Synthesis and characterization of technetium-99m-labeled tropanes as dopamine transporter-imaging agents

Article abstract of DOI:10.1021/jm960532j

In the development of novel Tc-99m-labeled tropane derivatives as dopamine transporter (reuptake site)-imaging agents, a series of neutral and lipophilic complexes containing bis-(aminoethanethiol) as a neutral complexing moiety for a [^99mTc]Tc

Full text of DOI:10.1021/jm960532j

J . Med. Chem. 1997, 40, 9-17
9
Syn th esis a n d Ch a r a cter iza tion of Tech n etiu m -99m -La beled Tr op a n es a s
Dop a m in e Tr a n sp or ter -Im a gin g Agen ts
Sanath K. Meegalla,† Karl Plo¨ssl,† Mei-Ping Kung,† Sumalee Chumpradit,† D. Andrew Stevenson,†
Steven A. Kushner,† William T. McElgin,‡ P. David Mozley,† and Hank F. Kung*,†,§
Departments of Radiology, Pharmacology, and Psychiatry, University of Pennsylvania, 3700 Market Street,
Philadelphia, Pennsylvania 19104
Received J uly 23, 1996X
In the development of novel Tc-99m-labeled tropane derivatives as dopamine transporter
(reuptake site)-imaging agents, a series of neutral and lipophilic complexes containing bis-
(aminoethanethiol) as a neutral complexing moiety for a [99mTc]TcO3+ center core was
successfully prepared. Biological evaluation of the Tc-99m-labeled complexes 13-16 as central
nervous system (CNS) dopamine transporter-imaging agents was reported. Synthesis of the
tropane derivatives was achieved by stepwise reactions adding two aminoethanethiol units.
The final free thiol ligands were obtained by deblocking the 4-methoxybenzyl protecting group
with Hg(OAc)2 to obtain trifluoroacetate salts. All of the Tc-99m complexes, with the exception
of 16, displayed good initial brain uptake and selective uptake in the striatal area, where
dopamine transporters are concentrated. One of the compounds, [2-[[2-[[[3-(4-chlorophenyl)-
8-methyl-8-azabicyclo[3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethyl]amino]ethanethiolato-
(3-)-N2,N2′,S2,S2′]oxo-[1R-(exo-exo)]- [99mTc]technetium, [99mTc]TRODAT-1 (13), displayed the
highest initial uptake in rat brain (0.4% at 2 min post iv injection); the striatal/cerebellar (ST/
CB) ratio reached 2.8 at 60 min after an iv injection. The specific uptake in rat brain can be
blocked by pretreating rats with a competing dopamine transporter binding agent, â-CIT (RTI-
55, 2â-carbomethoxy-3â-(4-iodophenyl)tropane; iv, 1 mg/kg), which reduced the regional brain
uptake ratio (ST/CB) to 1.2. In contrast, the specific striatal uptake was not affected by
pretreating rats with a noncompeting ligand, haldol (iv, 1 mg/kg). After an iv injection of 9
mCi of [99mTc]TRODAT-1 (13), in vivo images of baboon brain using single-photon emission-
computed tomography exhibited excellent localization in striatum (basal ganglia), where
dopamine neurons are known to be concentrated. This series of compounds may provide a
convenient source of short-lived imaging agents for routine diagnosis of CNS diseases (i.e.,
Parkinson’s disease) in which changes in the dopamine transporter concentration are implicated.
In tr od u ction
more suitable than GBR12,93516 for imaging dopamine
transporters. The dopamine transporter ligands are
useful in evaluating changes in dopamine transporters
in vivo and in vitro, especially for patients with Par-
kinson’s disease (PD), which is characterized by a
selective loss of dopamine neurons in the basal ganglia
and substantia nigra. Recent publications describing
the use of [11C]CFT7 (WIN35,428) and [123I]â-CIT10,17
suggest a strong correlation between the decrease in the
localization of dopamine transporter ligands in the
anterior putamen area and PD symptoms.7,8,17 The
results provide an impetus toward the further develop-
ment of these agents for diagnosing and monitoring the
treatment of PD patients. Currently, [123I]â-CIT is
being widely investigated for this purpose. However,
one of the drawbacks of [123I]â-CIT is the time required
(>18 h) for reaching an optimal uptake ratio in the
target area, the basal ganglia (BG), vs the nontarget
area, the frontal cortex (CX). Because of the need for
agents with faster equilibrium times, new ligands, such
as [123I]IPT14,18-20 and [123I]FP-CIT,13,15 both of which
reach maximum uptake in human brain within a few
hours, will play an important future role as the agents
of choice.
Recently, a large number of dopamine transporter
(reuptake site)-imaging agents based on cocaine or its
closely related congeners, tropane derivatives, have been
reported.1-4 The regional brain distribution of cocaine
is largely concentrated in the basal ganglia, where
dopamine neurons are located. [11C]N-Methyl-labeled
cocaine5,6 is a very useful PET (positron emission
tomography) ligand for studying the pharmacology and
drug effects of cocaine itself; however, additional modi-
fications of the cocaine molecule have led to the devel-
opment of PET imaging agents such as CFT7-9 (N-
methyl-2â-carbomethoxy-3â-(4-fluorophenyl)tropane,
WIN35,428) and single-photon emission-computed to-
mography (SPECT) imaging agents â-CIT10-13 (N-meth-
yl-2â-carbomethoxy-3â-(4-iodophenyl)tropane), IPT14 (N-
(3-iodopr open -2-yl)-2â-ca r bom et h oxy-3â-(4-ch lor o-
phenyl)tropane), FP-CIT15 (N-(3-fluoropropyl)-2â-car-
bomethoxy-3â-(4-iodophenyl)tropane), and other related
derivatives that display much higher binding affinity
and selectivity to dopamine transporters. These PET
and SPECT imaging agents displayed excellent specific
uptake in the striatum (basal ganglia) area and are
* Corresponding author: Hank F. Kung, Ph.D., 3700 Market St,
Room 305, Philadelphia, PA 19104. Tel: (215)662-3096. Fax: (215)-
349-5035. E-mail: kunghf@sunmac.spect.upenn.edu.
† Department of Radiology.
Technetium-99m (t1/2 ) 6 h, 140 keV) is the most
commonly used radionuclide in diagnostic nuclear medi-
cine.21,22 Its popularity is mainly due to the fact that
the radionuclide can be readily produced by a Mo-99/
Tc-99m generator, the medium γ-ray energy emitted by
‡ Department of Psychiatry.
§ Department of Pharmacology.
X Abstract published in Advance ACS Abstracts, December 15, 1996.
S0022-2623(96)00532-8 CCC: $14.00 © 1997 American Chemical Society

10 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1
Meegalla et al.
Tc-99m (140 keV) is suitable for γ-camera detection, and
the physical half-life is often compatible with the
biological localization and residence time required for
imaging. Currently, about 85% of routine nuclear
medicine procedures are performed with radiopharma-
ceuticals based on Tc-99m. In the past 10 years,
significant progress has been made on the chemistry of
technetium, using the chemical level of Tc-99 (t1/2 ) 2.1
× 105 years).21 Improvements in the understanding of
technetium chemistry have significantly enhanced the
development of a new generation of technetium radio-
pharmaceuticals for clinical use and potentially will
benefit millions of patients who receive Tc-99m agents
for routine nuclear medicine diagnosis. It is well
established that when [99mTc]pertechnetate (TcO4-), the
most commonly available starting material, is reduced
by a reducing agent, such as stannous chloride, in the
presence of “soft” chelating ligands, including N2S2 and
NS3, [TcvO]3+N2S2 or [TcvO]3+NS3 complexes are formed.
Several recent reports demonstrate that it is possible
to incorporate [TcvO]3+N2S2 into potential receptor-
selective imaging agents for muscarinic receptors,23
vesamicol sites,24 and steroid hormone receptors.25-30
The use of other chelating agents for preparing Tc-99m
muscarinic receptor-imaging agents has also been re-
ported.31 However, all of these central nervous system
(CNS) receptor-specific Tc-99m imaging agents have
demonstrated limited success in in vivo studies.
F igu r e 1. X-ray structure of [methyl 3-(4-chlorophenyl)-8-(2-
mercaptoethyl)-8-azabicyclo[3.2.1]octane-2-carboxylato-S][2,2′-
(methylimino)bis[ethanethiolato]](2-)-N,S,S′]oxorhenium.
Sch em e 1
Recently, a series of neutral and lipophilic complexes,
containing N-(alkylthiolato)tropane, aminobis(ethylthi-
olato), and a [99mTc]TcO3+ center core, was prepared and
evaluated as CNS dopamine transporter-imaging agents
in rats.32 One of the compounds, [methyl 3-(4-chlo-
rophenyl)-8-(2-mercaptoethyl)-8-azabicyclo[3.2.1]octane-
2-carboxylato-S][2,2′-(methylimino)bis[ethanethiolato]]-
(2-)-N,S,S′]oxo[99mTc]technetium, displayed low initial
uptake in rat brain (0.1% at 2 min post iv injection),
but the striatal/cerebellar (ST/CB) ratio reached 3.50
at 60 min after an iv injection. The corresponding
rhenium complex displayed the expected structure,
containing a square-pyramidal core with the RedO in
the axial position (Figure 1).
Another related Tc-99m-labeled tropane derivative,33
technepine, which was reported to have a high affinity
to the dopamine transporter, may also be useful as an
in vivo imaging agent. Both of the current examples of
Tc-99m-labeled tropane derivatives were modified at the
N-methyl position of the tropane ring to add a techne-
tium-carrying moiety. In order to increase the initial
brain uptake and limit the molecular size of the final
Tc-99m-labeled tropane derivative, a novel series of
derivatives with the bis(aminoethanethiol) ligand at-
tached at the 2â-position was prepared. In this report,
synthesis, radiolabeling, a preliminary biodistribution
study of the new Tc-99m complexes in rats, and a
SPECT imaging study in a baboon are described.
Resu lts
by the action of oxalyl chloride on carboxylic acids 4a -c
at room temperature, were not isolated but intercepted
with amine 2 to obtain amides 6a -c. Secondary amines
8 were prepared by diborane reduction of amides 6 and
converted to amides 9 by alkylations with alkyl chloride
7. The amide functions of compounds 9 were reduced
with BH3‚THF to yield compounds 11. Amines 11 and
amide 9 were deprotected with Hg(OAc)2 to obtain
Schemes 1-3 illustrate the synthetic strategy em-
ployed for the preparation of the compounds described
in this paper. Syntheses of the 3â-(p-chloro-, p-fluoro-,
and p-bromophenyl)tropane esters 3a -c and the cor-
responding carboxylic acids 4a -c were carried out
according to previously reported procedures.9,34 The
corresponding acyl chlorides 5a -c, which were prepared

New Tc-99m-Labeled DA Transporter-Imaging Agent
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1 11
Sch em e 2
trifluoroacetate salts of dithiols 10 and 12. Since the
instability of these disulfides prevented any further
purification, they were obtained as their trifluoroacetate
salts and used directly without further characterization
for the preparation of rhenium (17) and [99mTc]techne-
tium (13-16) complexes.
Rhenium oxo complexes of bisthiols 12a ,b were
prepared as surrogates for corresponding Tc complexes,
by employing [Bu4N][ReOCl4] as a rhenium starting
material in MeOH at room temperature in the presence
of triethylamine as an acid scavenger. Thin layer
chromatography, as well as NMR data of purified
rhenium complexes, indicated that there were two
isomers formed in each complexation reaction. Al-
though the NMR spectra of the rhenium complexes were
complicated due to the presence of isomers and the
diastereotopic nature of all methylene protons, IR and
high-resolution mass spectral data confirmed the pres-
ence of the expected rhenium oxo complexes (see Dis-
cussion and Figure 4).
their neutrality and lipophilicity. Tc-99m-labeled com-
plexes 13-15 displayed excellent medium range lipo-
philicity (partition coefficients between 1-octanol and pH
7.0 buffer of 99-262). Complex 16 was an exception; it
fell out of this range, with a PC of 1818 (Table 1).
Biodistribution studies of 13-16 in rats were per-
formed after an iv injection of a tracer dose. The results
showed a distribution pattern reflecting regional per-
fusion (i.e., high uptake in muscle, kidney, liver, brain
and skin; Tables 1 and 2). However, the brain uptake
is moderate, ranging from 0.43% to 0.11% dose/organ
at 2 min for the four complexes. The complex containing
an amide group, 16, showed the lowest brain uptake
(Table 1), despite its high lipophilicity (PC ) 1818). The
results suggest that an amide group in the technetium
N2S2 complexing moiety may hinder the initial brain
uptake.35 Increasing the lipophilicity of these com-
pounds does not appear to improve brain uptake in rats.
The most significant finding of this initial biodistribu-
tion study is that the brain uptake of these complexes,
except 16, was highly concentrated in the striatal area,
where the dopamine transporters are located, compared
to a region with no dopamine neurons (i.e., the cerebel-
lar region). At 60 min after an iv injection, the ratios
of ST/CB were found to be 2.66, 2.18, and 2.82 for 13,
14, and 15, respectively. However, complex 16 showed
The radiolabeling of 12a -c and 10 with sodium
[
99mTc]pertechnetate was successfully achieved using
stannous(II) glucoheptonate as the reducing agent to
produce 13-16 in good yield (80%) and high radio-
chemical purity (>95%). Key properties for these
potential dopamine transporter-imaging agents are

12 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1
Meegalla et al.
Sch em e 3
the rat striatum was specifically related to the dopamine
transporter in rat brain.
To evaluate further the potential of [99mTc]TRODAT-
1 (13), [2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo-
[3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethyl]ami-
n o]et h a n et h iola t o(3-)-N2,N2′,S 2,S 2′]oxo-[1R-(exo-
exo)][99mTc]technetium, as a dopamine transporter-
imaging agent, a SPECT imaging study was carried out
in a female baboon. To facilitate the identification of
anatomical localization, the coronal, transaxial, and
sagittal SPECT images (1.34 mm thick) obtained 60-
90 min postinjection were coregistered with the MRI
images of the same baboon (Figure 3). The fused images
displayed excellent consistency with the expected local-
ization of this agent in caudate and putamen areas,
where the dopamine transporters are known to be
located. The images also showed good correlation with
PET imaging agent [11C]CFT7,8 and SPECT imaging
agent [123I]â-CIT.10-12 In vitro binding studies of rhe-
nium complex 17a in rat striatal homogenates displayed
good binding affinity (Ki ) 14 nM, using [125I]IPT20 as
the ligand; Kd ) 0.2 nM), while the bis(ethanethiol) 12a
displayed a comparable affinity (Ki ) 7 nM). Although
the binding affinity of the corresponding rhenium
complex 17a is not as potent as other iodinated tropane
derivatives, the brain uptake and retention of [99mTc]-
TRODAT-1 (13) appears to be sufficient for in vivo
SPECT imaging in nonhuman primates.
Ta ble 1. Brain Uptake of Four [99mTc]TRODAT Derivatives (%
dose/organ)
ST/CB ratio
Discu ssion
compound
2 min
60 min
at 60 mina
PCb
Despite its attractive physical properties, technetium
is a difficult element for designing suitable SPECT
ligands. Technetium is a transition metal and requires
a complexing agent to stabilize it at different valence
states.22 Valence states can vary from 7+ (as pertech-
netate) to 0, depending on the reaction conditions and
chelating agents used during preparation.21 After com-
plexation, the molecules invariably become big and
bulky, which is the limiting factor in designing a
molecule targeted to (a) specific biological process(es).
Additional requirements for Tc-99m-labeled complexes
as CNS receptor-imaging agents are (i) small (molecular
weight < 750), with good lipophilicity (partition coef-
ficient ∼ 50-1000), (ii) high binding affinity (Ki < 10
nM) and high selectivity, and (iii) ideal brain uptake in
rats >0.5% dose/organ at 2 min post iv injection. This
novel series of compounds differs from those previously
reported in that the substitution of the bis(aminoet-
hanethiol) ligand is attached at the 2â-position of the
tropane core structure. It is apparent that by choosing
this position, the corresponding Tc-99m-labeled agent
displayed a 3-4-fold increase in initial brain uptake
(0.1% for N-substituted compound32 vs 0.3-0.4% in
brain at 2 min postinjection) and concomitantly retained
the specific uptake in the striatum area of the brain.
The present observation suggests that, in this series of
compounds, the 3â-p-fluoro is slightly less favorable
than the corresponding 3â-p-chlorophenyl derivative. As
previously reported for the same series of tropane
derivatives, the 3â-p-fluoro derivative displayed a lower
brain uptake, which may be due to its lower binding
affinity to dopamine transporters.3
13, TRODAT-1 0.43 ( 0.16 0.12 ( 0.001
2.66
2.18
2.82
1.17c
227
99
262
1818
14
15
16
0.37 ( 0.09 0.098 ( 0.014
0.41 ( 0.03 0.18 ( 0.01
0.11 ( 0.02 0.07 ( 0.014c
a
Striatum/cerebellum (ST/CB) ratio: percentage dose per gram
b
of striatum/percentage dose per gram of cerebellum. PC: mea-
sured between 1-octanol and pH 7.0 phosphate buffer. c Data were
from rats sacrificed at 30 min.
little specific uptake (ST/CB ) 1.17) at 60 min post iv
injection. The initial biodistribution study in rats
suggested that [99mTc]TRODAT-1 (13) is the best imag-
ing agent candidate in this series of complexes. It
displayed the highest initial brain uptake and high
retention in the target area (i.e., striatum) at later time
points; therefore, a more detailed biodistribution study
was carried out.
Specific uptake of [99mTc]TRODAT-1 (13) at different
time points displayed a prolonged retention, and the ST/
CB ratio in rat brain reached a maximum value of 4.07
at 4 h after injection (Table 3). Blocking studies in rats
were performed at 60 min postinjection to characterize
further the uptake and binding. In this series of
regional brain uptake studies in rats, the ST/CB ratio
at 60 min postinjection was 2.66 (Figure 1). The specific
uptake of [99mTc]TRODAT-1 (13) in rat striatum could
be blocked by pretreating rats with a dose of â-CIT (1
mg/kg, iv), a known dopamine transporter ligand. The
specific binding, as indicated by ST/CB, was reduced to
1.2 after pretreatment with â-CIT. The specific binding
was not reduced by pretreatment with haldol (1 mg/kg,
iv), an agent with a mixed pharmacological profile
(binding to various CNS receptors, but not to the
dopamine transporter); no blocking effect was observed.
The ST/CB ratio (2.6) was identical with that in control
rats (Figure 2). The results suggested that uptake in
The most promising compound in this series, [99mTc]-
TRODAT-1 (13), clearly displays the desired properties
described above. Even though the initial study reported

New Tc-99m-Labeled DA Transporter-Imaging Agent
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1 13
Ta ble 2. Biodistribution in Rats Post iv Injection of [99mTc]TRODAT Ligandsa
organ/region
13
14
15
16
2 min Postinjection
4.15 ( 0.40
blood
heart
muscle
lung
kidney
spleen
liver
4.94 ( 0.46
1.47 ( 0.16
10.84 ( 1.95
6.82 ( 0.37
5.13 ( 0.75
0.42 ( 0.16
16.62 ( 2.11
2.66 ( 0.35
0.43 ( 0.16
2.00 ( 0.28
1.51 ( 0.19
13.66 ( 2.15
6.46 ( 0.35
4.66 ( 0.64
0.52 ( 0.15
13.75 ( 1.26
2.54 ( 0.48
0.41 ( 0.03
5.19 ( 0.49
1.33 ( 0.09
9.67 ( 2.44
4.56 ( 0.65
3.46 ( 0.41
0.94 ( 0.34
25.05 ( 4.16
3.94 ( 0.63
0.106 ( 0.018
1.92 ( 0.10
15.42 ( 5.58
11.04 ( 1.41
6.88 ( 0.46
0.39 ( 0.09
18.5 ( 1.70
skin
brain
7.37 ( 0.36
0.37 ( 0.009
60 min Postinjection
1.24 ( 0.17
blood
heart
muscle
lung
kidney
spleen
liver
2.14 ( 0.17
0.19 ( 0.02
10.28 ( 1.36
1.86 ( 0.36
3.06 ( 0.14
0.45 ( 0.05
17.67 ( 3.42
3.96 ( 0.22
0.12 ( 0.00
0.92 ( 0.06
0.19 ( 0.01
9.47 ( 1.98
1.49 ( 0.09
1.68 ( 0.22
0.39 ( 0.04
22.80 ( 1.84
3.18 ( 0.40
0.18 ( 0.01
1.89 ( 0.20
0.36 ( 0.04
14.34 ( 1.76
3.00 ( 0.38
1.41 ( 0.06
0.67 ( 0.04
17.41 ( 0.75
4.43 ( 0.27
0.069 ( 0.014
0.71 ( 0.008
11.07 ( 0.45
1.54 ( 0.13
2.79 ( 0.09
0.61 ( 0.10
25.64 ( 2.12
9.10 ( 0.61
skin
brain
0.098 ( 0.014
Regional Brain Distribution (% dose/g) at 60 min Postinjection (Region/CB)b
cerebellum
striatum
0.057 ( 0.003
0.038 ( 0.001
0.070 ( 0.002
0.039 ( 0.006
(1.00)
0.046 ( 0.006
(1.17)
(1.00)
(1.00)
(1.00)
0.151 ( 0.002
(2.66)
0.083 ( 0.012
(2.18)
0.198 ( 0.013
(2.82)
hippocampus
cortex
0.096 ( 0.015
0.067 ( 0.004
0.114 ( 0.003
0.039 ( 0.000
(1.00)
0.051 ( 0.008
(1.30)
(1.69)
(1.76)
(1.61)
0.094 ( 0.012
(1.65)
0.052 ( 0.010
(1.37)
0.122 ( 0.014
(1.73)
a
b
Percentage dose/organ, average of three rats ( SD Region/CB ) percentage dose per gram of specific region/percentage dose per
gram of cerebellum.
Ta ble 3. Striatal/Cerebellar (ST/CB) Ratio in Rat Brain Post iv Injection of [99mTc]TRODAT-1, 13a
2 min
30 min
60 min
120 min
240 min
360 min
ST/CB
ratio
0.93 ( 0.23
2.04 ( 0.18
2.66 ( 0.01
3.90 ( 1.33
4.07 ( 0.54
2.97 ( 0.14
a
Striatal/cerebellar (ST/CB) ratio: percentage dose per gram of striatum/percentage dose per gram of cerebellum (average of three
rats ( SD).
F igu r e 3. Transaxial, coronal, and sagittal SPECT images
(1.34 mm thick) of baboon brain at 60-90 min post iv injection
of 9 mCi of [99mTc]TRODAT-1 (13). SPECT images were
acquired by a Picker T3000 scanner. The SPECT images were
coregistered with the same sections of MRI of the same baboon.
A high accumulation of [99mTc]TRODAT-1 (13) was observed
in caudate and putamen, areas of the brain where dopamine
transporters are concentrated.
F igu r e 2. Ratios of regional brain uptakes at 60 min post iv
injection of [99mTc]TRODAT-1 (13) in control rats and rats
pretreated with haldol (1 mg/kg, iv) or â-CIT (1 mg/kg, iv) 5
min prior to radiotracer injection. Values shown are means (
SD (n ) 3-4, p < 0.05, Student’s t-test). ST, striatum; HP,
hippocampus; CX, cortex; CB, cerebellum. The ST area, where
dopamine transporters are located, displayed selective regional
brain uptake, with the highest concentration ratio (ST/CB )
2.6). Pretreatment with â-CIT, which competes for dopamine
transporter binding, showed blocking of specific uptake of
utility. Optimized imaging protocol and reproducibility
of SPECT imaging of this new agent remain to be
investigated.
[
99mTc]TRODAT-1 (13) in the ST area.
in this paper strongly suggests that this compound may
potentially be useful for studying dopamine transport-
ers, there are a number of questions that remain
unanswered. Correlation of dopamine transporter up-
take as measured by SPECT imaging and the actual
neuronal integrity requires extensive kinetic modeling
studies to validate and estimate the potential clinical
Formation of ReO and TcO complexes with mono-N-
substituted N2S2 ligands can lead to four isomers. Two
enantiomers (R and S) are formed from the quaternary
amine nitrogen and two sets of diastereomers (syn and
anti) are formed by the orientation of the N-substituent
in relation to the metal-oxygen bond. In general, the
syn diastereomers are formed preferentially to the anti

14 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1
Meegalla et al.
values with chloroform or TMS as the internal reference.
Coupling constants are reported in hertz (Hz). The multiplic-
ity is defined by s (singlet), d (doublet), t (triplet), brs (broad
signal), dt (doublet of triplet), and m (multiplet). IR spectra
were recorded with a Mattson Polaris FT-IR spectrometer and
are reported in cm-1
. Melting points were determined on a
Meltemp apparatus (Cambridge, MA) and are uncorrected.
Elemental analyses were performed by Atlantic Microlabs
(Norcross, GA). High-resolution mass spectrometry was per-
formed by the Nebraska Center for Mass Spectroscopy,
University of Nebraska, Lincoln, NE. Compounds 1-4 were
synthesized according to the literature.9,34 [Bu4N][ReOCl4] was
prepared according to the literature method.41
P r ep a r a tion of Com p ou n d s 6a -c. Oxalyl chloride (2
mmol, 1 mL from 2 M solution in CH2Cl2) was added to tropane
acid 4 (1 mmol) in CH2Cl2 (10 mL) at room temperature under
N2. The resulting mixture was stirred for 1.5 h and concen-
trated in vacuo at 30 °C to obtain a viscous oil, which was
dried in a vacuum for 15 min. The acid chloride obtained was
dissolved in CH2Cl2 (10 mL) and cooled to -10 °C, and amine
2 (1 mmol, 197 mg) in CH2Cl2 (10 mL) was added followed by
Et3N (2 mmol, 0.28 mL) under N2. The resulting solution was
stirred at room temperature for 6 h. Water (20 mL) was then
added to the reaction mixture, and the product was extracted
into CH2Cl2 (3 × 20 mL). The CH2Cl2 layers were combined,
dried over Na2SO4, and concentrated in vacuo to produce an
oil that was chromatographed on silica gel (EtOAc:MeOH:NH4-
OH, 8.5:1:0.5) to obtain the title compound as a viscous oil.
F igu r e 4. (A) Comparison of HPLC profiles of [99mTc]TRO-
DAT-1 (13; lower profile, radioactivity trace) and Re-TRO-
DAT-1 (17a ; upper profile, UV-vis 254 nm) coinjected onto a
reversed-phase column (PRP-1) with acetonitrile:DMGA buffer
(pH 7) in a ratio of 80:20 and a flow rate of 1 mL/min. (B)
Comparison of HPLC profiles of [99mTc]TRODAT-1 (13; lower
profile, radioactivity trace) and Re-TRODAT-1 (17a ; upper
profile, UV-vis 254 nm) coinjected onto a Chiralpak AD
(chiral) column with hexane:EtOH in a ratio of 3:1 and a flow
rate of 1 mL/min. Generally, the retention times of Re
complexes are comparable to those of Tc-99m compounds.
3-(4-Ch lor op h en yl)-N-[2-[S-(4-m eth oxyben zyl)th io]et-
h yl]-8-a za b icyclo[3.2.1]oct a n e-2-ca r b oxa m id e-[1R-(exo-
exo)] (6a ): yield 63%; IR (CHCl3) 1656 cm-1; 1H NMR (CDCl3)
δ 1.55-1.78 (m, 3H), 2.05-2.6 (m, 9H), 3.1-3.4 (m, 5H), 3.69
(s, 2H), 3.79 (s, 3H), 6.84 (d, 2H, J ) 6 Hz), 7.09 (d, 2H, J )
6 Hz), 7.18 (d, 2H, J ) 6 Hz), 7.24 (d, 2H, J ) 6 Hz), 9.8 (brs,
1H); HRMS m/z 458.1794 calculated for C25H31O2N2SCl, M+
+ 1, 459.1994.
diastereomers. The formation of predominantly syn
diastereomers of TcO complexes with mono-N-substi-
tuted N2S2 ligands has been documented previously by
several research groups.27,36-38 Since the N2S2 ligands
12a -c carry a N-substitution group with an additional
chiral center (tropanyl moiety), all of the possible
metal-oxo complexes become diastereomers. However,
HPLC analyses of the crude reaction mixtures of the
3-(4-F lu or op h en yl)-N-[2-[S-(4-m eth oxyben zyl)th io]et-
h yl]-8-a za b icyclo[3.2.1]oct a n e-2-ca r b oxa m id e-[1R-(exo-
exo)] (6b): yield 59%; IR (CHCl3) 1653 cm-1; 1H NMR (CDCl3)
δ 1.59-1.76 (m, 3H), 1.97-2.4 (m, 6H), 2.4-2.5 (m, 3H), 3.09
(m, 1H), 3.3-3.38 (m, 4H), 3.68 (s, 2H), 3.78 (s, 3H), 6.8-6.9
(m, 4H), 7.08-7.13 (m, 2H), 7.2 (d, 2H, J ) 10 Hz), 9.8 (brs,
[
99mTc]TRODAT complexes (13) indicated the presence
of two major isomers in a ratio of about 1:0.8. Although
these isomers displayed similar elution profiles on
reversed-phase HPLC, they have different retention
times on HPLC profiles obtained using a chiral column
(Figure 4). Similarly, the corresponding ReO complexes
17a ,b also contained two major isomers, as evidenced
by TLC, 1H NMR, and HPLC (Figure 4), and they have
comparable retention times to the Tc-99m-oxo-TRODAT
complexes (13). To further resolve the structures of the
individual isomers, we are in the process of obtaining
suitable crystals of each rhenium isomer to be used for
X-ray structure determination.
1H); HRMS m/z 442.2090 calculated for C25H31O2N2SF, M+
1, 443.2161.
+
3-(4-Br om op h en yl)-N-[2-[S-(4-m eth oxyben zyl)th io]et-
h yl]-8-a za b icyclo[3.2.1]oct a n e-2-ca r b oxa m id e-[1R-(exo-
exo)] (6c): yield 53%; IR (CHCl3) 1651 cm-1; 1H NMR (CDCl3)
δ 1.53-1.78 (m, 3H), 1.93-2.5 (m, 6H), 2.6-2.7 (m, 3H), 3.05
(m, 1H), 3.2-3.38 (m, 4H), 3.64 (s, 2H), 3.78 (s, 3H), 6.84 (d,
2H, J ) 8.7 Hz), 7.03 (d, 2H, J ) 8.4 Hz), 7.23 (d, 2H, J ) 8.7
Hz), 7.32 (d, 2H, J ) 8.4 Hz), 9.8 (brs, 1H); HRMS m/z
502.1289 calculated for C25H31O2N2SBr, M+ + 1, 503.1164.
P r ep a r a tion of Com p ou n d s 8a -c. BH3‚THF (5 mmol,
5 mL of 1 M solution in THF) was added to compounds 6a -c
(1 mmol) in THF (10 mL) under N2, and the resulting mixture
was heated at reflux for 12 h. The reaction mixture was cooled
to 0 °C, and 1 N HCl was added dropwise until no more gas
evolution was observed; then the mixture was concentrated
in vacuo. HCl (1 N, 10 mL) was added to the viscous oil
obtained, and the resulting mixture was stirred at 90 °C for
30 min. The solution was then cooled to 0 °C and basified
with concentrated NH4OH. The product was extracted into
CH2Cl2 and purified by chromatography on silica gel (EtOAc:
MeOH:NH4OH, 8.5:1:0.5) to produce the title compound.
Con clu sion
A novel series of Tc-99m-labeled complexes was
reported. One of the compounds, [99mTc]TRODAT-1
(13), may potentially be useful for in vivo measurement
of dopamine neuronal loss related to changes in dopa-
mine transporters.39,40
Exp er im en ta l Section
Gen er a l. Reagents used in the syntheses were purchased
from Aldrich (Milwaukee, WI) or Fluka (Ronkonkoma, NY) and
used without further purification unless otherwise indicated.
Anhydrous Na2SO4 was used as a drying agent. Reaction
yields are reported without attempts at optimization. Thin
layer chromatography was performed on EM Science (Gibb-
stown, NJ ) precoated (0.2 mm) silica gel 60 plates, and the
spots were detected with iodine vapor and/or UV light. Silica
gel 60 (70-230 mesh), obtained from EM Science (Gibbstown,
NJ ), was used for column chromatography. 1H NMR spectra
were obtained on a Bruker spectrometer (Bruker AC 300). All
samples prepared for NMR analysis were dissolved in CDCl3,
purchased from Aldrich. Chemical shifts are reported as δ
3-(4-Ch lor oph en yl)-2-[[N-[2-[S-(4-m eth oxyben zyl)th io]-
eth yl]a m in o]m eth yl]-8-a za bicyclo[3.2.1]octa n e-[1R-(exo-
exo)] (8a ): yield 79%; IR (CHCl3) 1608 cm-1; 1H NMR (CDCl3)
δ 1.5-1.8 (m, 5H), 2.05-2.2 (m, 4H), 2.23 (s, 3H), 2.34-2.39
(m, 2H), 2.47-2.52 (m, 2H), 2.65 (dd, 1H, J 1 ) 5.3 Hz, J 2
)
7.8 Hz), 3.03 (td, 1H, J 1 ) 4 Hz, J 2 ) 8.8 Hz), 3.25 (m, 2H),
3.5 (s, 2H), 3.78 (s, 3H), 6.84 (d, 2H, J ) 6 Hz), 7.09 (d, 2H, J
) 6 Hz), 7.18 (d, 2H, J ) 6 Hz), 7.24 (d, 2H, J ) 6 Hz); HRMS
m/z 444.2002 calculated for C25H33ON2SCl, M+ + 1, 444.1963.
3-(4-Flu or op h en yl)-2-[[N-[2-[S-(4-m eth oxyben zyl)th io]-
eth yl]a m in o]m eth yl]-8-a za bicyclo[3.2.1]octa n e-[1R-(exo-

New Tc-99m-Labeled DA Transporter-Imaging Agent
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1 15
exo)] (8b): yield 83%; IR (CHCl3) 1600 cm-1; 1H NMR (CDCl3)
δ 1.59-1.76 (m, 5H), 2.0-2.8 (m, 12H), 3.09 (m, 1H), 3.2-
3.38 (m, 2H), 3.68 (s, 2H), 3.78 (s, 3H), 6.8-6.9 (m, 4H), 7.08-
7.13 (m, 2H), 7.2 (d, 2H, J ) 10 Hz); HRMS m/z 428.2297
calculated for C25H33ON2SF, M+ + 1, 429.1543.
3-(4-Br om op h en yl)-2-[[N-[2-[S-(4-m eth oxyben zyl)th io]-
eth yl]a m in o]m eth yl]-8-a za bicyclo[3.2.1]octa n e-[1R-(exo-
exo)] (8c): yield 77%; IR (CHCl3) 1600 cm-1; 1H NMR (CDCl3)
δ 1.59-1.76 (m, 5H), 2.0-2.8 (m, 12H), 3.03 (m, 1H), 3.2-
3.42 (m, 2H), 3.7 (s, 2H), 3.78 (s, 3H), 6.84 (d, 2H, J ) 8.7 Hz),
7.00 (d, 2H, J ) 8.4 Hz), 7.21 (d, 2H, J ) 8.7 Hz), 7.22 (d, 2H,
J ) 8.4 Hz); HRMS m/z 488.1496 calculated for C25H33ON2-
SBr, M+ + 1, 489.1543.
P r ep a r a tion of Com p ou n d s 9a -c. Amine 2 (12.5 mmol,
2.46 g) in CH2Cl2 (15 mL) was added dropwise to a stirred
solution of chloroacetyl chloride (12.5 mmol, 1 mL) in CH2Cl2
(15 mL) followed by Et3N (12.5 mmol, 1.7 mL) at -78 °C under
N2. The reaction mixture was allowed to warm to room
temperature and stirred for 1 h. Water (20 mL) was then
added, and the organic layer was separated and washed with
1 N HCl (20 mL), brine (20 mL), and water (20 mL). The
organic layer was concentrated in vacuo and dried in vacuo
for 30 min. The oil was dissolved in EtOAc (50 mL) and
hexane (100 mL). The turbid solution obtained was concen-
trated to one-half the volume and stored in a freezer for 4 h.
The solid formed was collected by suction filtration to produce
compound 7, which was used for further reactions without
additional purification.
Amines 8a -c (1 mmol), chloro compound 7 (2 mmol, 548
mg), and Et3N (2 mmol, 0.28 mL) in acetonitrile (10 mL) were
heated at reflux for 12 h. The reaction mixture was concen-
trated in vacuo, and the product was partitioned between
water (20 mL) and CH2Cl2 (20 mL). The oil obtained by
concentrating the organic phase was chromatographed on
silica gel. An impurity was washed out with MeOH:CH2Cl2
(1:9). The title compound was eluted with MeOH:CH2Cl2 (2:
8) and isolated as a yellow oil.
4H), 7.15-7.25 (m, 8H); HRMS m/z 667.3032 calculated for
C37H50O2N3S2Cl, M+ + 1, 668.3115.
2-[[2-[[[3-(4-Flu or oph en yl)-8-m eth yl-8-azabicyclo[3.2.1]-
oct-2-yl]m eth yl][[S-(4-m eth oxyben zyl)th io]eth yl]a m in o]-
eth yl]am in o]-S-(4-m eth oxyben zyl)eth an eth iol (11b): yield
1
63%; IR (CHCl3) 1603 cm-1; H NMR (CDCl3) δ 1.4-1.7 (m,
5H), 1.95-2.29 (m, 5H), 2.25-2 (m, 10H), 2.7-2.9 (m, 5H),
3.0 (td, 1H, J 1 ) 4 Hz, J 2 ) 8.8 Hz), 3.2 (m, 1H), 3.35 (m, 1H),
3.6 and 3.65 (s, 2H each), 3.7 and 3.77 (s, 3H each), 6.7-6.8
(m, 4H), 6.9-6.96 and 7.01-7.06 (m, 2H each), 7.16-7.22 (m,
4H); HRMS m/z 651.3328 calculated for C37H50O2N3S2F, M+
+ 1, 652.3145.
2-[[2-[[[3-(4-Br om oph en yl)-8-m eth yl-8-azabicyclo[3.2.1]-
oct-2-yl]m eth yl][[S-(4-m eth oxyben zyl)th io]eth yl]a m in o]-
eth yl]a m in o]-S-(4-m eth oxyben zyl)eth a n eth iol (11c): yield
1
68%; IR (CHCl3) 1603 cm-1; H NMR (CDCl3) δ 1.4-1.7 (m,
5H), 1.95-2.29 (m, 5H), 2.25-2 (m, 10H), 2.7-2.9 (m, 5H),
3.0 (td, 1H, J 1 ) 4 Hz, J 2 ) 8.8 Hz), 3.2 (m, 1H), 3.35 (m, 1H),
3.6 and 3.65 (s, 2H each), 3.7 and 3.77 (s, 3H each), 6.7-6.8
(m, 4H), 6.93 (d, 2H, J ) 8.4 Hz), 7.15-7.25 (m, 4H), 7.3 (d,
2H,
J ) 9.1 Hz); HRMS m/z 711.2528 calculated for
C37H50O2N3S2Br, M+ + 1, 712.2626.
Gen er a l P r oced u r e for Com p ou n d s 10 a n d 12a -c.
Substrate 9a or 11a -c (1 mmol) was dissolved in TFA (7.5
mL) and anisole (0.25 mL) at 0 °C, and Hg(OAc)2 (636 mg, 2
mmol) was added. The resulting mixture was stirred for 30
min and concentrated in vacuo to obtain a viscous oil that was
dried in vacuo for 30 min. Dry ether (10 mL) was then added
to the above oil, and the resulting suspension was sonicated
for 5 min. The colorless solid that formed was collected by
suction filtration, dried in vacuo for 20 min, and dissolved in
absolute EtOH (10 mL). H2S gas was passed through the
solution for 20 min, and the reaction mixture was filtered
through a pad of Celite. The filtrate was concentrated in vacuo
to obtain the trifluoroacetate salts of the title compounds,
which were used for further reactions without additional
purification.
2-[[[3-(4-Ch lor op h en yl)-8-m et h yl-8-a za b icyclo[3.2.1]-
oct-2-yl]m eth yl][2-[S-(4-m eth oxyben zyl)th io]eth yl]am in o]-
N-[2-[S-(4-m eth oxyben zyl)th io)eth yl]a cta m id e-[1R-(exo-
exo)] (9a ): yield 53%; IR (CHCl3) 1654 cm-1; 1H NMR (CDCl3)
δ 1.4-1.8 (m, 5H), 2.05-2.9 (m, 13H), 2.92-3.5 (m, 7H), 3.52
(s, 2H), 3.95 (s, 2H), 3.76 (s, 6H), 6.8-6.85 (m, 4H), 6.9 (d, 2H,
P r ep a r a tion of Com p ou n d s 17a ,b. A solution of com-
pound 12a or 12b (1 mmol) in MeOH (10 mL) was added to a
solution of [Bu4N][ReOCl4]41 (588 mg, 1 mmol) in MeOH (2
mL) under N2 at 0 °C. Et3N (0.5 mL, 4 mmol) was then added,
and the resulting mixture was stirred at room temperature
for 12 h. The reaction mixture was concentrated in vacuo. The
residue obtained was chromatographed on silica gel (EtOAc:
MeOH:NH4OH, 9.5:0.4:0.1) to obtain a purple solid, which was
recrystallized (MeOH:CH2Cl2) to give pinkish needles.
2â-{Oxo[N,N′-bis(2-m er captoeth yl)eth ylen ediam in ato]-
r h en iu m (V)m et h yl}-3â-(4-ch lor op h en yl)t r op a n e (17a ):
yield 43%; IR (KBr) 933 cm-1; HRMS m/z 627.1154, calculated
for C21H31ON3S2ClRe, M+ + 1, 628.1249.
2â-{Oxo[N,N′-bis(2-m er captoeth yl)eth ylen ediam in ato]-
r h en iu m (V)m et h yl}-3â-(4-flu or op h en yl)t r op a n e (17b ):
yield 49%; IR (KBr) 938 cm-1; HRMS m/z 611.1450, calculated
for C21H31ON3S2FRe, M+ + 1, 612.1569.
Ra d iola belin g w ith Tc-99m . The appropriate ligand
(12a -c or 10; 0.2-0.4 µmol) was dissolved in 100 µL of EtOH
and 100 µL of HCl (1 N). HCl (500 µL, 1 N), 1 mL of
Sn-glucoheptonate solution (containing 136 µg of SnCl2 and
200 µg of Na-glucoheptonate, pH 6.67), and 50 µL of EDTA
solution (0.1 N) were successively added. [99mTc]Pertechnetate
(100-200 µL, range of 1-20 mCi) in saline solution was then
added. The reaction mixture was heated for 30 min at 100 °C
(or heated at 125 °C in an autoclave for 30 min), cooled to room
J
) 6 Hz), 7.15-7.25 (m, 6H), 8.4 (brs, 1H); HRMS m/z
681.2825 calculated for C37H48O3N3S2Cl, M+ + 1, 682.2927.
2-[[[3-(4-F lu or op h en yl)-8-m et h yl-8-a za b icyclo[3.2.1]-
oct-2-yl]m eth yl][2-[S-(4-m eth oxyben zyl)th io]eth yl]am in o]-
N-[2-[S-(4-m eth oxyben zyl)th io]eth yl]a cta m id e-[1R-(exo-
exo)] (9b): yield 66%; IR (CHCl3) 1650 cm-1; 1H NMR (CDCl3)
δ 1.34-1.72 (m, 5H), 2.0-2.77 (m, 13H), 2.82-3.48 (m, 7H),
3.53 (s, 2H), 3.66 (s, 2H), 3.71 (s, 6H), 6.79-6.85 (m, 4H), 6.9-
7.03 (m, 2H), 7.18-7.45 (m, 6H), 8.4 (brs, 1H); HRMS m/z
665.3121 calculated for C37H48O3N3S2F, M+ + 1, 666.2927.
2-[[[3-(4-Br om oph en yl)-8-m eth yl-8-azabicyclo[3.2.1]oct-
2-yl]m et h yl][2-[S-(4-m et h oxyb en zyl)t h io]et h yl]a m in o]-
N-[2-[S-(4-m eth oxyben zyl)th io]eth yl]a cta m id e-[1R-(exo-
exo)] (9c): yield 56%; IR (CHCl3) 1648 cm-1; 1H NMR (CDCl3)
δ 1.38-1.76 (m, 5H), 2.0-2.81 (m, 13H), 2.88-3.46 (m, 7H),
3.58 (s, 2H), 3.68 (s, 2H), 3.77 (s, 6H), 6.76-6.82 (m, 4H), 6.93
(d, 2H, J ) 8.7 Hz), 7.13-7.21 (m, 4H), 7.34 (d, 2H, J ) 8.7
Hz), 8.4 (brs, 1H); HRMS m/z 725.2320 calculated for
C37H48O3N3S2Br, M+ + 1, 726.2110.
P r ep a r a tion of Com p ou n d s 11a -c. BH3‚THF (1.5 equiv)
was added to a solution of compounds 9a -c (1 mmol) in THF
(10 mL) under N2, and the resulting mixture was heated at
reflux for 12 h. Workup of this reaction mixture was carried
out as described for compounds 8a -c followed by chromatog-
raphy of the resulting product on a chromatotron (EtOAc:
MeOH:NH4OH, 8.5:1:0.5), producing the title compound.
2-[[2-[[[3-(4-Ch lor oph en yl)-8-m eth yl-8-azabicyclo[3.2.1]-
oct-2-yl]m eth yl][[S-(4-m eth oxyben zyl)th io]eth yl]a m in o]-
eth yl]a m in o]-S-(4-m eth oxyben zyl)eth a n eth iol (11a ): yield
temperature, and neutralized with
a saturated NaHCO3
solution. After the complex was extracted from the aqueous
reaction medium with ethyl acetate (1 × 3 mL, 2 × 1.5 mL)
and passed through a small column of Na2SO4, ethyl acetate
was removed under a flow of N2. The residue was dissolved
in 200 µL of EtOH and purified by HPLC (PRP-1 column, 250
× 4.1 mm, CH3CN/3,3-dimethylglutarate buffer, 5 mM, pH 7,
volume ratio 8:2, flow rate 1 mL/min). The retention times
for the stereoisomers of 13 were 10.5 and 11.5 min (radio-
chemical yield 88%, radiochemical purity >98%). All of the
complexes displayed in vitro stability at 4 and 24 h after
preparation. Little change in radiochemical purity was ob-
served. Identical labeling and HPLC conditions were used for
1
71%; IR (CHCl3) 1609 cm-1; H NMR (CDCl3) δ 1.5-1.8 (m,
5H), 2.05-2.3 (m, 5H), 2.3 (s, 3H), 2.3-2.6 (m, 7H), 2.7-2.9
(m, 5H), 3.1 (td, 1H, J 1 ) 4 Hz, J 2 ) 8.8 Hz), 3.35-3.45 (m,
2H), 3.59 (s, 2H), 3.65-3.7 (m, 2H), 3.76 (s, 6H), 6.8-6.85 (m,

16 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1
Meegalla et al.
the preparation of mixtures of 14-16 (retention times of 9.4
and 10.1, 14.2 and 15.6, and 7.8 and 8.9 min, respectively)
with radiochemical yields of 70%, 88%, and 80%, respectively,
and radiochemical purities of >98%.
scans of the same baboon. The coregistration program simul-
taneously displayed three orthogonal views of either the MRI
or the SPECT scans (coronal, transaxial, and sagittal views).
When alignment was achieved, images of SPECT and MRI
were displayed.
P a r tition Coefficien ts. Partition coefficients were mea-
sured by mixing each Tc-99m compound with 3 g each of
1-octanol and buffer (pH 7.0, 0.1 M phosphate) in a test tube.
The test tube was vortexed for 3 min at room temperature and
then centrifuged for 5 min. Two weighted samples (0.5 g each)
from the 1-octanol and buffer layers were counted in a well
counter. The partition coefficient was determined by calculat-
ing the ratio of cpm/g of 1-octanol to that of buffer. Samples
from the 1-octanol layer were repartitioned until consistent
partition coefficient values were obtained. The measurement
was repeated three times.
Biod istr ibu tion in Ra ts. Male Sprague-Dawley rats
(225-300 g), allowed free access to food and water, were used
for in vivo biodistribution studies.42,43 While under ether
anesthesia, 0.2 mL of a saline solution containing 13, 14, 15,
or 16 (50-100 µCi) was injected directly into the femoral vein
of rats, and the rats were sacrificed by cardiac excision at
various time points postinjection. The organs of interest were
removed and weighed, and the radioactivity was counted with
an automatic gamma counter (Packard 5000). The percentage
dose per organ was calculated by a comparison of the tissue
counts to suitably diluted aliquots of the injected material.
Total activities of blood and muscle were calculated under the
assumption that they were 7% and 40% of the total body
weight, respectively.
Regional brain distribution in rats was obtained after an
injection of 13, 14, 15, or 16. Samples from different brain
regions (cortex, striatum, hippocampus, and cerebellum) were
dissected, weighed, and counted, and the percentage dose per
gram of sample was calculated by comparing the sample
counts with the count of the diluted initial dose. The uptake
ratio of each region was obtained by dividing the percentage
dose per gram of that region by that of the cerebellum. For
blocking studies, rats were injected with either â-CIT or
haloperidol (iv, 1 mg/kg) 5 min prior to the injection of 13.
The rats were dissected, and brain tissue samples were
counted as described above. The specific uptake of the
compound was expressed as a ratio of ST/CB (percentage
dose/g of striatum divided by percentage dose/g of cerebellum).
SP ECT a n d MRI Im a gin g in a Ba boon . A baboon (∼15
kg) was the subject of a SPECT imaging study. Prior to
imaging, the animal was fasted, immobilized with ketamine
(10-20 mg/kg, im) and xylazine (2-3 mg/kg, im), intubated,
and maintained on an 1.5-2.0% isofluorane/98.5% oxygen
mixture (flow rate of 200-500 cc/min). The animal was
injected with glycopyrrolate (10 µg/kg, sc), an anticholinergic
drug that does not cross the blood-brain barrier, in order to
decrease digestive and respiratory secretions. Body temper-
ature was maintained using a hot water-circulating heating
pad and monitored with a rectal thermometer. The animal’s
head was immobilized using a vacuum-packed bean-bag device
that hardens upon evacuation when molded around the head.
No-carrier-added [99mTc]TRODAT-1 (13; 9 mCi) was adminis-
tered as an iv bolus in the saphenous vein of the baboon.
Immediately after injection, sequential 5 min/frame dynamic
SPECT scans were acquired on a triple-headed Picker Prism
3000 camera (fwhm: 7 mm) equipped with fan beam collima-
tors for 2 h. The acquisition parameters were a 20% energy
window at 140 keV, 120 projection angles over 360°, a 128 ×
128 matrix, and a zoom factor of 1.78 in a slice thickness of 2
mm cubic voxels. The projection data were reconstructed with
a count-dependent 3-D Wiener filter. Chang’s first-order
correction method was used to compensate for attenuation.
Magnetic resonance images (MRI) of the baboon brain were
acquired with a 1.5 T machine (GE Medical Systems, Milwau-
kee, WI). The spoiled GRASS acquisition parameters for T1
weighted images included a repetition time (TR) of 5 ms and
a flip angle of 35°, which produced 1 mm thick slices. The
data were reinterpolated in 256 × 256 matrices with cubic
voxels of 2 mm/side to match the SPECT images. The SPECT
images of [99mTc]TRODAT-1 (13) at 60-90 min postinjection
were summed and reformatted to coregister with the MRI
Ack n ow led gm en t. This work was supported by
grants awarded by the National Institutes of Health
(H.F.K.; NS24538 and NS18509) and the Department
of Energy (H.F.K.; ER61657). The authors thank Ms.
Susan West for her editorial assistance.
Refer en ces
(1) Carroll, F. I.; Scheffel, U.; Dannals, R. F.; Boja, J . W.; Kuhar,
M. J . Development of imaging agents for the dopamine trans-
porter. Med. Res. Rev. 1995, 15, 419-444.
(2) Carroll, F. I.; Mascarella, S. W.; Kuzemko, M. A.; Gao, Y.;
Abraham, P.; Lewin, A. H.; Boja, J . W.; Kuhar, M. J . Synthesis,
ligand binding, and QSAR (CoMFA and classical) study of 3â-
(3′-substituted phenyl)-, 3â-(4′-substituted phenyl)-, and 3â-(3′,4′-
disubstituted phenyl)tropane-2â-carboxylic acid methyl esters.
J . Med. Chem. 1994, 37, 2865-2873.
(3) Carroll, F. I.; Kotian, P.; Dehghani, A.; Gray, J . L.; Kuzemko,
M. A.; Parham, K. A.; Abraham, P.; Lewin, A. H.; Boja, J . W.;
Kuhar, M. J . Cocaine and 3â-(4′-substituted phenyl)tropane-2â-
carboxylic acid ester and amide analogues. New high-affinity
and selective compounds for the dopamine transporter. J . Med.
Chem. 1995, 38, 379-388.
(4) Volkow, N. D.; Fowler, J . S.; Gatley, S. J .; Logan, J .; Wang, G.-
J .; Ding, Y.-S.; Dewey, S. PET evaluation of the dopamine system
of the human brain. J . Nucl. Med. 1996, 37, 1242-1253.
(5) Yu, D.-W.; Gatley, S. J .; Wolf, A. P.; MacGregor, R. R.; Dewey,
S. L.; Fowler, J . S.; Schlyer, D. J . Synthesis of carbon-11-labeled
iodinated cocaine derivatives and their distribution in baboon
brain measured using positron emission tomography. J . Med.
Chem. 1992, 35, 2178-2183.
(6) Fowler, J . S.; Volkow, N. D.; MacGregor, R. R.; Logan, J .; Dewey,
S. L.; Gatley, S. J .; Wolf, A. P. Comparative PET studies of the
kinetics and distribution of cocaine and cocaethylene in baboon
brain. Synapse 1992, 12, 220-227.
(7) Frost, J . J .; Rosier, A. J .; Reich, S. G.; Smith, J . S.; Ehlers, M.
D.; Snyder, S. H.; Ravert, H. T.; Dannals, R. F. Positron emission
tomographic imaging of the dopamine transporter with [11C]-
WIN 35,428 reveals marked declines in mild Parkinson’s disease.
Ann. Neurol. 1993, 34, 423-431.
(8) Wong, D. F.; Yung, B.; Dannals, R. F.; Shaya, E. K.; Ravert, H.
T.; Chen, C. A.; Chan, B.; Folio, T.; Scheffel, U.; Ricaurte, G. A.;
Neumeyer, J . L.; Wagner, H. N., J r.; Kuhar, M. J . In vivo
imaging of baboon and human dopamine transporters by positron
emission tomography using [11C]WIN35,428. Synapse 1993, 15,
130-142.
(9) Meltzer, P. C.; Liang, A. Y.; Brownell, A.-L.; Elmaleh, D. R.;
Madras, B. K. Substituted 3-phenyltropane analogs of cocaine:
synthesis, inhibition of binding at cocaine recognition sites, and
positron emission tomography imaging. J . Med. Chem. 1993, 36,
855-862.
(10) Innis, R. B.; Seibyl, J . P.; Scanley, B. E.; Laruelle, M.; Abi-
Dargham, A.; Wallace, E.; Baldwin, R. M.; Zea-Ponce, Y.; Zoghbi,
S.; Wang, S.; et al. Single photon emission computed tomographic
imaging demonstrates loss of striatal dopamine transporters in
Parkinson’s disease. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
11965-11969.
(11) Seibyl, J . P.; Laruelle, M. A.; Van Dyck, C. H.; Wallace, E.;
Baldwin, R. M.; Zoghbi, S. S.; Zea-Ponce, Y.; Neumeyer, J . L.;
Charney, D. S.; Hoffer, P. B.; Innis, R. B. Reproducibility of
iodine-123-â-CIT SPECT brain measurement of dopamine trans-
porters. J . Nucl. Med. 1996, 37, 222-228.
(12) Kuikka, J . T.; Akerman, K.; Bergstrom, K. A.; Karhu, J .;
Hiltunen, J .; Haukka, J .; Heikkinen, J .; Tiihonen, J .; Wang, S.;
Neumeyer, J . L. Iodine-123-labeled N-(2-fluoroethyl)-2â-car-
bomethoxy-3â-(4-iodophenyl)nortropane for dopamine trans-
porter imaging in the living human brain. Eur. J . Nucl. Med.
1995, 22, 682-686.
(13) Neumeyer, J . L.; Wang, S.; Gao, Y.; Milius, R. A.; Kula, N. S.;
Campbell, A.; Baldessarini, R. J .; Zea-Ponce, Y.; Baldwin, R. M.;
Innis, R. B. N-ω-Fluoroalkyl analogs of (1R)-2â-carbomethoxy-
3â-(4-iodophenyl)-tropane (â-CIT): radiotracers for positron
emission tomography and single photon emission computed
tomography imaging of dopamine transporters. J . Med. Chem.
1994, 37, 1558-1561.
(14) Mozley, P. D.; Stubbs, J . B.; Kim, H.-J .; McElgin, W.; Kung, M.-
P.; Meegalla, S.; Kung, H. F. Dosimetry of an iodine-123-labeled
tropane to image dopamine transporters. J . Nucl. Med. 1996,
37, 151-159.

New Tc-99m-Labeled DA Transporter-Imaging Agent
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1 17
(15) Abi-Dargham, A.; Gandelman, M. S.; DeErausquin, G. A.; Zea-
Ponce, Y.; Zoghbi, S. S.; Baldwin, R. M.; Laruelle, M.; Charney,
D. S.; Hoffer, P. B.; Neumeyer, J . L.; Innis, R. B. SPECT imaging
of dopamine transporters in human brain with iodine-123-
fluoroalkyl analogs of â-CIT. J . Nucl. Med. 1996, 37, 1129-1133.
(16) Kilbourn, M. R. In vivo binding of [18F]GBR13119 to the brain
dopamine uptake system. Life Sci. 1988, 42, 1347-1353.
(30) O’Neil, J . P.; Wilson, S. R.; Katzenellenbogen, J . A. Preparation
and structural characterization of monoamine-monoamide bis-
(thiol) oxo complexes of technetium(V) and rhenium(V). Inorg.
Chem. 1994, 33, 319-323.
(31) J urisson, S. S.; Pirro, J .; Di Rocco, R. J .; Rosenspire, K. C.;
J agoda, E.; Nanjappan, P.; Eckelman, W. C.; Nowotnik, D. P.;
Nunn, A. D. Boronic acid adducts of technetium dioxime (BATO)
complexes derived from quinuclidine benzilate (QNB) boronic
acid stereoisomers: syntheses and studies of their binding to
the muscarinic acetylcholine receptor. Nucl. Med. Biol. 1995, 22,
269-281.
(32) Meegalla, S. K.; Plo¨ssl, K.; Kung, M.-P.; Stevenson, D. A.; Liable-
Sands, L. M.; Rheingold, A. L.; Kung, H. F. First example of a
Tc-99m complex as a dopamine transporter imaging agent. J .
Am. Chem. Soc. 1995, 117, 11037-11038.
(33) Madras, B. K.; J ones, A. G.; Mahmood, A.; Zimmerman, R. E.;
Garada, B.; Holman, B. L.; Davison, A.; Blundell, P.; Meltzer,
P. C. Technepine: a high affinity technetium-99m probe to label
the dopamine transporter in brain by SPECT imaging. Synapse
1996, 22, 239-246.
(34) Carroll, F. I.; Abraham, P.; Lewin, A. H.; Parham, K. A.; Boja,
J . W.; Kuhar, M. Isopropyl and phenyl esters of 3â-(4-substituted
phenyl)tropane-2â-carboxylic acids. Potent and selective com-
pounds for the dopamine transporter. J . Med. Chem. 1992, 35,
2497-2500.
(35) Oya, S.; Plo¨ssl, K.; Kung, M.-P.; Stevenson, D. A.; Kung, H. F.
Small and neutral TcO(V) BAT, bisaminoethanethiol (N2S2)
complexes for developing new brain imaging agents. Nucl. Med.
Biol. 1996, in press.
(36) Lever, S. Z.; Baidoo, K. E.; Mahmood, A. Structure proof of syn/
anti isomerism in N-alkylated diaminedithiol (DADT) complexes
of technetium. Inorg. Chim. Acta 1990, 176, 183-184.
(37) Mahmood, A.; Halpin, W. A.; Baidoo, K. E.; Swigart, D. A.; Lever,
S. Z. Synthesis and characterization of N-ethyl-diaminedithiol
oxotechnetate (V): a potential lung imaging agent. In Techne-
tium and Rhenium in Chemistry and Nuclear Medicine; Nicolini,
M., Bandoli, G., Mazzi, U., Eds.; Raven Press: New York, 1990;
p 113.
(17) Innis, R. B. Single photon emission tomography imaging of
dopamine terminal innervation:
a potential clinical tool in
Parkinson’s disease [editorial; review]. Eur. J . Nucl. Med. 1994,
21, 1-5.
(18) Goodman, M. M.; Kung, M.-P.; Kabalka, G. W.; Kung, H. F.;
Switzer, R. Synthesis and characterization of radioiodinated
N-(3-iodopropen-1-yl)-2â-carbomethoxy-3â-(4-chlorophenyl)tro-
panes: potential dopamine reuptake site imaging agents. J . Med.
Chem. 1994, 37, 1535-1542.
(19) Malison, R. T.; Vessotskie, J . M.; Kung, M.-P.; McElgin, W.;
Romaniello, G.; Kim, H.-J .; Goodman, M. M.; Kung, H. F.
Striatal dopamine transporter imaging in nonhuman primates
with iodine-123-IPT SPECT. J . Nucl. Med. 1995, 36, 2290-2297.
(20) Kung, M.-P.; Essman, W. D.; Frederick, D.; Meegalla, S.;
Goodman, M.; Mu, M.; Lucki, I.; Kung, H. F. IPT: a novel
iodinated ligand for the CNS dopamine transporter. Synapse
1995, 20, 316-324.
(21) J urisson, S. S.; Berning, D.; J ia, W.; Ma, D.-S. Coordination
compounds in nuclear medicine. Chem. Rev. 1993, 93, 1137-
1156.
(22) Steigman, J .; Eckelman, W. C. The Chemistry of Technetium In
Medicine;. National Academy: Washington, DC, 1992.
(23) Lever, S. Z.; Baidoo, K. E.; Mahmood, A.; Matsumura, K.;
Scheffel, U. A.; Wagner, H. N., J r. Novel technetium ligands with
affinity for the muscarinic cholinergic receptor. Nucl. Med. Biol.
1994, 21, 157-164.
(24) Del Rosario, R. B.; J ung, Y.-W.; Baidoo, K. E.; Lever, S. Z.;
Wieland, D. M. Synthesis and in vivo evaluation of
99m/99Tc]-DADT-benzovesamicol: a potential marker for cholin-
ergic neurons. Nucl. Med. Biol. 1994, 21, 197-203.
a
[
(25) Chi, D. Y.; O’Neil, J . P.; Anderson, C. J .; Welch, M. J .; Katzenel-
lenbogen, J . A. Homodimeric and heterodimeric bis(aminothiol)
oxometal complexes with rhenium(V) and technetium(V). Control
of heterodimeric complex formation and an approach to metal
complexes that mimic steroid hormones. J . Med. Chem. 1994,
37, 928-937.
(38) Wolff, J . A. Massachusetts Institute of Technology Thesis, MIT,
MA, 1991.
(39) Kung, H. F.; Kim, H.-J .; Kung, M.-P.; Meegalla, S. K.; Plo¨ssl,
K.; Lee, H.-K. Imaging of dopamine transporters in humans with
technetium-99m TRODAT-1. Eur. J . Nucl. Med. 1996, 23, 1527-
1530.
(26) Chi, D. Y.; Katzenellenbogen, J . A. Selective formation of
heterodimeric bis-bidentate aminothiol-oxometal complexes of
rhenium(V). J . Am. Chem. Soc. 1993, 115, 7045-7046.
(27) DiZio, J . P.; Fiaschi, R.; Davison, A.; J ones, A. G.; Katzenellen-
bogen, J . A. Progestin-rhenium complexes: metal-labeled ste-
roids with high receptor binding affinity, potential receptor-
directed agents for diagnostic imaging or therapy. Bioconjugate
Chem. 1991, 2, 353-366.
(28) DiZio, J . P.; Anderson, C. J .; Davison, A.; Ehrhardt, G. J .;
Carlson, K. E.; Welch, M. J .; Katzenellenbogen, J . A. Techne-
tium- and rhenium-labeled progestins: synthesis, receptor bind-
ing and in vivo distribution of an 11-â-substituted progestin
labeled with technetium-99 and rhenium-186. J . Nucl. Med.
1992, 33, 558-569.
(40) Ell, P. J . Milestones [editorial]. Eur. J . Nucl. Med. 1996, 23,
1441.
(41) Alberto, R.; Schibli, R.; Egli, A.; Schubiger, P. A.; Herrmann,
W. A.; Artus, G.; Abram, U.; Kaden, T. A. Metal carbonyl
syntheses XXII. Low pressure carbonylation of [MOCl4]- and
[MO4]-: the technetium(I) and rhenium(I) complexes [NEt4]2-
[MCl3(CO)3]. J . Organomet. Chem. 1995, 493, 119-127.
(42) Kung, H. F.; Molnar, M.; Billings, J .; Wicks, R.; Blau, M.
Synthesis and biodistribution of neutral lipid-soluble Tc-99m
complexes that cross the blood-brain barrier. J . Nucl. Med. 1984,
25, 326-332.
(43) Kung, H. F.; Yu, C. C.; Billings, J .; Molnar, M.; Blau, M.
Synthesis of new bis-aminoethanethiol (BAT) derivatives: pos-
sible ligands for Tc-99m brain imaging agents. J . Med. Chem.
1985, 28, 1280-1284.
(29) O’Neil, J . P.; Carlson, K. E.; Anderson, C. J .; Welch, M. J .;
Katzenellenbogen, J . A. Progestin radiopharmaceuticals labeled
with technetium and rhenium: synthesis, binding affinity, and
in vivo distribution of a new progestin N2S2-metal conjugate.
Bioconjugate Chem. 1994, 5, 182-193.
J M960532J