Technetium-99m SPECT imaging agent which targets the dopamine transporter in primate brain

Article abstract of DOI:10.1021/jm960868t

The dopamine transporter (DAT), located presynaptically on dopamine neurons, provides a marker for certain neurological diseases. In particular, the DAT is depleted in Parkinson's disease, and the extent of depletion correlates with the loss of dopamine.

Full text of DOI:10.1021/jm960868t

J . Med. Chem. 1997, 40, 1835-1844
1835
A Tech n etiu m -99m SP ECT Im a gin g Agen t Wh ich Ta r gets th e Dop a m in e
Tr a n sp or ter in P r im a te Br a in
Peter C. Meltzer,*,† Paul Blundell,† Alun G. J ones,‡ Ashfaq Mahmood,‡ Basem Garada,‡ Robert E. Zimmerman,‡
Alan Davison,§ B. Leonard Holman,‡ and Bertha K. Madras|
Organix Inc., 65 Cummings Park, Woburn, Massachusetts 01801, Department of Psychiatry,
Harvard Medical School and New England Regional Primate Center, Southborough, Massachusetts 01772,
Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115,
and Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received April 2, 1997X
The dopamine transporter (DAT), located presynaptically on dopamine neurons, provides a
marker for certain neurological diseases. In particular, the DAT is depleted in Parkinson’s
disease, and the extent of depletion correlates with the loss of dopamine. Herein we describe
the design, synthesis, and biological evaluation of technepine, the first 99mTc-labeled SPECT
imaging agent which targets the dopamine transporter in striatum. We have demonstrated
that the DAT can accommodate a chelating unit attached to the 8-amine function of a tropane
skeleton. Further, we have demonstrated for the first time that a molecule can be designed to
carry the radionuclide 99mTc across the blood-brain barrier in sufficient quantity to obtain in
vivo images of the striatum in monkeys. This advance will undoubtedly lead to the design of
new receptor and transporter-mediated 99mTc agents which can label specific transporter and
receptor targets in the central nervous system.
In tr od u ction
eV γ emission). 99mTc offers important advantages,
particularly the ability to generate the isotope in the
laboratory from a commercially available kit and with-
out need of a cyclotron. Consequently, 99mTc-based
compounds are widespread in clinical use17,18 as, for
example, blood perfusion agents19 and cardiovascular
imaging agents.20 However, while iodine can be at-
tached covalently to the specific ligand, 99mTc must be
The dopamine transporter (DAT), located presynap-
tically on dopamine neurons, is a principal target of
cocaine in the mammalian brain.1,2 The stimulant and
reinforcing properties of cocaine have been ascribed to
cocaine’s ability to inhibit the DAT.3-6 The DAT also
provides a marker for certain neurological diseases. In
particular, Madras and others have shown in primates,
as well as in in vitro studies of human Parkinsonian
patients,7,8 that the DAT is depleted in Parkinson’s
disease (PD) and the extent of depletion correlates with
the loss of dopamine.
Considerable research has focused on the design of
positron emission tomography (PET) and single photon
emission computed tomography (SPECT) imaging agents
which could provide a quantitative measure of the DAT
density in living brain.9-13 The lead compound and a
widely used PET imaging agent is WIN35,428.8,9,14
SPECT imaging agents, however, have greater clinical
potential than do PET agents, and in 1991 the synthesis
of [123I]RTI55 (âCIT), an analog of WIN35,428, was
reported.15 RTI55 has an affinity (IC50) of 1.08 nM and
selectivity for the DAT over the serotonin (5HT) trans-
porter (SERT) of 2.12
bound by means of a chelating agent. Consequently,
99m
notwithstanding considerable effort21-26 to design a
-
Tc-chelated imaging agent which can image specific
receptor or transporter systems, no such agent had been
demonstrated to be successful in vivo.44 Herein we
report the design and synthesis of the first transporter-
mediated technium-99m-based in vivo SPECT imaging
agent, [N-[2-((3′-N′-propyl-3′′â-(4-fluorophenyl)tropane-
2′′â-carboxylic acid methyl ester)(2-mercaptoethyl)ami-
no)acetyl]-2-aminoethanethiolato] technetium-99m(V)
oxide, technepine, 1 (Figure 1), which labels the DAT
in primates. Technepine was first disclosed in a com-
munication in 1996.27
Ch em istr y
The imaging agent 1 (technepine; O-861T) is com-
posed of three parts: (i) a potent and selective tropane,
(ii) a chelating unit to which the radionuclide 99mTc is
bound, and (iii) a tether which connects the chelating
entity to the tropane skeleton. The synthetic route
developed to obtain the target compound 1 is presented
in Scheme 1.
The final compounds reported here are, with the
exception of technepine itself, rhenium complexes. Rhe-
nium is an excellent model for the radioactive 99mTc. It
forms square pyramidal complexes very similar to those
formed by 99mTc and behaves similarly to technetium.
Its N2S2 complexes are equally stable.28 Furthermore,
the lipophilicity and the biological availability of rhe-
nium chelates has proved similar to that of 99mTc
chelates.28 Consequently, it has been utilized in place
We recently introduced altropane,11,16 an N-[123I]-
iodoallyl analog of WIN35,428, as a novel SPECT
imaging agent. This compound binds potently (IC50
6.9 nM) and selectively (DAT:SERT ) 28) to the DAT.
It has an in vivo striatum to cerebellum ratio of 8 in
monkey brains and provides excellent SPECT images
in humans. Altropane is currently in human trials.
)
The most widely accepted radionuclide in nuclear
medicine is technetium-99m (99mTc; T1/2 ) 6.0 h, 140 K
† Organix, Inc.
‡ Brigham and Women’s Hospital.
§ Massachusetts Institute of Technology.
| Harvard Medical School and New England Regional Primate
Center.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(96)00868-0 CCC: $14.00 © 1997 American Chemical Society

1836 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Meltzer et al.
Ta ble 1. Inhibition of [3H]-3â-(4-Fluorophenyl)tropane-2â-
carboxylic Acid Methyl Ester Binding to the Dopamine
Transporter (DAT) and [3H]Citalopram Binding to the
Serotonin Transporter (SERT) by Rhenium Analogs 11-13
in Cynomolgus Monkey Caudate-Putamena
F igu r e 1. Structure of technepine, 1, and the three “guiding
ligands”.
IC50 (nM)
dopamine
serotonin
transporter
Sch em e 1
transporter
selectivity
compd
O-861, 11
[3H]WIN35,428 [3H]citalopram DAT/SERT
5.99 ( 0.81
7.38 ( 1.33
4.04 ( 0.98
37.2 ( 3.4
616 ( 88
482
124 ( 17
66.9 ( 3.0
299 ( 23
264 ( 16
55200 ( 20000
21
9
74
7.1
90
O-927, 11a
O-928, 11b
O-863,b 12
O-864,b 13
O-918, 13a
O-919, 13b
O-862,b 14
(2R-COOCH3)
343
2960 ( 157
5020 ( 1880
1.7
a
Each radioligand was incubated with tissue (4 mg/mL original
wet tissue weight) and 7-14 concentrations of a cocaine congener
as described in the experimental section. Nonspecific binding of
[3H]WIN35,428 was measured with 30 µM (-)-cocaine and of
[3H]citalopram with 1 µM fluoxetine. IC50 values were computed
by the EBDA computer program and are the means ((SD) or SEM
of one to four independent experiments, each conducted in
b
triplicate. Madras et al. Synapse 1996, 22, 239-246.
The N2S2 ligand (MAMA′) 15, as described by O’Neil
et al.30 and DiZio et al.,29,31 was attached to 1-chloro-
3-propyl triflate in methylene chloride to provide 16 in
72% yield. Rhenium could then be introduced directly
upon reaction of the bistrityl-protected compound 16
with sodium perrhenate under reductive conditions
(SnCl2) in ethanol. A 48% yield of the chelate 18 was
obtained after flash column chromatography. The IR
stretching frequency for 18 shows a strong band at 952
cm-1. This compares favorably with a stretching fre-
quency of 955 cm-1 reported by O’Neil et al.30 for their
N-benzyl syn analog (syn with respect to the RedO core).
In contrast, the anti N-benzyl analog shows a strong
of 99mTc in studies which do not require the presence of
a radiolabel.29 A critical advantage is that rhenium can
be handled under normal laboratory conditions since it
is not radioactive.
Synthesis of the final compounds 11-14 (Table 1)
reported here was therefore facilitated by the use of cold
rhenium. Three pathways were elaborated in order to
establish routes for rapid introduction of rhenium and/
or technetium-99m (Scheme 1).
band at 980 cm-1 32
No anti isomer was evident for 18.
.
1H-NMR decoupling experiments confirmed the syn
relationship of the N-alkyl linker of 18. Thus the triplet
at δ 3.64 (J ) 6 Hz) assigned to the methylene protons
R to the chlorine at C9 was irradiated and confirmed
the position of the C10 protons (m, δ 2.1-2.5, 2H) (see
Figure 2 for compound numbering). The protons at C10

Technetium-99m SPECT Imaging
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1837
to the anisotropic effect of the RedO bond and the
quaternary nature of the nitrogen (N12). The diaste-
reotopy of the C13 protons is evidenced in that the C13â
proton manifests a four-line multiplet [11a , δ 4.79 (d, J
) 16 Hz, 1H H-13â), and 11b, 4.77 (d, J ) 16 Hz, 1H,
H-13â]. The corresponding 13R proton also shows a
four-line multiplet [11a , δ 4.07 (d, J ) 16 Hz, 1H,
H-13R); 11b, 4.04 (d, J ) 16 Hz, 1H, H-13R) with almost
0.8 ppm upfield shift from the 13â.
The effect of the RedO bond is also apparent on the
16R,â protons. The 16R is a four-line multiplet for 11a
at δ ∼4.02 and the 16â is a four line multiplet at δ
∼4.57. In the diastereomeric mixture of 11, the 16â
protons overlap, resulting in a six-line multiplet at δ
∼4.55. It is also interesting that the carbomethoxy
moieties in the 2â position (11, 12, and 13) all show
different chemical shifts (two singlets) for the two
different diastereomers (11, δ 3.44, 3.49; 12, δ 3.50, 3.55;
13, δ 3.65, 3.70). When the carbomethoxy group is in
the 2R configuration, as in 14, both diastereomers have
the same chemical shift, and a singlet (δ 3.52) is seen.
This implies that the chelate in 14 is no longer in a
position to influence the carbomethoxy group. The
X-ray analysis of 11a confirmed (Figure 4) this interac-
tion as, in this case, the 2-CO2Me group is forced toward
the chelate.
F igu r e 2. The two diastereoisomers, 11a and 11b, of the
rhenium analog of technepine, 1.
were then irradiated, and this provided a singlet for
those at C9 at δ 3.64 and simplified the downfield
multiplet at δ 3.7-4.2. (Note: the C11 methylene
protons of the precursor 16 resonate at δ 2.35.) This is
consistent with the findings of Lever et al.32 who report
the N-CH3 syn downfield at δ 3.4 ppm and the N-CH3
anti at δ 1.9, thus implying the C11 syn relationship with
the RedO core, which correlated well with data reported
by O’Neil et al.30 for an N-benzyl analog.
The absolute structure (Figure 4) of diastereoisomer
11a was established by X-ray crystallography and
confirmed that the propyl group attached at N12 bears
a syn relationship to the RedO bond. Further, the
absolute structure of 11a is as shown in Figure 2. The
chirality of the tropane moiety is derived from (1R)-
cocaine and is therefore fixed. However, the introduc-
tion of a chiral rhenium chelate leads to the formation
of these diastereoisomers. Therefore, diastereoisomer
11a is the 1R,12S isomer [(1R,12S)-N-(2-((3′-N′-propyl-
3′′â-(4-fluorophenyl)tropane-2′′â-carboxylic acid methyl
ester)(2-mercaptoethyl)amino)acetyl)-2-aminoethanethi-
olato]rhenium(V) oxide and diastereoisomer 11b is the
1R,12R isomer [(1R,12R)-N-(2-((3′-N′-propyl-3′′â-(4-fluo-
rophenyl)tropane-2′′â-carboxylic acid methyl ester)(2-
mercaptoethyl)amino)acetyl)-2-aminoethanethiolato]rhe-
nium(V) oxide.
The X-ray structural analysis (see below) of 11a shows
the N-alkyl subsitituent with syn geometry and thus
confirms the syn relationship in the intermediate, 18.
The preformed rhenium chelate 18 could then be
attached to an appropriate nortropane. Thus, the
nortropanes12,33 (exemplified in Scheme 1 for WIN35,-
428) were then alkylated by this preformed N2S2 chelate
attached to the propyl halide, to provide the novel
rhenium chelates, 11-14 (Table 1). In all cases, none
of the anti isomers could be detected.
Alternately, the chloropropyl chelator 16 could be
attached directly to the appropriate nortropane by
reaction in acetonitrile in the presence of potassium
carbonate and potassium iodide to provide the bistrityl-
protected chelator 17 in 72% yield. Introduction of
rhenium (or 99mTc) could then be effected. Thus 17 was
treated with sodium perrhenate under reductive condi-
tions (SnCl2) in ethanol. A 30% yield of the chelate 11
was obtained after flash column chromatography.
Compound 11 exists in two diastereomeric forms
(Figure 2), 11a and 11b. These two diastereomers
coelute on TLC in a variety of solvent systems, and
therefore chromatographic separation is difficult. How-
ever, column chromatography with a ratio of silica gel
to compound (ca. 40 g:171 mg) and collection of multiple
small fractions allowed pooling of early versus late
fractions to provide purified samples of each diastere-
oisomer. Diastereomers 13a and 13b could be similarly
isolated. However, in this case TLC could be used to
monitor separation.
Biology
The final compounds reported here are, with the
exception of technepine itself, rhenium complexes.
Since, as noted above, rhenium is an excellent model
for the radioactive 99mTc, the in vitro biological data
reported here were obtained for the rhenium chelates.
Since 99mTc will be introduced in a final preparative step
for routine use, a diastereomeric mixture of the imaging
agents is likely to be used. Therefore the biological
activity of the diastereomeric mixture of 11, the model
for technepine itself, was obtained. In addition, binding
constants for each of the diastereoisomers 11a and 11b
were also measured.
The affinities (IC50) of compound 11-14 for the
dopamine (DAT) and serotonin (SERT) transporters
were determined in competition studies using [3H]-3â-
(4-fluorophenyl)tropane-2â-carboxylic acid methyl ester
([3H]WIN35,428 or [3H]CFT) to label the dopamine
transporter5 and [3H]citalopram to label the serotonin
transporter.27 Studies were conducted in cynomolgus
monkey striatum since these compounds are part of an
Structural assignment of the rhenium chelates was
again facilitated by NMR analysis. The presence of
diastereomeric mixtures was clearly evidenced in the
1H-NMR spectra of these compounds. In this regard,
the C13 protons are particularly diagnostic for the RedO
complexes. For example, in the ligand 16 these protons
appear as a singlet at δ 2.85. In the RedO chelate 11,
both C13 protons appear considerably downfield in the
region of δ 4.0-4.8. This downfield shift is due, in part,

1838 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Meltzer et al.
F igu r e 3. SPECT and MRI image of a rhesus monkey with technepine, 1, as imaging agent, processed 1-1.5 h after injection
of the radioactive probe. Panel 1 shows an MRI image. Panel 2 shows a SPECT image. Panel 3 shows coregistration of the MRI
and SPECT images.
of 1. The first panel shows an MRI image of the brain,
the second shows accumulation of 1 in the striatum, and
the third shows a coregistration of MRI and SPECT
images. Coregistration of the SPECT images obtained
in the coronal plain with coronal images generated by
MRI confirmed the location of the radiolabeled probe
within the striatum at several anterior-to-posterior
planes. The selectivity of technepine was confirmed by
a comparison of striatum versus cerebellum radioactive
labeling and found to be 2:1 in the female monkey and
3:1 in the male monkey.
Discu ssion
F igu r e 4. ORTEP diagram of diastereoisomer 11a .
The focus of this work is the design and synthesis of
99mTc-labeled ligand which targets the DAT in primate
ongoing investigation of structure-activity relationships
at the dopamine transporter in this tissue5,12,33 and
meaningful comparisons with an extensive data base
and in vivo imaging data can be made. Competition
studies were conducted with a fixed concentration of
radioligand and a range of concentrations of the test
drug. All drugs inhibited [3H]WIN35,428 and [3H]-
citalopram binding in a concentration-dependent man-
ner. Binding constants are presented in Table 1.
It is apparent that the WIN35,428 template provides
a potent compound (11: IC50 ) 5.99 ( 0.81 nM). Of
the two diastereomeric forms of O-861 (11), 11b (O-928)
was more potent and conferred the higher level of
transporter selectivity on the molecule. In contrast, 11a
(O-927) was of reduced potency and transporter selec-
tivity.
a
brain. In order to achieve this, four rhenium-labeled
model ligands were prepared, and their binding poten-
cies and selectivities to neurotransmitter uptake sys-
tems (DAT, SERT, NET) were measured. The most
potent and selective of these models was then labeled
with the SPECT radionuclide, 99mTc, to provide tech-
nepine (O-861T; 1), and successful images in primate
brain were obtained. Herein we discuss the design of
technepine as well as the biology of the rhenium
complexes, 11-14. Technepine is the first 99mTc-labeled
chemical entity to enable an in vivo SPECT image in
primate brain. Although others26 have reported the
development of a 99mTc agent that binds with high
affinity to the dopamine transporter, SPECT images
have not yet been published for this compound. These
authors26 later reported that their compound failed to
accumulate in sufficient quantity to obtain an in vivo
image.34
Syn th esis of [99m Tc]Tech n ep in e
Synthesis of technepine, 11 (O-861T: M ) 99mTc),
as a 1:1 diastereomeric mixture, was achieved by
deprotection of the bistritylated compound 17 with
trifluoroacetic acid under cation trapping conditions
(triethylsilane). The bisthiol obtained was treated with
technetium-99m glucoheptonate. The product was pu-
rified by HPLC to provide 6 mCi of the ligand 1 for in
vivo studies.
Design of Tech n ep in e. The stereoselectivity of
transporter systems, and in particular the DAT, imposes
specific constraints on the design of transporter-targeted
imaging agents. Not least among these is the steric
impediment offered by a bulky 99mTc chelating unit
attached to a ligand selected to guide the imaging agent
to its biological target. It may be anticipated that, in
the absence of careful design, the sterically sensitive
uptake mechanism may preclude potent binding and
selectivity for the DAT. The design of technepine
hinged upon the careful selection of a “guiding” tropane
moiety to which a neutral 99mTc chelate could be
attached by a methylene chain linking group. The
ligand is therefore composed of three parts: (i) a tropane
skeleton specifically selected to be both potent and
selective for the DAT (the “guiding ligand”), (ii) a
SP ECT Im a gin g Da ta
SPECT images in rhesus monkeys were obtained over
3 h. The striatum was localized by coregistration of the
SPECT images with Magnetic Resonance Imaging
(MRI). Radioligand accumulated within the striatum
within minutes and was detectable in this region for a
period of 3 h. Figure 3 shows a lateral view of a SPECT
image taken 60-95 min after administration of 7.5 mCi

Technetium-99m SPECT Imaging
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1839
Ta ble 2. Inhibition of [3H]-3â-(4-Fluorophenyl)tropane-2â-
carboxylic Acid Methyl Ester Binding to the Dopamine
Transporter (DAT) and [3H]Citalopram Binding to the
Serotonin Transporter (SERT) by Arylalkyl Analogs 5-10
in Cynomolgus Monkey Caudate-Putamena
chelating unit to which the radionuclide 99mTc (or Re)
is bound (the “chelator”), and (iii) a “tether” which
connects the chelating entity to the tropane skeleton.
(A) Gu id in g Liga n d . Selection of a potent guiding
ligand was aided by structure-activity relationships
(SAR)12,33 of the tropane family of compounds. Specif-
ically, SAR of the tropanes indicated that three tem-
plates might be appropriate (see Figure 1). First, the
3,4-dichloro analog 3 (dichloropane: IC50 ) 0.9 nM)12
of WIN35,428, 2, might provide a ligand of high potency,
albeit low selectivity. Second, the biological data ob-
tained for difluoropine, 4 (IC50 ) 10.9 nM; DAT:SERT
) 325),33 indicated that this compound might provide a
potent and selective guiding ligand for use in a 99mTc-
chelated SPECT agent. Third, WIN35,428, the proto-
typical tropane for DAT inhibition (IC50 ) 11.0 nM;
DAT:SERT ) 15), had already been demonstrated to
provide an exceptional template from which to construct
SPECT imaging agents.11,16 Upon assessment of all
three ligands, the optimum, designated technepine, was
based upon the WIN35,428 template.
IC50 (nM)
dopamine
serotonin
transporter
transporter
selectivity
compd series n [3H]WIN35,428 [3H]citalopram DAT/SERT
O-755, 5
O-747, 6
O-764, 7
O-934, 8
O-932, 9
O-933, 10
Ab
Ab
Ab
B
B
B
1
3
5
1
3
5
223 ( 53
4,970 ( 700
19.7 ( 0.9
550 ( 63
22
0.9
6
18
95
15
22.0 ( 11.9
99.0 ( 28
58.9 ( 1.65
1.4 ( 0.20
3.4 ( 0.83
1073 (n ) 1)
133 ( 7
(B) P oin t of Atta ch m en t. Upon examination of the
parent compounds, 2-4, it is apparent that there are
two obvious potential points of attachment which are
chemically readily accessible. First, the 2-carbomethoxy
moiety which has been demonstrated to tolerate wide
molecular modification35 and, second, the 8-nitrogen
which had not yet been explored as to transporter
tolerance for the substantial bulk envisioned for a
technetium chelate. In order to avoid the potential of
a labile label placed directly at the 2-carbomethoxy
(hydrolyzable ester) position, we elected, first, to focus
attention on the 8-amino moiety.
A model study of both WIN35,428 and difluoropine36
was conducted to explore the available transporter space
in the region of the 8-amine. Six N-arylalkyl analogs,
5-10, were prepared (Table 2). Potency at the dopam-
ine transporter was measured in the striatum of cyno-
molgus monkey brain using [3H]WIN35,428 to label the
sites. Affinity for the serotonin transporter was mea-
sured in the same tissue using [3H]citalopram to label
those sites. In both the WIN35,428 and the difluoropine
family, it was evident that highest affinity for the DAT
was obtained with a propyl linker (IC50 ) 22.0 nM for
6, and IC50 ) 1.4 nM 9). However transporter selectiv-
ity was low in the difluoropine analog (6), but favorable
for the WIN35,428 analog (9, DAT:SERT ) 95). From
these model studies we inferred that reasonable binding
potency could be expected for a suitably selected 99mTc
chelate if it were bound to the tropane by a propyl
tether. We reasoned that the metal chelate, which is
larger than the phenyl group of the model, would then
reside in the transporter space occupied by the phenyl
group of a pentane-linked model, and thus effect binding
minimally.
49.9 ( 10.2
a
Each radioligand was incubated with tissue (4 mg/mL original
wet tissue weight) and 7-14 concentrations of a cocaine congener
as described in the experimental section. Nonspecific binding of
[3H]WIN 35,428 was measured with 30 µM (-)-cocaine and of
[3H]citalopram with 1 µM fluoxetine. IC50 values were computed
by the EBDA computer program and are the means ((SD) of two
to four independent experiments, each conducted in triplicate.
b
Meltzer, P. C.; Liang, A. Y.; Madras, B. K. J . Med. Chem. 1996,
39, 371-379.
ability to cross the blood-brain barrier and bind selec-
tively to its biological target. In contrast, it was
demonstrated that use of a simple N2S2 chelating
unit38,39 provided a reasonable match of lipophilicity for
the steroid. These tetradentate N2S2 ligands give rise
to some interesting characteristics such as syn and
anti geometry, and in the case of a chiral ligand
such as a tropane, the existence of diastereomers as
discussed earlier. We reasoned that the alternate
symmetrical tridentate (and therefore diastereomer-
free) SNS ligands41 require that the tropane be linked
to the chelate via the remaining electron donor. This
was anticipated to lead to a potential lack of in vivo
stability of the chelated ligand, since the exogenous
sulfur could readily be replaced by an endogenous sulfur
(e.g. cysteine). We therefore selected the N2S2 ligand,
MAMA′,30 for use in our studies. Indeed, since comple-
tion of this work, it has been reported that an SNS
ligand has failed to provide an approach for primate
imaging since the amount of label in brain proved too
low (0.1% dose/organ at 2 min post iv injection).34
The affinities (IC50) of compounds 11-14 for the
dopamine (DAT) and serotonin (SERT) transporters are
presented in Table 1. The most potent compound is the
12R diastereomer, 11b, which has an IC50 of 4.04 nM
and a selectivity of 74 (DAT:SERT). Of interest is the
fact that the two diastereomers 11a and 11b, while
similar in DAT potency (11a , IC50 ) 7.38 nM; 11b, IC50
) 4.04 nM), differ markedly in selectivity for the DAT
(9 vs 74). Most important, the mixture 11 is sufficiently
potent and selective (DAT:SERT ) 21) to provide a
promising SPECT ligand.
(C) Ch ela t or . Substantial work has been carried
out on the design and synthesis of 99mTc brain per-
fusion imaging agents. These agents need to cross
the blood-brain barrier, and it has been estab-
lished17-19,24,28,30,31,37-42 that to do so the ligand should
be neutral and should have sufficient lipid solubility.
The selection of the 99mTc chelate utilized here
(MAMA′) was based upon the work of DiZio et al.29,31
They demonstrated that, for a lipophilic steroid, selec-
tion of a highly lipophilic chelator detracted from its
The 3,4-dichloro analog 12 is of intermediate potency
(DAT: IC50 ) 37.2 ( 3.4 nM) and of moderate selectivity

1840 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Meltzer et al.
pram (86.8 Ci/mmol) were purchased from DuPont-New
England Nuclear (Boston, MA). (R)-(-)-Cocaine hydrochloride
for the pharmacological studies was donated by the National
Institute on Drug Abuse (NIDA). Fluoxetine was donated by
E. Lilly & Co.
(DAT:SERT ) 7). In comparison, SAR of the classical
tropane series of compounds5,12 has shown that the 3,4-
dichloro analog of WIN35,428 is among the most potent
of DAT inhibitors (IC50 ) 0.9 ( 0.02 nM). Although
highly selective for the DAT over the SERT, the least
potent compound proved to be the difluoropine analogs
13 (IC50 ) 616 ( 88 nM; selectivity, DAT:SERT ) 90).
It is noteworthy that, as for the tropane series in
general, biological selectivity is maintained for the 2â-
COOCH3 (11) vs the 2R-COOCH3 (14; DAT: IC50 ) 2960
nM) compound.
Finally, norepinephrine binding of 11 evidenced con-
siderable selectivity for the DAT over the NET (11:
DAT:NET ) 6700). On the basis of these data, com-
pound 11 was selected for introduction of 99mTc and
subsequent SPECT imaging studies.
The rank order of potency at the DAT for each of the
diastereomeric pairs is 11 > 12 > 13 > 14. This is in
contrast to the rank order for the parent compounds: 3
(IC50 ) 0.9 nM) > 4 (IC50 ) 10.9 nM) ≈ 2 (IC50 ) 11.0
nM). This difference in rank order indicates that the
bulky N-substituent causes the C3 aromatic ring to
interact differently at the transporter binding site. This
difference is likely a π-stacking or steric bulk (protru-
sion) effect rather than an electronic one. Indeed, from
these data in conjunction with those obtained from a
family of oxytropanes (compounds in which oxygen has
been substituted for nitrogen43 where the differences
encountered on modification of the aromatic substitu-
ents are considerably more marked) it is increasingly
evident that the aromatic system can play a dominant
role in transporter-ligand interactions.
N-[[[2-[(Tr ip h en ylm eth yl)th io]eth yl]a m in o]ca r bon yl]-
m eth yl]-N-(3′-ch lor op r op yl)-S-(tr ip h en ylm eth yl)-2-a m i-
n oeth a n eth iol (16). The amine N-[[[2-[(triphenylmethyl)-
thio]ethyl]amino]carbonyl]methyl]-S-(triphenylmethyl)-2-
aminoethanethiol, 1530 (10.86 g, 16 mmol), was dissolved in
dry methylene chloride (10 mL), and to this solution was added
the 3-chloropropyl triflate (1.81 g, 8 mmol, prepared from
3-chloropropanol). The resulting solution was stirred at room
temperature for 2 h, at which point a further 90 mL of
methylene chloride was added. The solution was filtered to
remove excess amine triflate salt, and the filtrate was chro-
matographed (SiO2, ethyl acetate/hexanes, 1:1). In this man-
ner 4.36 g (72% based on the triflate) of the desired product,
16, was isolated as a white foam: mp 55-56 °C; Rf 0.5 (EtOAc/
1
hexanes, 1:1); IR (KBr disk) 1640 cm-1; H NMR (100 MHz,
CDCl3) δ 1.6-1.9 (m, 2H), 2.2-2.55 (m, 8H), 2.85 (s, 2H), 3.02
(q, 2H, J ) 6 Hz), 3.45 (t, 2H, J ) 6 Hz), 7.1-7.5 (m, 30H);
HRCIMS calculated for C47H48ClN2S2O [MH]+ 755.2896, found
755.2899.
[N-[2-[(3′-Ch lor opr opyl)(2-m er captoeth yl)am in o]acetyl]-
2-a m in oeth a n eth iola to]r h en iu m (V) Oxid e (18). N-[[[[2-
(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-(3′-
chloropropyl)-S-(triphenylmethyl)-2-aminoethanethiol, 16 (2.5
g, 3.3 mmol), was dissolved in boiling ethanol (50 mL). To
this was added a solution of tin(II) chloride (690 mg, in 6.25
mL of 0.05 M HCl), followed immediately by a solution of
sodium perrhenate (1 g in 6.25 mL of 0.05 M HCl). Reflux
was continued overnight, after which boiling acetonitrile (200
mL) was added and the resulting solution filtered through a
pad of Celite. The cake was further washed with boiling
acetonitrile (2 × 200 mL). To the filtrate was added silica gel
(30 g), and the solvent was evaporated. The solid was then
layered onto a silica gel column and eluted with ethyl acetate.
A racemate, 18, was isolated in 48% yield (740 mg): mp 218.4-
Con clu sion s
218.8° C; Rf 0.32 (EtOAc); IR (KBr disk) 1640, 952 cm-1
;
Herein we describe the design, synthesis, and biologi-
cal evaluation of the first 99mTc-labeled SPECT imaging
agent, technepine, 1, for the dopamine transporter in
striatum. We have demonstrated that there is sufficient
space in the transporter to accommodate a bulky chelat-
ing unit in the region of the 8-amine function of the
tropane skeleton. Further, we have demonstrated for
the first time that a molecule can be designed to carry
the radionuclide 99mTc across the blood-brain barrier
in sufficient quantity to obtain in vivo images of the
striatum in monkeys. This critical advance will un-
doubtedly lead to the design of new receptor and
transporter-mediated 99mTc agents which can label
specific transporter and receptor targets in the central
nervous system.44
1H NMR (100 MHz, CDCl3) δ 1.67 (dt, 1H, J ) 12, 4.6 Hz),
2.1-2.5 (m, 2H), 2.90 (dd, 1H, J ) 13, 4.6 Hz), 3.1-3.5 (m,
4H), 3.64 (t, 2H, J ) 6 Hz), 3.7-4.2 (m, 3H), 4.09 (d, 1H, J )
16 Hz), 4.5-4.7 (m, 1H), 4.68 (d, 1H, J ) 16 Hz); HRCIMS
calculated for C9H17ClN2S2O2Re [MH]+ 470.9950 found
470.9971. Anal. (C9H17ClN2S2O2Re‚0.2 CH3CO2C3H5) C, H,
N.
[N-[2-[(3′-N′-P r op yl-3′′â-(4-flu or op h en yl)t r op a n e-2′′â-
ca r boxylic a cid m eth yl ester )(2-m er ca p toeth yl)a m in o]-
a cet yl]-2-a m in oet h a n et h iola t o]r h en iu m (V) oxid e (11).
Rou te A. To a solution of nor-3â-(4-fluorophenyl)tropane-2â-
carboxylic acid methyl ester12,33 (253 mg, 0.96 mmol) in dry
acetonitrile (10 mL) was added in succession [N-[2-[(3′-chlo-
ropropyl)(2-mercaptoethyl)amino]acetyl]-2-aminoethanethiola-
to]rhenium(V) oxide, 18 (451 mg, 0.96 mmol), potassium iodide
(159 mg, 0.96 mmol), and potassium carbonate (1.3 g, 9.6
mmol). The resulting slurry was then brought to reflux
overnight. Upon completion of reaction (as shown by TLC)
the solution was allowed to cool to room temperature, silica
gel (10 g) was added, and the solvent was evaporated. The
resulting solid was layered onto a silica gel column and eluted
with 0.5% ammonium hydroxide in ethyl acetate. In this
manner the title compound, 11, was obtained as a mixture of
diastereomers in 90% yield (608 mg); mp 101.9 °C; Rf 0.55 (94%
EtOAc/5% MeOH/1% NH4OH); IR (KBr disk) 1720, 1666, 957
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.14-7.24 (m, 2H), 6.92-
6.96 (m, 2H), 4.79, 4.77 (2d, 1H, J ) 16 Hz), 4.52-4.59 (m,
1H), 3.95-4.11 (m, 3H), 3.55-3.75 (m, 2H), 3.35-3.55 (m, 2H),
3.44, 3.49 (2s, 3H), 3.10-3.30 (m, 3H), 2.95-3.05 (m, 1H),
2.80-2.90 (m, 2H), 2.35-2.60 (m, 2H), 2.15-2.30 (m, 1H),
1.95-2.10 (m, 2H), 1.05-1.95 (m, 6H); HRCIMS calcd for
C24H34FN3S2O4Re [MH]+ 698.1505, found 698.1557. Com-
pound 11 was converted to the hydrochloride for elemental
analysis. Anal. (C24H33FN3O4S2Re‚HCl‚2H2O) C, H, N.
Rou te B. N-[2-[(3′-N′-Propyl-3′′â-(4-fluorophenyl)tropane-
2′′â-carboxylic acid methyl ester)[2-[(triphenylmethyl)thio]-
Exp er im en ta l Section
NMR spectra were recorded on either a Bruker 100, a
Varian XL 400, or a Bruker 300 NMR spectrometer. TMS was
used as internal standard. Melting points are uncorrected and
were measured on a Gallenkamp melting point apparatus.
Thin layer chromatography (TLC) was carried out on Baker
Si 250F plates. Visualization was accomplished with either
iodine vapor, UV exposure, or treatment with phosphomolybdic
acid (PMA). Preparative TLC was carried out on Analtech
uniplates silica gel GF 2000 µm. Flash chromatography was
carried out on Baker silica gel 40 µM. Elemental analyses
were performed by Atlantic Microlab, Atlanta, GA. A Beck-
man 1801 scintillation counter was used for scintillation
spectrometry; 0.1% bovine serum albumin and (-)-cocaine
were purchased from Sigma Chemicals. All reactions were
carried out under an inert atmosphere (N2).
[3H]WIN35,428 ([3H]CFT, 2â-carbomethoxy-3â-(4-fluorophe-
nyl)-N-[3H]methyltropane, 79.4-87.0 Ci/mmol) and [3H]citalo-

Technetium-99m SPECT Imaging
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1841
16 Hz), 6.85-7.30 (m, 4H); HRCIMS calcd for C24H34N3S2O4-
ethyl]amino]acetyl]-S-(triphenylmethyl)-2-aminoethanethiol,
17 (98 mg, 0.1 mmol), was dissolved in boiling ethanol (1.5
mL). To this was added a solution of tin(II) chloride (21 mg
in 200 mL of 0.05 M HCl), followed immediately by a solution
of sodium perrhenate (30 mg in 200 mL of 0.05 M HCl).
Boiling was continued overnight, after which boiling acetoni-
trile (10 mL) was added and the resulting solution filtered
through a pad of Celite. The cake was further washed with
boiling acetonitrile (2 × 20 mL). To the filtrate was added
silica gel (1 g), and the solvent was removed on a rotary
evaporator. The resultant solid was layered onto a silica gel
column and eluted with ethyl acetate. Compound 11 was
obtained as a mixture of diastereomers in 30% yield (21 mg)
and was identical (TLC, NMR) to that prepared by route A
above.
Re [MH - F]+ 679.1521, found 679.1569. Anal. (C24H33
FN3O4S2Re‚1.5CHCl3) C, H, N.
-
2â-Ca r bom eth oxy-3â-(4-flu or op h en yl)-8-[3-[N-[2-[[[[2-
[(t r ip h en ylm et h yl)t h io]et h yl]a m in o]ca r bon yl]m et h yl]-
N -[S -(t r ip h e n ylm e t h yl)t h io]e t h yl]a m in o]p r op yl]n or -
tr op a n e (17). Rou te A. To a solution of nor-3â-(4-fluorophe-
nyl)tropane-2â-carboxylic acid methyl ester12 (52.6 mg, 0.2
mmol) in dry acetonitrile (10 mL) was added in succession
N-[[[[2-[(triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-
N′-(3′-chloropropyl)-S-(triphenylmethyl)-2-aminoethanethiol,
16 (151 mg, 0.2 mmol), potassium iodide (33 mg, 0.2 mmol),
and potassium carbonate (280 mg, 2.0 mmol). The resulting
slurry was then boiled overnight. Once the reaction was
complete, the solution was allowed to cool to room temperature,
and then 2 g of silica gel was added and the solvent evaporated.
The resulting solid was layered onto a silica gel column and
eluted with 0.5% ammonium hydroxide in a 1:1 solution of
ethyl acetate and hexanes. In this manner compound 17
was recovered as a foam in 72% yield (141 mg). This was
converted to the dihydrochloride: mp 166-168 °C; Rf 0.42
(EtOAc/hexanes (1:1) + 0.5% NH4OH); IR (KBr disk)1666
Sep a r a tion of Dia ster eiosom er s 11a a n d 11b. A mix-
ture of 11a and 11b (171 mg) was chromatographed (40 g of
SiO2 prewashed with Et3N (4 mL in 100 mL EtOAc); eluent
EtOAc with 0.5% NH4OH). Compound 11a eluted first (31.5
mg) followed by a mixture of 11a and 11b (112.7 mg) and
finally by 11b (26.8 mg) [TLC: Rf for both 11a and 11b is 0.55
(94% ethyl acetate, 5% MeOH, 1% NH4OH)].
1
1
cm-1; H NMR (100 MHz, CDCl3) δ 1.8-3.8 (m, 24H), 3.3 (s,
11a : mp 261-262 °C; H NMR (400 MHz, CDCl3) δ 1.50-
1.92 (m, 6H), 1.94-2.10 (m, 2H), 2.16-2.24 (m, 1H), 2.38-
2.46 (m, 1H), 2.50-2.60 (m, 1H), 2.80-2.90 (m, 2H), 2.94-
3.02 (m, 1H), 3.10-3.28 (m, 3H), 3.36-3.48 (m, 2H), 3.44 (s,
3H, 2′â-CO2CH3), 3.54-3.60 (m, 1H), 3.64-3.74 (m,1H), 3.92-
4.08 (m, 2H), 4.07 (d, 1H, J ) 16 Hz, H-6R), 4.54-4.60 (m,
1H, H-4â), 4.79 (d, 1H, J ) 16 Hz, H-6â), 6.90-7.00 (m, 2H),
7.10-7.30 (m, 2H).
3H), 3.9-4.0 (m, 1H), 4.2-4.3 (m, 1H), 4.4-4.5 (m, 1H), 6.9-
7.4 (m, 34H), 8.7-8.9 (m, 1H), 9.3-9.4 (m, 1H). Anal.
(C62H64N3O3S2F‚2HCl‚2H2O) C, H, N.
Rou te B. Nor-3â-(4-fluorophenyl)tropane-2â-carboxylic acid
methyl ester12 (52.6 mg, 0.2 mmol) was dissolved in dry
methylene chloride (1 mL). To this solution was added the
3-chloropropyl triflate (22.6 mg, 0.1 mmol, prepared from
3-chloropropanol). The resulting solution was stirred at room
temperature for 2 h at which point a further 10 mL of
methylene chloride was added. The solution was filtered to
remove excess amine triflate salt, and the filtrate was evapo-
rated and immediately dissolved in dry acetonitrile (10 mL).
To this solution was added, in succession, N-[[[[2-(triphenyl-
methyl)thio]ethyl]amino]carbonyl]methyl]N-(3′-chloropropyl)-
S-(triphenylmethyl)-2-aminoethanethiol, 16 (136 mg, 0.2 mmol),
potassium iodide (33 mg, 0.2 mmol), and potassium carbonate
(280 mg, 2.0 mmol). The resulting slurry was then maintained
at reflux overnight. Upon completion of reaction (by TLC),
the solution was allowed to cool to room temperature, 2 g of
silica gel was added, and the solvent was evaporated. The
resulting solid was layered onto a silica gel column and eluted
with 0.5% ammonium hydroxide in ethyl acetate/hexanes (1:
1). In this manner compound 17 was obtained as a foam (98
mg; 50%) and was identical (TLC, NMR) to that prepared by
route A.
11b: 1H NMR (100 MHz, CDCl3) δ 1.50-3.80 (m, 21H), 3.49
(s, 3H, 2′â-CO2CH3), 3.85-4.30 (m, 2H), 4.04 (d, 1H, J ) 16
Hz, H-6R), 4.40-4.70 (m, 1H, H-4â), 4.77 (d, 1H, J ) 16 Hz,
H-6â), 6.80-7.40 (m, 4H).
[N-[2-[(3′-N′-p r op yl-3′′â-(3,4-d ich lor op h en yl)t r op a n e-
2′′â-ca r boxylic a cid m eth yl ester )(2-m er ca p toeth yl)a m i-
n o]acetyl]-2-am in oeth an eth iolato]r h en iu m (V) Oxide (12).
This compound was prepared as described above for route A
and purified by chromatography: 59% yield; mp 108 °C dec;
Rf 0.18 (EtOAc); IR (KBr disk) 1724, 1653, 957 cm-1; 1H NMR
(100 MHz, CDCl3) δ 1.5-3.9 (m, 22H), 3.55, 3.50 (2s, 3H), 3.9-
4.2 (m, 2H), 4.5-4.7 (m, 1H), 4.80 (d, 1H, J ) 16.4 Hz), 7.0-
7.4 (m, 3H); HRCIMS calcd for C24H33Cl2N3S2O4Re [MH]+
748.0819, found 748.0856. Anal. (C24H33Cl2N3O4S2Re) C, H,
N.
[N-[2-[(3′-N′-P r op yl-3′′r-(bis(4-flu or op h en yl)m eth oxy)-
tr op a n e-2′′â-ca r boxylic a cid m eth yl ester )(2-m er ca p to-
et h yl)a m in o]a cet yl]-2-a m in oet h a n et h iola t o]r h en iu m -
(V) Oxid e (13). This compound was prepared as described
above for route A and purified by column chromatography:
68% yield; mp 102 °C dec; Rf 0.47 (EtOAc); IR (KBr disk) 1709,
1640, 957 cm-1; 1H NMR (100 MHz, CDCl3) δ 1.4-2.4 (m, 9H),
2.6-4.3 (m, 15H), 3.70, 3.65 (2s, 3H), 4.4-4.7 (m, 1H), 4.85,
4.69 (2d, 1H, J ) 16 Hz), 5.35 (s, 1H), 6.8-7.4 (m, 8H);
HRCIMS calcd for C31H39F2N3S2O5Re [MH]+ 822.1829, found
822.1818. Anal. (C31H38F2N3O5S2Re‚2H2O) C, H, N.
Sep a r a tion of Dia ster eoisom er s 13a a n d 13b. The
diastereomeric mixture of 13a and 13b was separated by silica
gel column chromatography (eluent 2% NH4OH/EtOAc). Com-
pound 13a eluted first (Rf 0.51 (EtOAc)) and 13b second (Rf
0.43 (EtOAc)).
Gen er a l P r oced u r e for P r ep a r a tion of Ar yla lk yltr o-
p a n es. The arylalkyl halide was brought to reflux overnight
with nor-3â-(4-fluorophenyl)tropane-2â-carboxylic acid methyl
ester12 in acetonitrile in the presence of potassium iodide (1
equiv) and potassium carbonate (10 equiv). The solvent was
evaporated and the residue purified by chromatography. The
following compounds were obtained by this standard proce-
dure.
(1R)-N-(3′-Ben zyl)-3â-(4-flu or op h en yl)tr op a n e-2â-ca r -
boxylic a cid m eth yl ester (8): yield 93%; mp 132-133 °C;
1
Rf 0.56 (20% EtOAc/hexanes + 0.5% NH4OH); H NMR (100
MHz, CDCl3) δ 1.6-2.4 (m, 6H), 2.7-3.2 (m, 4H), 3.4 (s, 3H),
3.4-3.7 (m, 2H), 6.8-7.5 (m, 9H). Anal. (C22H24FNO2‚0.5H2O)
C, H, N.
13a : 1H NMR (100 MHz, CDCl3) δ 1.4-2.4 (m, 9H), 2.6-
4.3 (m, 15H), 3.70 (s, 3H), 4.4-4.7 (m, 1H), 4.85 (d, 1H, J )
16 Hz), 5.35 (s, 1H), 6.8-7.4 (m, 8H).
(1R)-N-(3′-P h en ylp r op yl)-3â-(4-flu or op h en yl)tr op a n e-
2â-ca r b oxylic a cid m et h yl est er (9): yield 99%; oil; Rf
0. 42 (20% EtOAc/hexanes + 0.5% NH4OH); 1H NMR (100
MHz, CDCl3) δ 1.5-2.2 (m, 7H), 2.3 (t, 2H, J ) 7 Hz), 2.4-3.2
(m, 5H), 3.45 (br s, 1H), 3.53 (s, 3H), 3.75 (br s, 1H), 6.8-7.4
13b: 1H NMR (100 MHz, CDCl3) δ 1.4-2.4 (m, 9H), 2.6-
4.3 (m, 15H), 3.65 (s, 3H), 4.4-4.7 (m, 1H), 4.69 (d, 1H, J )
16 Hz), 5.35 (s, 1H), 6.8-7.4 (m, 8H).
[N-[2-[(3′-N′-P r op yl-3′′â-(4-flu or op h en yl)t r op a n e-2′′r-
ca r boxylic a cid m eth yl ester )(2-m er ca p toeth yl)a m in o]-
a cet yl]-2-a m in oet h a n et h iola t o]r h en iu m (V) Oxid e (14).
This compound was prepared as described above for route A
and purified by column chromatography: 70% yield; mp 98.6-
99.6 °C; Rf 0.29 (93% EtOAc/5% MeOH/2% NH4OH); IR (KBr
disk) 1716, 1646, 957 cm-1; 1H NMR (100 MHz, CDCl3) δ 1.5-
2.1 (m, 7H), 2.57 (t, 2H, J ) 7 Hz), 2.8-4.0 (m, 13H), 3.52 (s,
3H), 4.0-4.3 (m, 2H), 4.5-4.7 (m, 1H), 4.75, 4.74 (2d, 1H, J )
(m, 9H); analyzed as the hydrochloride. Anal. (C24H28
-
FNO2‚1.5HCl‚1H2O) C, H, N.
(1R)-N-(3′-P h en ylp en tyl)-3â-(4-flu or op h en yl)tr op a n e-
2â-ca r boxylic a cid m eth yl ester (10): yield 66%; oil; Rf 0.
31 (20% EtOAc/hexanes + 0.5% NH4OH); 1H NMR (100 MHz,
CDCl3) δ 1.2-2.3 (m, 13H), 2.3-3.2 (m, 5H), 3.4 (br s, 1H),
3.5 (s, 3H), 3.7 (br s, 1H), 6.8-7.4 (m, 9H); analyzed as the
hydrochloride. Anal. (C26H32FNO2‚2HCl‚1.5H2O) C, H, N.

1842 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Meltzer et al.
[N-[2-[(3′-N′-p r op yl-3′′â-(4-flu or op h en yl)t r op a n e-2′′â-
ca r boxylic a cid m eth yl ester )(2-m er ca p toeth yl)a m in o]-
a cetyl)-2-a m in oeth a n eth iola to]tech n etiu m -99m (V) Ox-
id e (1). 2â-Carbomethoxy-3â-(4-fluorophenyl)-8-[3-[N-[2-[[[[2-
[(triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[S-
(triphenylmethyl)thio]ethyl]amino]propyl]nortropane, 17 (1.0
mg), was dissolved in 0.2 mL of anhydrous anisole and cooled
in an ice bath to 5 °C. Anhydrous trifluoroacetic acid (10 mL)
was added to obtain a bright yellow solution which was stirred
at 5 °C for 5 min. The reaction mixture was then titrated with
triethylsilane until the disappearance of the yellow color. The
solution was evaporated to dryness by rotary evaporation at
room temperature and dried under high vacuum drying for 1
h. The dry solid was reconstituted in 0.5 mL of distilled water.
A portion of of this aqueous solution (100 µL) was then added
to 300 µL of a 58 mCi 99mTc-glucoheptonate solution (Glucos-
can kits from Du Pont, Billerica, MA) and incubated for 45
min at 40-45 °C. HPLC separation on a C8 reverse phase
column equipped with a C18 guard column, eluted with 0.1 M
ammonium acetate and acetonitrile under a linear gradient,
provided the major 99mTc-labeled product having a retention
time of 21.5 min. The major peak was collected in a round
bottom flask and evaporated on a high-vacuum rotary evapo-
rator at room temperature. The product was subsequently
reconstituted in 1 mL of sterile saline for injection.
two values. Competition experiments to determine the affini-
ties of other drugs at [3H]WIN35,428 binding sites were
conducted using procedures similar to those outlined above.
Stock solutions of water-soluble drugs were dissolved in water
or buffer, and stock solutions of other drugs were made in a
range of ethanol/HCl solutions. Several of the drugs were
sonicated to promote solubility. The stock solutions were
diluted serially in the assay buffer and added (0.2 mL) to the
assay medium as described above.
Ser oton in Tr a n sp or ter Assa y. The serotonin transporter
was assayed in caudate-putamen membranes using conditions
similar to those for the dopamine transporter. The affinity of
[3H]citalopram (spec. act.: 82 Ci/mmol, DuPont-NEN) for the
serotonin transporter was determined in experiments by
incubating tissue with a fixed concentration of [3H]citalopram
and a range of concentrations of unlabeled citalopram. The
assay tubes received, in Tris‚HCl buffer (50 mM, pH 7.4 at
0-4 °C; NaCl 100 mM), the following constituents at a final
assay concentration: citalopram, 0.2 mL (1 pM to 100 or 300
nM), [3H]citalopram (1 nM); membrane preparation 0.2 mL
(4 mg original wet weight of tissue/mL). The 2 h incubation
(0-4 °C) was initiated by addition of membranes and termi-
nated by rapid filtration over Whatman GF/B glass fiber filters
presoaked in 0.1% polyethylenimine. The filters were washed
twice with 5 mL of Tris‚HCl buffer (50 mM) and incubated
overnight at 0-4 °C in scintillation fluor (Beckman Ready-
Value, 5 mL), and radioactivity was measured by liquid
scintillation spectrometry (Beckman 1801). Cpm were con-
verted to dpm following determination of counting efficiency
(>45%) of each vial by external standardization. Total binding
was defined as [3H]citalopram bound in the presence of
ineffective concentrations of unlabeled citalopram (1 or 10 pM).
Nonspecific binding was defined as [3H]citalopram bound in
the presence of an excess (10 µM) of fluoxetine. Specific
binding was the difference between the two values. Competi-
tion experiments to determine the affinities of other drugs at
[3H]citalopram binding sites were conducted using procedures
similar to those outlined above.
Biology. Animals used in this study were maintained in
accordance with the guidelines of the Committee on Animals
of the Harvard Medical School and of the “Guide for Care and
Use of Laboratory Animals” of the Institute of Laboratory
Animal Resources, National Research Council, Department of
Health, Education and Welfare, Publication No. (NIH)85-23,
revised 1985.
Tissu e Sou r ces a n d P r ep a r a tion . Brain tissue from
adult male and female cynomolgus monkeys (Macaca fascicu-
laris) was stored at -85 °C in the primate brain bank at the
New England Regional Primate Research Center. The cau-
date-putamen was dissected from coronal slices and yielded
1.4 ( 0.4 g tissue. Membranes were prepared as described
previously.5,45 Briefly, the caudate-putamen was homog-
enized in 10 volumes (w/v) of ice-cold Tris‚HCl buffer (50 mM,
pH 7.4 at 4 °C) and centrifuged at 3800g for 20 min in the
cold. The resulting pellet was suspended in 40 volumes of
buffer, and the entire was procedure was repeated twice. The
membrane suspension (25 mg original wet weight of tissue/
mL) was diluted to 12 mL/ml for [3H]WIN 35,428 or [3H]-
citalopram assay in buffer just before assay and was dispersed
with a Brinkmann Polytron homogenizer (setting 5) for 15 s.
All experiments were conducted in triplicate, and each experi-
ment was repeated in each of two or three preparations from
individual brains.
Dop a m in e Tr a n sp or ter Assa y. The dopamine trans-
porter was labeled with [3H]WIN35,428 ([3H]CFT, 2â-car-
bomethoxy-3â-(4-fluorophenyl)-N-[3H]methyltropane, 81-84
Ci/mmol, DuPont-NEN).45 The affinity of [3H]WIN35,428 for
the dopamine transporter was determined in experiments by
incubating tissue with a fixed concentration of [3H]WIN35,-
428 and a range of concentration of unlabeled WIN35,428. The
assay tubes received, in Tris‚HCl buffer (50 mM, pH 7.4 at
0-4 °C; NaCl 100 mM), the following constituents at a final
assay concentration: WIN35,428, 0.2 mL (1 pM to 100 or 300
nM), [3H]WIN35,428 (0.3 nM); membrane preparation 0.2 mL
(4 mg original wet weight of tissue/ml). The 2 h incubation
(0-4 °C) was initiated by addition of membranes and termi-
nated by rapid filtration over Whatman GF/B glass fiber filters
presoaked in 0.1% bovine serum albumin (Sigma Chemical
Co.). The filters were washed twice with 5 mL of Tris‚HCl
buffer (50 mM) and incubated overnight at 0-4 °C in scintil-
lation fluor (Beckman Ready-Value, 5 mL), and radioactivity
was measured by liquid scintillation spectrometry (Beckman
1801). Cpm were converted to dpm following determination
of counting efficiency (>45%) of each vial by external stan-
dardization. Total binding was defined as [3H]WIN35,428
bound in the presence of ineffective concentrations of unlabeled
WIN35,428 (1 or 10 pM). Nonspecific binding was defined as
[3H]WIN35,428 bound in the presence of an excess (30 µM) of
(-)-cocaine. Specific binding was the difference between the
Da ta An a lysis. Data were analyzed by the EBDA com-
puter software programs (Elsevier-Biosoft, U.K.). Final esti-
mates of IC50 values were computed by the EBDA program.
Baseline values for the individual drugs were established from
the competition curves, and these generally were similar to
baseline values established by 30 µM (-)-cocaine or 1 µM
fluoxetine.
Sin gle-Cr ysta l X-r a y An a lysis of Dia ster isom er 11a .
Orthorhombic crystals of the purified 11a were obtained by
slow growth at the interface of a hexane/anhydrous ethanol
suspension maintained at room temperature. A representative
crystal was selected, and a 1.451 78 Å data set was collected
at room temperature. Pertinent crystal, data collection, and
refinement parameters: crystal size, 0.40 × 0.38 × 0.14 mm;
cell dimensions, a ) 7.4147(5) Å, b ) 15.9123(11) Å, c )
22.2645(14) Å, R ) 90°, â ) 90°, γ ) 90°; formula, C24H33FN3O4-
ReS2; formula weight ) 696.85; volume ) 2626.9 (3) Å3;
calculated density ) 1.762 g cm-3; space group ) P212121;
number of reflections ) 2723 of which 2512 were considered
independent (Rint ) 0.0232). Refinement method was full-
matrix least-squares on F2. The final R indices were [I > 2σ
(I)] R1 ) 0.0322, wR2 ) 0.0836.
Coordinates, anisotropic temperature factors, distances, and
angles are available as Supporting Information.
Sin gle-P h ot on E m ission Com p u t ed Tom ogr a p h y.
SPECT and MRI imaging was performed on male and female
rhesus monkeys. The SPECT imaging technique was similar
to that used for human studies with HMPAO and ECD.46 The
monkey was placed within the gantry of the dedicated brain
imaging instrument (Ceraspect). Sixty-four slices of 128 × 128
pixels (voxel size 1.67 mm on a side) were obtained. Recon-
struction was by conventional filtered backprojection with a
Butterworth filter with power 10 and cutoff of 0.95 cm.
MRI images were obtained using standard sequences for T1,
T2, and proton density images. Image registration was
performed using the techniques described by Holman et al.47

Technetium-99m SPECT Imaging
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1843
(16) Fischman, A. J .; Bonab, A. A.; Babich, J . W.; Alpert, N. M.;
Elmaleh, D. R.; Barrow, S. A.; Graham, W.; Meltzer, P. C.;
Hanson, R. N.; Madras, B. K. SPECT imaging of the dopamine
transporter with [123I]-2â-carbomethoxy-3â-(4-fluorphenyl)-N-(1-
iodopropen-3-yl)nortropane ([123I]IACFT): initial experience in
humans. Neuroscience-Net, in press.
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Technetium(V) and rhenium(V) complexes of 2,3-bis(mercap-
toacetamido)propanoate. Chelate ring stereochemistry and influ-
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1990, 112, 5798-5804.
Ack n ow led gm en t. This work was supported by the
National Institute on Neurological Diseases and Stroke
(NINDS34611 (P.M.), NINDS30556 (B.K.M.) and The
National Institute on Drug Abuse (NIDA4-8309 (P.M.),
NIDA06303 (B.K.M.), NIDA 09462 (B.K.M.), RR00168
(B.K.M.)) and USPHS R37CA34970 (A.G.J .). The au-
thors thank Dr. Clifford George (Naval Research Labo-
ratory, Washington, D.C.) for conducting the X-ray
structural analysis. Technepine has been licensed to
Boston Life Sciences Inc.
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anisotropic temperature factors, distances, and angles (8
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J M960868T