at least three experiments. b Ki VAChT is measured using Torpedo synaptic vesicles. c σ1binding is
measured using guinea pig brain membranes. d σ2 binding is measured using rat liver preparations. e Calculated value at pH 7.4 by ACD/I-Lab, version 7.0
(Advanced Chemistry Development, Inc., Canada). f Reference 43. g Reference 62. h Reference 51.
Table 2. Biodistribution of (-)-[18F]9e, (+)-[18F])9e, and (()-[18F]9e in Male Sprague Dawley Ratsa
compd
5 min
30 min
1 h
2 h
blood
lung
(-)-[18F]9e
(+)-[18F]9e
(()-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(()-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(()-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(()-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(()-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(()-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(()-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(()-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(()-[18F]9e
0.073 ( 0.010
0.136 ( 0.020
0.122 ( 0.023
1.577 ( 0.224
0.904 ( 0.161
1.389 ( 0.247
0.321 ( 0.027
0.194 ( 0.006
0.328 ( 0.014
0.091 ( 0.018
0.047 ( 0.010
0.079 ( 0.015
0.132 ( 0.095
0.052 ( 0.017
0.088 ( 0.026
1.506 ( 0.186
1.849 ( 0.566
1.939 ( 0.382
2.018 ( 0.448
0.791 ( 0.123
1.644 ( 0.232
0.334 ( 0.047
0.187 ( 0.015
0.307 ( 0.011
0.823 ( 0.091
0.350 ( 0.040
0.606 ( 0.100
0.037 ( 0.002
0.087 ( 0.016
0.071 ( 0.009
0.481 ( 0.088
0.332 ( 0.073
0.553 ( 0.056
0.109 ( 0.014
0.094 ( 0.013
0.141 ( 0.005
0.080 ( 0.008
0.051 ( 0.010
0.071 ( 0.006
0.207 ( 0.018
0.108 ( 0.053
0.177 ( 0.039
1.769 ( 0.179
1.239 ( 0.271
1.504 ( 0.271
1.896 ( 0.333
0.416 ( 0.048
1.042 ( 0.205
0.157 ( 0.004
0.277 ( 0.080
0.445 ( 0.032
0.226 ( 0.014
0.071 ( 0.012
0.153 ( 0.044
0.029 ( 0.005
0.048 ( 0.007
0.043 ( 0.003
0.327 ( 0.022
0.188 ( 0.035
0.346 ( 0.045
0.066 ( 0.008
0.051 ( 0.005
0.094 ( 0.008
0.056 ( 0.010
0.034 ( 0.008
0.076 ( 0.012
0.241 ( 0.021
0.154 ( 0.065
0.196 ( 0.035
2.214 ( 0.323
0.814 ( 0.120
1.402 ( 0.102
1.298 ( 0.372
0.291 ( 0.032
0.990 ( 0.321
0.148 ( 0.015
0.399 ( 0.078
0.623 ( 0.080
0.124 ( 0.013
0.041 ( 0.002
0.095 ( 0.012
0.020 ( 0.002
0.027 ( 0.002
0.027 ( 0.004
0.199 ( 0.032
0.112 ( 0.023
0.223 ( 0.014
0.047 ( 0.009
0.031 ( 0.006
0.056 ( 0.007
0.035 ( 0.007
0.022 ( 0.005
0.047 ( 0.006
0.249 ( 0.036
0.150 ( 0.062
0.173 ( 0.042
1.791 ( 0.297
0.692 ( 0.081
1.188 ( 0.158
1.339 ( 0.160
0.194 ( 0.027
0.853 ( 0.171
0.138 ( 0.006
0.532 ( 0.096
0.739 ( 0.044
0.095 ( 0.016
0.022 ( 0.004
0.059 ( 0.008
heart
muscle
fat
liver
kidney
bone
brain
a %ID/g values (mean ( SD) with n ) 4 rats per group.
uptake than the plus isomer (+)-[18F]9e. Modest but progressive
accumulation of radioactivity was observed in bone tissue for
racemic (()-[18F]9e and (+)-[18F]9e in rats. The progressive
increase in bone uptake was most likely the result of defluori-
nation. For (+)-[18F]9e, bone uptake increased from 0.187
%ID/g at 5 min to 0.532 %ID/g at 2 h p.i. At the same time,
bone uptake following injection of racemic (()-[18F]9e increased
from 0.31 %ID/g at 5 min to 0.739 %ID/g at 2 h p.i. In contrast,
(-)-[18F]9e washed out of the bone between 5 min and 2 h p.i.
(bone uptake was 0.334 %ID/g at 5 min and 0.138 %ID/g at
2 h) and thus does not appear to undergo defluorination in vivo.
For (-)-[18F]9e, the initial uptake of the blood was low and
the clearance of this radiotracer from the blood was quite rapid,
yielding %ID/g values of 0.077, 0.037, 0.029, 0.020 at 5 min,
30 min, 1 h, and 2 h p.i., respectively. Apart from the lung,
liver, and kidney, where the initial tracer uptakes 5 min p.i.
were 1.58, 1.51, and 2.02 %ID/g, respectively, all other tissues
displayed initial uptake values below 1.0 %ID/g.
Following injection of (-)-[18F]9e, radioactivity in the brain
reached a level of 0.823 %ID/g at 5 min p.i.; thereafter, the
level declined to 0.226, 0.124, and 0.095 %ID/g at 30 min, 1 h,
and 2 h p.i., respectively. In contrast, the levels of radioactivity
obtained in the brain following injection of (+)-[18F]9e were
0.350, 0.071, 0.041, 0.022 %ID/g at 5 min, 30 min, 1 h, and
2 h, respectively. Therefore, both the initial accumulation and
subsequent retention are greater for (-)-[18F]9e than for (+)-

18F-Labeled PET Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1363
Figure 3. Regional brain uptake of (-)-[18F]9e and (+)-[18F]9e in male Sprague-Dawley rats from 5 min to 2 h p.i. The active enantiomer,
(-)-[18F]9e, has higher striatal uptake at all time points.
Table 3. Ratioa of Striatum/Nonstriatal Tissue for (-)-[18F]9e and (+)-[18F]9e in Male Sprague-Dawley Rats
compd
5 min
30 min
1 h
2 h
striatum/blood
(-)-[18F]9e
(+)-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
(-)-[18F]9e
(+)-[18F]9e
12.55 ( 0.77
2.67 ( 0.57
1.00 ( 0.05
0.92 ( 0.02
1.09 ( 0.01
0.99 ( 0.04
1.23 ( 0.11
1.15 ( 0.04
1.11 ( 0.04
1.02 ( 0.03
8.86 ( 0.29
0.95 ( 0.18
1.39 ( 0.05
1.08 ( 0.09
1.36 ( 0.12
0.93 ( 0.03
1.85 ( 0.06
1.3 ( 0.07
5.97 ( 1.12
0.9 ( 0.23
5.17 ( 0.73
0.87 ( 0.11
1.09 ( 0.15
0.91 ( 0.15
1.11 ( 0.18
0.99 ( 0.08
1.32 ( 0.07
1.14 ( 0.09
1.08 ( 0.10
1.04 ( 0.06
striatum/cortex
1.36 ( 0.23
0.95 ( 0.09
1.35 ( 0.11
0.98 ( 0.14
1.64 ( 0.18
1.17 ( 0.12
1.38 ( 0.13
1.02 ( 0.09
striatum/hippocampus
striatum/cerebellum
striatum/total brain
1.44 ( 0.07
1.13 ( 0.04
a Calculated from %ID/g striatum divided by %ID/g nonstriatal tissue with n ) 4 rats per group.
[18F]9e. The regional distribution of (-)-[18F]9e within the brain
also suggests that it is selectively retained in regions (i.e.,
striatum) with a high VAChT density while (+)-[18F]9e
displayed a relatively homogeneous uptake (Figure 3), consistent
with the enantioselectivity observed previously with other
benzovesamicol analogues and in our own in vitro studies (Table
1).
Brain dissection studies revealed that (-)-[18F]9e uptake was
high in the striatum, moderate in the cortex and hippocampus,
and low in the cerebellum. Initial brain uptake of (-)-[18F]9e
at 5 min p.i. was relatively homogeneous with 0.911 %ID/g
for striatum, 0.911 %ID/g for cortex, 0.837 %ID/g for hippoc-
ampus, and 0.743 %ID/g for cerebellum (Figure 3). However,
the differences in regional tracer uptake became more pro-
nounced over time, resulting in striatum/blood, striatum/cortex,
striatum/hippocampus, striatum/cerebellum, and striatum/total
brain ratios of 8.86, 1.39, 1.36, 1.88, and 1.44, respectively, at
30 min following tracer injection (Table 3). Therefore, the
regional distribution of (-)-[18F]9e in the brain also mirrors
VAChT density in this organ.56-58
Ex Vivo Autoradiography of Rat Brain. In order to confirm
the striatal uptake of (-)-[18F]9e in rat brain, (-)-[18F]9e was
injected iv into a male Sprague-Dawley rat, which was
sacrificed 1 h p.i., and the brain was quickly removed and sliced
into 1 mm coronal sections for digital autoradiography. Exami-
Figure 4. Ex vivo electronic autoradiography on a 1.0 mm slice
through the striatum. The rat was sacrificed 60 min p.i. with 500 µCi
(-)-[18F]9e. The left is the photographic picture of the slice acquired
on a flatbed scanner. The right is the electronic autoradiography picture.
Thesedatademonstratestriatallocalizationof(-)-[18F]9einSprague-Dawley
rats.
nation of the autoradiogram (Figure 4) reveals that (-)-[18F]9e
has a high accumulation in the striatum and moderate accumula-
tion in the cortex, a finding that is consistent with the data

1364 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Tu et al.
Figure 5. Representative microPET (top), MRI (bottom), and coregistered images (middle). PET images are summed images (0-120 min) over
a 2 h dynamic scan, obtained by injection of (-)-[18F]9e into a rhesus monkey. High uptake of (-)-[18F]9e is observed in the putamen and caudate.
Figure 6. (A) Tissue time-activity curves of (-)-[18F]9e in the monkey brain. Each point shows the SUV mean ( SEM of three monkeys. The
uptake of (-)-[18F]9e in the brain reaches peak accumulation in the caudate and putamen at 30 min p.i. (B) Average striatum to cerebellum ratio
of (-)-[18F]9e in three monkey brains. The ratio of striatum to cerebellum displays increasing activity in the striatum over time. At 2 h p.i., the
striatum to cerebellum ratio reaches 2.1- to 2.3-fold.
obtained from tissue dissection studies in the rat and the known
relative density of VAChT in the brain.
putamen occurred at about 30 min post-iv injection. Figure 6B
indicates that the putamen/cerebellum ratio increases with time,
reaching a value of 2.1 at 2 h. These data indicate that (-)-
[18F]9e is a promising and novel PET radiotracer for imaging
the VAChT. Further studies will be required to determine if
(-)-[18F]9e can be used in clinical imaging studies.
MicroPET Imaging Study of Rhesus Monkeys. The results
of the rodent brain distribution studies and ex vivo autoradio-
graphic studies suggest that (-)-[18F]9e may be a suitable ligand
for studying cholinergic innervations via the VAChT in humans.
To confirm the feasibility of using (-)-[18F]9e for measuring
the density of VAChT in vivo, PET imaging studies with (-)-
[18F]9e were conducted in three male rhesus monkeys (Figures
5 and 6) on a microPET Focus 220 scanner (Concorde/CTI/
Siemens Microsystems, Knoxville, TN). A representative summed
image from a microPET study of a male rhesus macaque injected
with (-)-[18F]9e demonstrated high uptake of (-)-[18F]9e in
the caudate and putamen (Figure 5, upper), which is a region
expressing a high density of the VAChT. These data indicate
that (a) (-)-[18F]9e enters the brain readily and (b) the
distribution of (-)-[18F]9e is consistent with the distribution of
the VAChT in brain. The tissue time-activity curves (Figure
6A) indicate that peak uptake of (-)-[18F]9e in caudate and
Conclusion
In the present study, we synthesized a new class of com-
pounds targeting the VAChT by replacing the phenyl group in
benzovesamicol with a substituted benzoyl moiety. In vitro
binding assays of this new class of compounds suggested that
this modification of vesamicol yields analogues with not only
an increased affinity for VAChT but also high selectivity for
VAChT versus σ receptors. For example, compound 9b has very
high affinity for VAChT (Ki ) 0.25 ( 0.02 nM) and very low
affinity for σ receptors (Ki ) 297.7 ( 18.7 nM for σ1, Ki )
592.6 ( 95.2 nM for σ2). In vivo evaluation of (()-[18F]9d,
(+)-[18F]9e, and (-)-[18F]9e demonstrated that (-)-[18F]9e not

18F-Labeled PET Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1365
only is more potent than (+)-[18F]9e but is also retained in target
regions of the brain. The synthesis and radiosynthesis are
straightforward: (-)-[18F]9e was easily produced with good yield
and high specific activity in a simple one-step reaction. More
importantly, (-)-[18F]9e not only demonstrated a high initial
uptake (0.911 %ID/g for striatum at 5 min p.i.) and high target
to nontarget ratio (1.88-fold at 30 min) in rat brain but also
displayed a clear image of striatal tissue in rhesus monkey brain.
In conclusion, the initial studies suggest that (-)-[18F]9e has
excellent potential to be a novel PET tracer for clinical imaging
studies of the VAChT.
N2. Evaporation of solvent and other volatiles under reduced
pressure provided the crude acyl chloride which was redissolved
in dry benzene (25 mL). To this solution, AlCl3 (6.8 g, 51.0 mmol)
was added portionwise over 30 min under N2. The reaction mixture
was then refluxed overnight, allowed to cool, and poured onto
crushed ice with vigorous stirring. The resulting mixture was
extracted with methylene chloride (3 × 100 mL). The organic layer
was dried with anhydrous Na2SO4 and then concentrated to yield
the crude product. The crude product was purified by column
chromatography on silica gel with 3:7 acetone/hexane as mobile
1
phase to give 3a (6 g, 60%). H NMR (CDCl3): δ 1.70-4.85 (m,
9H), 7.30-7.96 (m, 10H).
1-Benzoyl-4-(4-bromobenzoyl)piperidine (3b). Compound 3b
was prepared from compound 2 as described in procedure A using
bromobenzene in place of benzene (10.8 g, 85%). 1H NMR
(CDCl3): δ 1.50-4.20 (m, 8H), 4.40-4.90 (s, 1H), 7.20-7.48 (m,
7H), 7.50-7.84 (m, 2H).
Experimental Section
Materials. All reagents and chemicals were purchased from
commercial suppliers and used without further purification unless
otherwise stated. Tetrahydrofuran (THF) was distilled from sodium
hydride immediately prior to use. Anhydrous toluene was distilled
from sodium/toluene shortly before use.
1-Benzoyl-4-(4-phthalimidobenzoyl)piperidine (4). A mixture
of 3b (16.0 g, 43.0 mmol), potassium phthalimide (9.0 g, 51.63
mmol), and copper(I) iodide (16.0 g, 51.63 mmol) in freshly distilled
dimethylacetamide (100 mL) was refluxed under N2 for 72 h. After
cooling to room temperature, the reaction mixture was treated with
0.6 N HCl (50 mL). The resulting precipitate was removed by
filtration. The filtrate was extracted with ethyl acetate (3 × 60 mL)
and methylene chloride (3 × 100 mL). The combined organic
solution was dried with anhydrous sodium sulfate and concentrated.
The residue was purified on silica gel column with 40% ethyl acetate
and 60% hexane as mobile phase to give 4 (18.0 g, 95%). 1H NMR
(CDCl3): δ 1.75-4.90 (m, 9H), 7.30-7.60 (s, 5H), 7.60-8.20 (m,
8H).
General. All anhydrous reactions were carried out in oven-dried
glassware under an inert nitrogen atmosphere unless otherwise
stated. When the reactions involved extraction with methylene
chloride (CH2Cl2), chloroform (CHCl3), ethyl acetate (EtOAc), or
ethyl ether (Et2O), the organic solutions were dried with anhydrous
Na2SO4 and concentrated with a rotary evaporator under reduced
pressure. Flash column chromatography was conducted using silica
gel 60A, “40 Micron Flash” [32-63 µm] (Scientific Adsorbents,
Inc.); the mobile phase used is reported in the experimental
procedure for each compound. Melting points were determined
using the MEL-TEMP 3.0 apparatus and left uncorrected. 1H NMR
spectra were recorded at 300 MHz on a Varian Mercury-VX
spectrometer with CDCl3 as solvent and tetramethylsilane (TMS)
as the internal standard. All chemical shift values are reported in
parts per million (ppm) (δ).The mass spectra were recorded on a
JKS-HX 11UHF/HX110 HF tandem mass spectrometer in the fast
atom bombardment (FAB+) mode. Elemental analyses (C, H, N)
were determined by Atlantic Microlab, Inc.
1-Benzoyl-4-(4-aminobenzoyl)piperidine (3c). A solution of 4
(0.7 g, 1.6 mmol) in 32% HBr-HOAc (60 mL) was stirred at 45
°C over 48 h until TLC demonstrated that the reaction was
complete. The excess acid was removed by coevaporation with
water (50 mL). The residue was treated with 30 mL of 0.1 N NaOH
aqueous solution and then extracted consecutively with methylene
chloride (3 × 30 mL) and ethyl acetate (2 × 30 mL). The combined
organic solution was dried over anhydrous sodium sulfate and
[18F]Fluoride was produced in our institution by 18O(p, n)18F
reaction through proton irradiation of enriched 18O water (95%)
using a JSW BC-16/8 (Japan Steel Works), CS-15 cyclotron
(Cyclotron Corp., Berkeley, CA), or a RDS111 cyclotron (Siemens/
CTI Molecular Imaging, Knoxville, TN). [18F]Fluoride is first passed
through an ion-exchange resin and then is extracted with 0.02 M
potassium carbonate (K2CO3) solution. The radiochemical and
chemical purities were analyzed by reversed-phase C-18 HPLC with
UV-visible and NaI detectors.
1
concentrated to generate 3c (0.45 g, 92%). H NMR (CDCl3): δ
1.75-4.00 (m, 9H), 4.20-4.80 (d, 2H), 7.30-7.50 (s, 5H),
6.50-7.90 (d, 4H).
1-Benzoyl-4-(4-nitrobenzoyl)piperidine (3d). A solution of 3b
(0.5 g, 1.62 mmol) in chloroform (20 mL) was added slowly to a
refluxed solution of technical grade (55-75%) m-CPBA (5 g, 15.52
mmol) in chloroform (50 mL). The reaction mixture was refluxed
until TLC demonstrated the reaction was complete. The reaction
mixture was cooled to room temperature and treated with 10%
NaHSO3 (50 mL). The resulting mixture was washed with 0.2 N
of NaOH aqueous solution. The organic layer was dried with
anhydrous sodium sulfate and concentrated to a residue. The product
was purified by radial flow chromatography with 15% of acetone
1-Benzoylpiperidine-4-carboxylic Acid (2). Benzoyl chloride
(21.1 g, 150 mmol) was added dropwise with stirring to a solution
of ethyl isonipecotate (1) (23.58 g, 150 mmol) and triethylamine
(22.8 g, 225 mmol) in methylene chloride (250 mL). Following
the addition, the reaction mixture was stirred overnight, then treated
with water (30 mL) and stirred for an additional 10 min. The layers
were separated, and the organic extract was washed consecutively
with 0.6 M HCl and saturated sodium bicarbonate. After drying
over anhydrous sodium sulfate, the solution was concentrated to
yield 39 g of ester. 1H NMR (CDCl3): δ 1.20-1.26 (t, 3H),
4.09-4.19 (q, 2H), 1.60-4.75 (m, 9H), 7.26-7.50 (m, 5H).
The ester was redissolved in 70% aqueous ethanol (150 mL).
Then NaOH pellets (15 g, 375 mmol) were added into the above
solution. The reaction mixture was stirred overnight, neutralized
with glacial acid, and concentrated to a residue which was
partitioned between methylene chloride and water. The organic
extract was dried over anhydrous sodium sulfate and concentrated
1
and 85% of hexane as mobile phase to give 3d (0.33 g, 60%). H
NMR (CDCl3): δ 1.60-4.90 (m, 9H), 7.30-7.60 (m, 5H),
8.00-8.40 (d, 4H). EIMS calcd, 338.1267 (M+); found, 338.1275
(M+).
Procedure B: Compounds 5a-d. 4-Benzoylpiperidine (5a).
A solution of compound 3a (5.6 g, 19.1 mmol) was refluxed in 6
N HCl (60 mL) overnight and monitored by TLC to demonstrate
that the hydrolysis was complete. The reaction mixture was cooled
to room temperature and extracted with methylene chloride (2 ×
50 mL) to remove benzoic acid. The aqueous layer was concentrated
to a residue which was treated with 0.1 N NaOH (50 mL). The
resulting mixture was extracted with methylene chloride (3 × 60
mL) and ethyl acetate (2 × 50 mL). The combined organic extracts
were dried over anhydrous sodium sulfate and concentrated to give
5a (3.2 g, 95%). 1H NMR (CDCl3): δ 1.40-3.60 (m, 10H),
7.40-8.00 (m, 5H).
1
to give 2 (34 g, 97%). H NMR (CDCl3): δ 1.50-4.70 (m, 9H),
7.29-8.01 (m, 5H), 10.89 (s, 1H). The latter was used without
further purification.
Procedure A: Compounds 3a-b. 1-Benzoyl-4-benzoylpiperi-
dine (3a). Oxalyl chloride (12.2 mL, 35.0 mmol) was added to a
solution of compound 2 (7.92 g, 34.0 mmol) in methylene chloride,
and the solution was stirred overnight at room temperature under
4-(4-Bromobenzoyl)piperidine (5b). Compound 5b was pre-
pared from compound 3b as described in procedure B (4.8 g, 93%).
1H NMR (CDCl3): δ 1.40-3.60 (m, 10H), 7.40-8.00 (m, 4H).

1366 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
4-(4-Aminobenzoyl)piperidine (5c). Compound 5c was prepared
Tu et al.
hexane, 5.0 mL/min, UV wavelength 254 nm) to give 110 mg of
(+)-9d (retention time, 22.3 min; enantiomeric purity, 99%) and
109 mg of (-)-9d (retention time, 38.13 min; enantiomeric purity,
99%). The specific rotation was determined on an automatic
polarimeter (Autopol 111, Rudolph Research, Flanders, NJ). For
the (+)-9d, the optical rotation [R]D is +37.0° with concentration
as 5.68 mg/mL in methanol. For the (-)-9c, the optical rotation
[R]D is -32.7° with concentration as 5.40 mg/mL in methanol.
(()-trans-2-Hydroxy-3-(4-(4-fluorobenzoyl)piperidino)tetra-
lin (9e). Compound 9e was prepared from compound 5e as
described in procedure C (1.0 g, 61%). 1H NMR [free base]
(CDCl3): δ 1.60-2.10 (m, 4H), 2.42 (m, 1H), 2.70-3.60 (m, 8H),
3.88 (m, 1H), 6.90-7.30 (m, 6H), 7.97 (m, 2H). Anal.
(C22H24FNO2 ·HCl·0.25H2O) C, N, H.
Resolution of Compound (()-9e. Chromatographic resolution
of compound 9e (0.170 g) was accomplished by HPLC using a
250 mm × 10 mm i.d. Chiralcel OD column (10:90 isopropanol/
hexane, 5.0 mL/min, UV wavelength as 254 nm) to give 92 mg of
(+)-9e (retention time, 14.9 min; enantiomeric purity, 99%) and
65 mg of (-)-9e (retention time, 25.2 min; enantiomeric purity,
99%). The optical rotation of (+)-9e is +40.6° with concentration
of 5.4 mg/mL in methanol. The optical rotation of (-)-9d is -43.6°
with concentration of 5.22 mg/mL in methanol. The purity of (+)-
9e was 99% (determined by HPLC). The purity of (-)-9e was 99%
(determined by HPLC).
X-Ray Diffraction study of (-)-9e. Crystals of appropriate
dimension were obtained by slow evaporation of ethanol/dichlo-
methane solutions at 23 °C. A crystal with approximate dimensions
of 0.05 × 0.12 × 0.37 mm3 was mounted on glass fibers in a
random orientation. Preliminary examination and data collection
were performed using a Bruker Kappa Apex II charge coupled
device (CCD) detector system single crystal X-ray diffractometer
equipped with an Oxford Cryostream LT device. All data were
collected using graphite monochromated Mo KR radiation (λ )
0.710 73 Å) from a fine focus sealed tube X-ray source. Preliminary
unit cell constants were determined with a set of 36 narrow frame
scans. Intensity data set included combinations of ω and φ scan
frames with scan width of 0.5° and counting time of 20 s/frame at
a crystal to detector distance of 4.0 cm. The collected frames were
integrated using an orientation matrix determined from the narrow
frame scans. Apex II and SAINT software packages (Bruker
Analytical X-Ray, Madison, WI, 2006) were used for data collection
and data integration. Analysis of the integrated data did not show
any decay. Final cell constants were determined by global refine-
ment of xyz centroids of 1393 reflections from the complete data
set. Collected data were corrected for systematic errors using
SADABS (Blessing, R. H. Acta Crystallogr. 1995, A51, 33-38)
based on the Laue symmetry using equivalent reflections.
Crystal data and intensity data collection parameters are listed
in Supporting Information (Table 4).
1
from compound 3c as described in procedure B (3.0 g, 65%). H
NMR (DMSO-d6 + CD3OD): δ 1.30-3.50 (m, 9H), 3.90-4.30
(s, 3H), 6.40-7.80 (d, 4H).
4-(4-Nitrobenzoyl)piperidine (5d). Compound 5d was prepared
1
from compound 3d as described in procedure B (2.5 g, 70%). H
NMR (CDCl3): δ 1.60-1.80 (m, 4H), 1.90-2.00 (s, 1H), 2.70-2.85
(m, 2H), 3.10-3.25 (m, 2H), 3.25-3.40 (m, 1H), 8.06-8.10 (m,
2H), 8.30-8.40 (m 2H).
3-Bromo-1,2,3,4-tetrahydronaphthalen-2-ol (7). 1,4-Dihy-
dronaphthanene, 6 (5.12 g, 39.3 mmol), and N-bromosuccinimide
(8.0 g, 44.9 mmol) were placed into 50 mL of 20% THF aqueous
solution. The reaction mixture was stirred overnight and then
extracted with methylene chloride (3 × 20 mL). The organic
solution was combined and dried by anhydrous sodium sulfate. After
concentration, the crude product was recrystallized from ethyl
1
acetate and hexane to give 7 (8.0 g, 90%). H NMR (CDCl3): δ
3.17 (s, 1H), 3.24 (s, 2H), 3.31 (s, 1H), 3.36-3.37 (d, 1H), 3.49
(s, 2H), 7.04-7.09 (m, 2H), 7.13-7.18 (m, 2H).
1,2,3,4-Tetrahydronaphthanlen-2,3-oxirane (8). Compound 7
(2.16 g, 9.5 mmol) was dissolved in a 20 mL solution of 2.0 g of
sodium hydroxide in 10 mL of water and 30 mL of chloroform.
The reaction mixture was refluxed over 2 h until TLC indicated
that the reaction was complete. The reaction mixture was extracted
with methylene chloride (3 × 15 mL). The combined organic
solution was dried by anhydrous sodium sulfate and concentrated.
The crude product was purified on silica gel column with 10% of
ethyl acetate and 90% of hexane mobile phase to give 8 (0.5 g,
35%). 1H NMR (CDCl3): δ 3.17 (s, 1H), 3.24 (s, 2H), 3.31 (s, 1H),
3.36-3.37 (d, 1H), 3.49 (s, 2H), 7.04-7.09 (m, 2H), 7.13-7.18
(m, 2H).
Procedure C: General Method for the Synthesis of Tetra-
lins (9a-e). (()-trans-2-Hydroxy-3-(4-benzoylpiperidino)tetra-
lin Hydrochloride (9a). To a solution of 8 (1.3 g, 8.8 mmol) in
ethanol (50 mL), 5a (155 g, 8.2 mmol) and sodium carbonate (4.0
g, 37.7 mmol) were added. The reaction mixture was refluxed over
42 h and then cooled to room temperature. The reaction mixture
was filtered, and the precipitate was washed with a small amount
of ethanol. The filtrate was concentrated to a residue. The residue
was redissolved in methylene chloride (60 mL) and washed with
brine. The organic solution was dried with anhydrous sodium sulfate
and concentrated to a residue. The crude product was purified by
1
radial flow chromatography to give 9a (1.0 g, 35%). H NMR
(CDCl3): δ 1.60-3.30 (m, 14H), 3.30-3.40 (s, 1H), 3.80-4.00 (s,
1H), 6.90-7.30 (m, 4H), 7.40-8.10 (m, 5H). The hydrochloride
was obtained from the methanolic hydrogen chloride solution and
recrystallized from isopropyl alcohol/ethyl acetate, mp 242-243
°C. Anal. (C22H25NO2.HCl) C, H, N.
(()-trans-2-Hydroxy-3-(4-(4-bromobenzoyl)piperidino)tetra-
lin Hydrochloride (9b). Compound 9b was prepared from com-
1
pound 5b as described in procedure C (2.4 g, 71%). H NMR
Structure solution and refinement were carried out using the
SHELXTL-PLUS software package (Sheldrick, G. M., Bruker
Analytical X-Ray Division, Madison, WI, 2006). The structure was
solved by direct methods and refined successfully in the space
group, P21. Full matrix least-squares refinement was carried out
(CDCl3): δ 1.60-3.40 (m, 14H), 3.75-4.00 (s, 1H), 4.00-4.50 (s,
1H), 6.90-7.30 (m, 4H), 7.50-7.98 (d, 4H). The free base was
converted to the hydrochloride by dissolving in methanolic HCl
solution and recrystallized from isopropyl alcohol/diethyl ether, mp
271-273 °C. Anal. (C22H24BrNO2 ·HCl) C, H, N.
by minimizing ∑w(Fo - Fc2)2. The non-hydrogen atoms were
2
refined anisotropically to convergence. All hydrogen atoms were
treated using appropriate riding model (AFIX m3). The final residual
values and structure refinement parameters are available in Sup-
porting Information (Table 4). Complete listings of positional and
isotropic displacement coefficients for hydrogen atoms and aniso-
tropic displacement coefficients for the non-hydrogen atoms are
available in Supporting Information (Tables 5-9).
(()-trans-2-Hydroxy-3-(4-(4-aminobenzoyl)piperidino)tetra-
lin (9c). Compound 9c was prepared from compound 5c as
1
described in procedure C (0.6 g, 48%), mp 70-73 °C. H NMR
(CDCl3): δ 1.70-3.80 (m, 8H), 3.80-4.00 (m, 1H), 4.00-4.70 (s,
3H), 6.90-7.60 (m, 4H), 6.50-8.40 (d, 4H). EIMS calcd, 350.1994
(M+); found, 350.1994 (M+).
(()-trans-2-Hydroxy-3-(4-(4-nitrobenzoyl)piperidino)tetra-
lin (9d). Compound 9d was prepared from compound 5d as
described in procedure C (0.2 g, 48%). 1H NMR (CDCl3): δ
1.50-3.70 (m, 8H), 3.70-4.00 (m, 1H), 4.00-4.70 (s, 1H),
6.90-7.50 (m, 4H), 7.90-8.45 (d, 4H). The purity of 9d was greater
than 95% (determined by HPLC).
Radiochemistry. Procedure D: General Method for Radio-
labeling trans-2-Hydroxy-3-(4-(4-fluorobenzoyl)piperidino)te-
tralin with [ 18F]Fluoride ([18F]9e). (()-trans-2-Hydroxy-3-(4-
(4-[18F]fluorobenzoyl)piperidino)tetralin ((()-[18F]9e). A sample
of 100-150 mCi [18F]/fluoride was added to a reaction vessel
containing 1.0 mg of K2CO3 and Kryptofix (5-6 mg), and the
solution was evaporated under argon (block temperature of 110
°C). Acetonitrile (3 × 1.0 mL) was added and evaporated to ensure
Resolution of Compound (()-9d. Chromatographic resolution
of compound 9d (0.230 g) was accomplished by HPLC using a
250 mm × 10 mm i.d. Chiralcel OD column (18:82 isopropanol/

18F-Labeled PET Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1367
In Vitro Biological Evaluation: Receptor Binding
Assays. The compounds were dissolved in DMF, DMSO, or ethanol
and then diluted in 50 mM Tris-HCl buffer containing 150 mM
NaCl and 100 mM EDTA at pH 7.4 prior to performing the σ1 and
σ2 receptor binding assays. The procedures for isolating the
membrane homogenates and performing the σ1 and σ2 receptor
binding assays have been described in detail previously.59,60
complete removal of water. After all the water was removed,
1.5-2.0 mg of the corresponding nitrobenzoylbenzovesamicol, (()-
9d, was dissolved in DMSO (300 µL). The precursor solution was
transferred into the reaction vessel containing [18F]fluoride/Kryptofix
and K2CO3. The reaction tube was capped and briefly mixed and
then subjected to microwave irradiation for 40 s at medium power
(60 W) until TLC scanner indicated a suitable incorporation yield.
TLC mobile phase was 25:75 methanol/methylene chloride. The
Rf value of the product is approximately 0.90.
The reaction mixture was diluted with 3.0 mL of HPLC mobile
phase and passed through a light alumina Neutral Sep-Pak cartridge.
The crude product was then loaded onto a C-18 Econosil semi-
preparative HPLC column with UV wavelength of 254 nm. The
HPLC system used a 5 mL injection loop. With 23:77 THF/0.1 M
formate buffer as eluent at 3.5 mL/min flow rate, the retention time
of the product was 12.6 min. The retention time of the precursor
was approximately 19.6 min. After evaporation of the HPLC
solvent, the radiotracer was dissolved in 3-5 mL of saline and
filtered using a 0.22 µm sterile syringe filter.
σ
Briefly, the σ1 receptor binding assays were conducted in 96-
well plates using guinea pig brain membrane homogenates (∼300
µg of protein) and ∼5 nM [3H](+)-pentazocine (34.9 Ci/mmol,
Perkin-Elmer, Boston, MA). The total incubation time was 90 min
at room temperature. Nonspecific binding was determined from
samples that contained 10 µM cold haloperidol. After 90 min, the
reaction was terminated by the addition of 150 µL of ice-cold wash
buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) using a 96-channel
transfer pipet (Fisher Scientific, Pittsburgh, PA). The samples were
harvested and filtered rapidly through a 96-well fiber glass filter
plate (Millipore, Billerica, MA) that had been presoaked with 100
µL of 50 mM Tris-HCl buffer at pH 8.0 for 1 h. Each filter was
washed 3 times with 200 µL of ice-cold wash buffer and the filter
counted in a Wallac 1450 MicroBeta liquid scintillation counter
(Perkin-Elmer, Boston, MA).
Quality control of the product was performed on an analytical
HPLC system that consisted of an Alltech Econosil reversed-phase
C-18 column (250 mm × 4.6 mm) with a mobile phase of 37:63
acetonitrile/0.1 M ammonium formate buffer (pH 4.0-4.5). At a
flow rate of 1.5 mL/min, the (()-[18F]9e sample was co-injected
with cold standard of 9e and monitored with a variable-wavelength
detector set at 254 nm with sodium iodide scintillation detector.
The retention time of the product is 6.37 min. Under the same
conditions, the retention time of the precursor is 9.10 min. The
labeling yield was 50-60% (decay corrected). Both the radio-
chemical purity and chemical purity are also greater than 99%. The
specific activity was >2000 Ci/mmol. The entire procedure took
∼2 h.
The σ2 receptor binding assays were conducted using rat liver
membrane homogenates (∼300 µg of protein) and ∼5 nM [3H]DTG
(58.1 Ci/mmol, Perkin-Elmer, Boston, MA) in the presence of 1
µM (+)-pentazocine to block σ1 sites. The incubation time was
2 h at room temperature. Nonspecific binding was determined from
samples that contained 10 µM cold haloperidol. All other procedures
were identical to those described for the σ1 receptor binding assay
above.
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 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 Prusoff61 and are presented as the mean
( 1 SEM. For these calculations, we used a Kd value of 7.89 nM
for [3H](+)-pentazocine and guinea pig brain and a Kd value of
30.73 nM for [3H]DTG and rat liver.60
(+)-trans-2-Hydroxy-3-(4-(4-[18F]fluorobenzoyl)piperidino)te-
tralin ((+)-[18F]9e). Compound (+)-[18F]9e was prepared from (+)-
9d as described in procedure D. The solution of the corresponding
precursor (+)-9d in DMSO was transferred into the reaction vessel
containing predried [18F]fluoride/Kryptofix and K2CO3. The reaction
vessel was capped and then irradiated for 40 s by microwave at
medium power (60 W). After the reaction vessel was cooled, the
purification was performed as described above on the same HPLC
system. The labeling yield was 50-60% (decay-corrected). The
radiochemical purity is greater than 99% radiochemical purity, and
chemical purity is also greater than 99%. The specific activity was
>2000 Ci/mmol. The entire procedure took ∼2 h.
In Vivo Biodistribution and ex Vivo Autoradiography
Studies. All animal experiments were conducted in compliance with
the Guidelines for the Care and Use of Research Animals
established by Washington University’s Animal Studies Committee.
For the biodistribution studies, 25-35 µCi of (()-[18F]9e, (+)-
[18F]9e, or (-)-[18F]9e in 100-150 µL of saline was injected via
the tail vein into mature male Sprague-Dawley rats (250-400 g)
under 2-3% isoflurane/oxygen anesthesia. Groups of at least four
rats were used for each time point. At 5 min, 30 min, 1 h, and 2 h
p.i., the groups of rats were again anesthetized and euthanized. The
whole brain was quickly removed and dissected into segments
consisting of hippocampus, striatum, cortex, and cerebellum. The
remainder of the brain was also collected in order to determine
total brain uptake. At the same time, samples of blood, lung, liver,
kidney, muscle, fat, heart, and bone 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 nontarget tissue.
(-)-trans-2-Hydroxy-3-(4-(4-[18F]fluorobenzoyl)piperidino)te-
tralin ((-)-[18F]9e). Compound (-)-[18F]9e was prepared from (-)-
9d as described in procedure D. The solution of the corresponding
precursor (-)-9d in DMSO was transferred into the reaction vessel
containing predried [18F]fluoride/Kryptofix and K2CO3. The reaction
vessel was capped and then irradiated for 40 s by microwave at
medium power (60 W). After the reaction vessel was cooled, the
purification was performed as described above on the same HPLC
system. The labeling yield was 50-60% (decay-corrected). The
radiochemical purity is greater than 99%, and chemical purity is
also greater than 99%. The specific activity was >2000 Ci/mmol.
The entire procedure took ∼2 h.
In Vitro Biological Evaluation: Vesicular Acetylcholine
Transporter Binding. Dissociation constants of novel compounds
were determined by competition against the binding of 5 nM
[3H]vesamicol to postnuclear supernatant prepared from PC12 cells
stably expressing human VAChT according to the methods utilized
by Parsons and colleagues.47 Compounds were assayed in incre-
ments of 10-fold from 0.1 to 10 000 nM. The surfaces of containers
were precoated with Sigmacote (Sigma-Aldrich, MO). Samples
containing 200 µg of postnuclear supernatant in 200 µL were
incubated at 22 °C for 24 h in 0.02% sodium azide. 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. Averaged data
were fit by regression with a rectangular hyperbola to estimate IC50.
All compounds were independently assayed at least two times.
For the ex vivo electronic autoradiography study, a mature male
Sprague-Dawley rat was injected with ∼500 µCi of (-)-[18F]9e
via the tail vein under isoflurane/oxygen anesthesia, allowed to
recover, and then euthanized after 1 h as described above. The brain
was quickly removed and placed in a metal brain matrix with slots
spaced at 1 mm intervals. The brain was then frozen in the matrix
and sectioned into 1 mm coronal slices. Brain slices were carefully
removed from the brain matrix razor blades, placed on a clear sheet,
covered with film, and counted using the Packard InstantImager.
The photographic image was obtained using a flatbed scanner.

1368 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
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MicroPET Image Processing and Analysis. Acquired list mode
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Acknowledgment. This work was supported by a Wash-
ington University Alzheimer Disease Research pilot grant (Grant
NIA P50 AG05681-21 and NIH Grant 1P30NS048056-01). The
authors gratefully thank William H. Margenau and Robert
Dennett for their excellent technical assistance. Mass spectrom-
etry was provided by the Washington University Mass Spec-
trometryResource,anNIHResearchResource(GrantP41RR0954).
Optical rotation was determined in the laboratory of Dr. Douglas
F. Covey in the Department of Molecular Biology and Phar-
macology of Washington University. X ray crystallography was
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Supporting Information Available: Crystal structure data for
(-)-9e (final residual values, structure refinement parameters,
complete listings of positional and isotropic displacement coef-
ficients for hydrogen atoms, anisotropic displacement coefficients
for the non-hydrogen atoms) and analytical data of new compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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