Synthesis, Radiosynthesis, and Biological Evaluation of Carbon-11 and Iodine-123 Labeled 2β-Carbomethoxy-3β-[4′ -((Z)-2-haloethenyl)phenyl]tropanes: Candidate Radioligands for in Vivo Imaging of the Serotonin Transporter

Article abstract of DOI:10.1021/jm030384e

2β-Carbomethoxy-3β-[4′-((Z)-2-iodoethenyl)phenyl]tropane (ZIET) and 2β-carbomethoxy-3β-[4′ -((Z)-2-bromoethenyl)phenyl]tropane (ZBrET) were synthesized as well as their nortropane congeners ZIENT and ZBrENT. Binding affinities of these compounds were dete

Full text of DOI:10.1021/jm030384e

1122
J . Med. Chem. 2004, 47, 1122-1135
Syn th esis, Ra d iosyn th esis, a n d Biologica l Eva lu a tion of Ca r bon -11 a n d
Iod in e-123 La beled 2â-Ca r bom eth oxy-3â-[4′-((Z)-2-h a loeth en yl)p h en yl]tr op a n es:
Ca n d id a te Ra d ioliga n d s for in Vivo Im a gin g of th e Ser oton in Tr a n sp or ter
Christophe Plisson, J onathan McConathy, Laurent Martarello, Eugene. J . Malveaux, Vernon M. Camp,
Larry Williams, J ohn R. Votaw, and Mark M. Goodman*
Department of Radiology, Emory University, Atlanta, Georgia 30320
Received August 8, 2003
2â-Carbomethoxy-3â-[4′-((Z)-2-iodoethenyl)phenyl]tropane (ZIET) and 2â-carbomethoxy-3â-
[4′-((Z)-2-bromoethenyl)phenyl]tropane (ZBrET) were synthesized as well as their nortropane
congeners ZIENT and ZBrENT. Binding affinities of these compounds were determined in cells
transfected to express human SERT, DAT, and NET using [3H]citalopram, [125I]RTI-55, and
[3H]nisoxetine, respectively. Both ZIET and ZBrET displayed high affinity for the SERT (Ki )
0.11 and 0.08 nM, respectively).The affinities of ZIET and ZBrET for the DAT were 200 and
38-fold lower, respectively, than for the SERT. [11C]ZIET and [11C]ZBrET were prepared by
alkylation of their corresponding nortropanes with [11C]methyl iodide in approximately 30%
radiochemical yield (decay-corrected to end of bombardment, EOB). High specific activity
[
123I]ZIET was synthesized in 33% radiochemical yield (decay-corrected) by treating the
2â-carbomethoxy-3â-[4′-((Z)-2-trimethylstannylethenyl)phenyl]tropane (3) with no carrier-added
sodium [123I]iodide and hydrogen peroxide in ethanolic HCl. Biodistribution studies in rats
indicated that [123I]ZIET enters the brain readily and accumulates in SERT-rich regions.
Blocking studies performed in rats demonstrated that [123I]ZIET was selective and specific for
SERT-rich regions (e.g. thalamus, brainstem, and striatum). MicroPET brain imaging studies
in monkeys demonstrated that [11C]ZIET and [11C]ZBrET uptakes were selectivity localized in
the putamen, midbrain, caudate, thalamus, pons, and medulla. Radioactivity in the regions of
high SERT density of monkey brain was displaceable with citalopram except in the putamen
and caudate. Radioactivity uptake in these DAT-rich regions was significantly displaceable
either by preadministration of citalopram followed by injection of RTI-113 (or vice-versa) or by
administration of a mixture of DAT and SERT ligands. In conclusion, the high yield, high
specific activity, one-step radiolabeling method, high selectivity and favorable kinetics, and
the good results obtained with [123I]ZIET in rats support the candidacy of [11C]ZIET for in vivo
visualization and quantification of brain SERT.
Sch em e 1
In tr od u ction
The serotonin transporter (SERT) is a protein residing
on the presynaptic serotonergic neuron that regulates
the serotonin concentration by removing serotonin from
the extracellular space back to the presynaptic neuron.
Because of its localization, the SERT is a specific marker
for serotonergic neurons.1-4 Serotonergic neurons origi-
nate primarily from neuronal cell bodies of the medial
and dorsal raphe in the brainstem and innervate
discrete areas that include the hypothalamus, thalamus,
striatum, and cerebral cortex.5-7 Therefore, in vivo
imaging of the SERT in humans with PET or SPECT
would permit the study of the density and integrity of
serotonin neurons and help to understand how alter-
ations in SERT levels are related to numerous neuro-
psychiatric disorders such as depression,8 Alzheimer’s
disease,9 Parkinson’s disease10,11 and the neurotoxic
effects of illicit drugs (e.g. MDMA).12
the SERT in the central nervous system (CNS).13,6,14
Most of them appeared to be unsuitable for imaging due
to high lipophilicity and/or high nonspecific binding and/
or inappropriate in vivo kinetics.15 The first imaging
agent successfully employed in humans for studying the
SERT was the cocaine analogue [123I] 2â-carbomethoxy-
3â-(4′-iodophenyl)tropane ([123I]â-CIT) (Scheme 1). How-
ever, because of its lack of selectivity for the SERT
versus DAT (dopamine transporter), the evaluation of
the SERT sites is limited to areas outside the DAT-rich
striatum or requires preblocking of the DAT sites.16 The
most widely used PET imaging agent of the SERT
currently available is a pyrroloisoquinoline derivative,
(+)-[11C]McN-5652.17-19 This radioligand, which has
been used in human studies of SERT density, has
significant limitations such as modest signal to back-
A number of useful radioligands for both PET and
SPECT have been developed and evaluated for imaging
* Corresponding author:Mark M. Goodman, Ph. D. Emory Univer-
sity, Department of Radiology 1364 Clifton Road, NE, Atlanta, GA
30322; Phone: (404) 727-9366; Fax: (404) 727-3488; e-mail: mgoodma@
emory.edu.
10.1021/jm030384e CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/03/2004

Radioligands for in Vivo Imaging
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1123
Ta ble 1. In Vitro Evaluation of Various Ligands in Competition Assays with Human Monoamine Transportersa
compound
ZIENTb
ZIET
ZIENT
ZBrET
Ki for hSERT
Ki for hDAT
Ki for hNET
hSERT/hDAT ratio
0.05
3.5
22 ( 9
24
450 ( 24
N.D.
111 ( 12
2.5
5.6
161 ( 17
36
70
200
375
38
97
70
5
0.3
2
0.11 ( 0.006
0.04 ( 0.01
0.08 ( 0.01
0.04 ( 0.007
0.08 ( 0.006
1.35 ( 0.10
4.2
15 ( 1.4c
3.1 ( 0.3
3.9 ( 0.6
5.6 ( 3
ZBrENT
8
9
6.73 ( 0.63
1.3
0.69
â-CITd
nor-â-CITd
RTI-113e
R,S-citalopramf
reboxetineg
0.36
7.5
390 ( 34.26
1.6 ( 0.1
129 ( 13
5.25 ( 0.76
16 540 ( 3795
>10 000
242.29 ( 29.78
6190 ( 818
1.1 ( 0.2
0.013
10337
a
In vitro evaluation of candidate SERT ligands in competition assays using cells transfected with human monoamine transporters. All
Ki values are reported with nanomolar (nM) units. At least three separate Ki determinations were performed for each compound unless
otherwise indicated. The data are expressed as the geometric mean ( standard deviation. The hSERT/hDAT ratios reflect the affinity
b
(1/Ki) for hSERT divided by the affinity for hDAT. Previous values from competition assays performed using cells transfected with
hSERT, hDAT, or hNET.28 hDAT competition assays were performed using [3H]WIN 35,428 as the radiotracer, while in the current
d
study [125I]RTI-55 was used. c Average value of two separate Ki determinations. Literature values (IC50) from competition assays performed
with rat brain homogenates using [3H]paroxetine for SERT, [3H]WIN 35,428 for DAT, and [3H]nisoxetine for NET.35 e Literature values
(Ki) from competition assays performed with rat brain homogenates using [3H]paroxetine for SERT, [3H]WIN 35,428 for DAT, and
[3H]nisoxetine for NET.36 f Literature values (Ki) from competition assays performed using cells transfected with hSERT, hDAT or hNET
g
using [3H]citalopram, [125I]RTI-55, and [3H]nisoxetine.37 Literature values (Ki) from competition assays performed with rat brain
membranes labeled by [3H]paroxetine for SERT, [3H]WIN 35,428 for DAT, and [3H]nisoxetine for NET.38
ground due to high nonspecific binding. More recent-
ly a new class of radiotracers from the diaryl sulfide
class has been introduced such as [123I]ADAM,20 [11C]-
DASB,15,21,22 [11C]AFM,15,23 [11C]EADAM,24 and [18F]-
ACF.25 Compounds from this class have been labeled
with 123I for SPECT and 11C or 18F for PET. They
demonstrated high in vitro affinity and selectivity to the
SERT with low affinity for the DAT and NET (norepi-
nephrine transporter). The carbon-11 series of diaryl
sulfides showed higher in vitro selectivity but higher
lipophilicity and lower affinity to SERT than the tro-
panes reported in this article. Evaluation of these
radiotracers in humans and/or monkeys showed that
they are good candidates for imaging the SERT sites.
We hereby report the synthesis, in vitro characteriza-
tion, radiosynthesis, in vivo regional brain distribution
in rats of [123I]ZIET and microPET imaging in nonhu-
man primates of [11C]ZIET and [11C]ZBrET. The devel-
opment of these radiotracers as well as [123I]ZIET was
based on previous work with tropane derivatives with
unsaturated functional groups in the 4′-position of the
phenyl ring. A series of nortropanes was reported
containing alkenyl substituents at the 4′-position of the
phenyl ring of 2â-carbomethoxy-3â-arylnortropanes.26,27
In this series, the cis and trans propenyl derivatives
were potent and selective for the SERT versus the DAT
in vitro. Goodman and colleagues28 prepared and evalu-
ated the 2-iodoethenyl derivative, 2â-carbomethoxy-3â-
[4′-((Z)-2- iodoethenyl)phenyl]nortropane (ZIENT), which
possessed very high affinity and selectivity for the
hSERT as shown in Table 1. Additionally, SPECT
imaging studies conducted with [123I]ZIENT in monkeys
demonstrated uptake of radioactivity in SERT-rich
regions which was displaced with the selective SERT
ligand, citalopram, demonstrating the potential of this
tropane for visualizing the SERT.
reaction processing through the conjugated addition of
the phenyl ring, then nitration at the 4′-position of the
ring, reduction of the nitro, and finally diazotation
followed by treatment with cuprous bromide,29 or as a
two-step reaction involving the Grignard addition of
4-trimethylsilylphenylmagnesium bromide followed by
bromodesilylation with N-bromosuccinimide.30 In our
hands, 2 was prepared directly by 1,4-addition of freshly
made 4-bromophenylmagnesium bromide on the ecog-
nine methyl ester (1).31 Tropane 2 was then converted
to the 2â-carbomethoxy-3â-[4′-((Z)-2-trimethylstan-
nylethenyl) phenyl]tropane (3) via a Stille coupling
reaction using (Z)-1,2-trimethylstannylethylene32 (4)
and tetrakis(triphenylphosphine)palladium (0) as cata-
lyst in toluene. This methodology was developed simul-
tanously by Blough et al.33 The tropane 3 was used as
precursor in the radiosynthesis of [123I]ZIET. ZIET and
ZBrET were prepared by halodestannylation of 3 with
iodine monochloride and N-bromosuccinimide, respec-
tively. The preparation of [11C]ZIET and [11C]ZBrET,
based on an N-methylation reaction, involved the syn-
thesis of the corresponding desmethyl precursors 2â-
carbomethoxy-3â-[4′-((Z)-2-iodoethenyl)phenyl]nortro-
pane (ZIENT) and 2â-carbomethoxy-3â-[4′-((Z)-2-bro-
moethenyl)phenyl]nortropane (ZBrENT). Chronologi-
cally, the synthesis of ZIENT and ZBrENT was first
carried out from aldehyde 5 (Scheme 3): ZIENT was
prepared by treatment with triphenylphosphonium-
iodomethylene ylid then cleavage of the Boc group.28
Treatment of the aldehyde 5 with carbon tetrabromide
and triphenylphosphine gave the N-(tert-butoxycarbon-
yl)-2â-carbomethoxy-3â-[4′-(2,2′-dibromo)ethenyl phen-
yl]nortropane (6) which was converted to ZBrENT by
palladium-catalyzed hydrogenolysis using tetrakis-
(triphenylphosphine)palladium (0) and tributyltin hy-
dride34 followed by deprotection of the amine. Most
recently, ZIENT and ZBrENT were prepared from 2
according to an improve reaction sequence outlined in
Scheme 4. The conversion of compound 2 to its corre-
sponding carbamate using trichloroethylchloroformate
followed by Zn-acetic acid reduction gave the nortro-
pane 10, from which were obtained ZIENT and ZBrENT
Resu lts a n d Discu ssion
Ch em istr y. ZIET and ZBrET were prepared from the
same intermediate, the 2â-carbomethoxy-3â-(4′-bro-
mophenyl)tropane (2) (Scheme 2). The synthesis of this
compound was previously described as a four-step

1124 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Plisson et al.
Sch em e 2a
a
Reagents: (i) 4-BrPhMgBr, -40 °C then TFA, -78 °C. (ii) 4, Pd(PPh3)4, toluene, 100 °C. (iii) ICl, DCM, rt. (iv) NBS, DCM, rt.
Sch em e 3a
a
Reagents: (i) CBr4, PPh3, DCM. (ii) nBu3SnH, Pd(PPh3)4, toluene. (iii) TFA, DCM. iv) MeI, DMF, 90 °C.
according to the same pathway described above. To
evaluate the binding potency of the 2â-carbomethoxy-
3â-[4′-(2,2′-dibromo)ethenylphenyl]nortropane (8) and
its N-methyl analogue 9, the dibromo derivative 6 was
hydrolyzed using trifluoroacetic acid in dichloromethane
and the resulting nortropane 8 was alkylated with
methyl iodide in N,N-dimethylformamide to afford the
2â-carbomethoxy-3â-[4′-(2,2′-dibromo)ethenylphenyl]-
tropane (9) (Scheme 3).
Scheme 5. The entire procedure required approximately
45 min from the end of bombardment. Productions of
[11C]labeled tropanes provided an average 30% decay-
corrected yield ([11C]ZIET: n ) 18, [11C]ZBrET: n ) 20).
Analytical HPLC demonstrated that the radiolabeled
products were over 99% radiochemically pure and the
specific activity of the products was ranging from 0.4
to 0.6 Ci/µmol at time of injection (3.4 to 5.2 Ci/µmol
decay-corrected to end-of-bombardment).
Ra d ioch em istr y. The preparation of [11C]ZIET and
[11C]ZBrET was accomplished through N-alkylation of
the nortropane precursors, ZIENT and ZBrENT, with
[11C]CH3I in N,N-dimethylformamide as shown in
The radioiodinated 2â-carbomethoxy-3â-[4′-((Z)-2-
[
123I]iodoethenyl)phenyl]tropane [123I]ZIET was pre-
pared by treating the tin precursor 3 with 123I+, gener-
ated from no-carrier-added Na[123I]I and hydrogen

Radioligands for in Vivo Imaging
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1125
Sch em e 4a
a
Reagents: (i) TrocCl, toluene, reflux then Zn, AcOH, rt. (ii) (Z)-Me3SnCHdCHSnMe3, Pd(PPh3)4, toluene, 100 °C. (iii) ICl, DCM, rt.
(iv) NBS, DCM, rt.
Ta ble 2. Brain Distribution of Activity in Sprague-Dawley
Sch em e 5
Rats after iv Injection of [123I]ZIETa
tissue
5 min
30 min
60 min
120 min
blood
0.45 ( 0.11 0.53 ( 0.03 0.43 ( 0.03 0.38 ( 0.02
hypothalamus 0.36 ( 0.13 0.68 ( 0.03 0.69 ( 0.06 0.60 ( 0.04
brainstem
cerebellum
frontal cortex
occipital cortex 0.45 ( 0.17 0.76 ( 0.04 0.63 ( 0.07 0.42 ( 0.05
striatum
hippocampus
rest of brain
thyroid
0.35 ( 0.13 0.65 ( 0.02 0.54 ( 0.04 0.41 ( 0.02
0.34 ( 0.12 0.42 ( 0.01 0.27 ( 0.03 0.14 ( 0.02
0.45 ( 0.16 0.82 ( 0.04 0.71 ( 0.06 0.48 ( 0.04
0.39 ( 0.14 0.76 ( 0.03 0.71 ( 0.08 0.52 ( 0.03
0.38 ( 0.14 0.72 ( 0.03 0.63 ( 0.05 0.47 ( 0.03
0.38 ( 0.13 0.66 ( 0.03 0.54 ( 0.06 0.39 ( 0.03
0.08 ( 0.03 0.74 ( 0.14 1.11 ( 0.16 2.12 ( 0.26
a
Values are reported as the mean percent dose per gram of
tissue ( standard error. n ) 5 at all time points except at 120
min where n ) 4.
affinities, the hDAT binding affinities of the 3â-[4′-
(haloethenyl)phenyl]nortropanes and their associated
N-methyltropanes are approximately the same and
ranged from 3.5 to 6.7 nM. The measured binding
affinity of ZIET for hDAT was significantly lower with
a Ki of 22 nM. Consequently, the nortropanes exhibited
a higher selectivity for the hSERT versus hDAT than
corresponding tropanes. Nevertheless, ZBrET and es-
pecially ZIET showed higher affinity for the hSERT
than the hDAT. The affinity for the hDAT of ZBrET and
ZIET was 38- and 200-fold lower, respectively, than for
the hSERT. These analyses demonstrated that ZIET
and ZBrET possess high affinity and selectivity for
hSERT versus hDAT and hNET and that bromo deriva-
tives ZBrET and ZBrENT have higher affinities for both
hSERT and hDAT than their iodo analogues.
Rod en t Biod istr ibu tion Stu d ies. A regional dis-
tribution study in the brain of male Sprague-Dawley
rats was performed with iodine-123 labeled ZIET. [123I]-
ZIET allowed rodent studies to be conducted over a
longer period of time and at higher specific activity than
would be possible with [11C]ZIET. The distribution of
radioactivity was expressed as percent dose per gram
in various tissues of brain male rats. The results shown
in Table 2 demonstrated that the highest [123I]ZIET
uptake was observed at 30 min postinjection in all brain
regions except the hypothalamus which showed the
same activity of 0.69% dose/g at 60 min postinjection.
peroxide. The entire procedure required approximately
120 min. The radiolabeled product [123I]ZIET was
obtained in an average 33% decay-corrected yield (n )
2). The radiolabeled product was obtained in over 99%
chemical and radiochemical purity, as demonstrated by
analytical HPLC of the formulated solution. The specific
activity of the product at time of injection was over 3.8
Ci/µmole.
Biologica l Resu lts. In Vitr o Com p etition Assa ys.
The affinities of tropanes (ZIET, ZBrET and 9) and
nortropanes (ZIENT, ZBrENT and 8) for the hSERT,
hDAT, and hNET were determined through in vitro
competition assays. These data are shown in Table 1
along with the previously determined Ki values for
ZIENT.
[3H]Citalopram (SERT ligand), [125I]RTI-55 (DAT
ligand), and [3H]nisoxetine (NET ligand) were used as
radiotracers during the in vitro displacement experi-
ments. The rank of order of hSERT affinities was
ZBrENT g ZIENT > ZBrET g 8 > ZIET > nor-â-CIT
> 9 > â-CIT > RTI-113. As previously reported in the
literature,35 these results indicate that nortropanes have
higher affinities for the hSERT than their corresponding
tropanes. The rank of order of hDAT affinities was nor-
â-CIT > â-CIT > RTI-113 > ZBrET g ZBrENT > 8 >
9 > ZIENT > ZIET. In comparison with the hSERT

1126 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Plisson et al.
Ta ble 3. Ratios of Uptake of Radioactivity in Various Brain
Regions versus Cerebellum after iv Injection of [123I]ZIET
brain region
5 min
30 min
60 min
120 min
hypothalamus
brainstem
frontal cortex
occipital cortex
striatum
1.1
1.0
1.3
1.3
1.1
1.1
1.1
1.6
1.5
2.0
1.8
1.8
1.7
1.6
2.6
2.0
2.6
2.3
2.6
2.3
2.0
4.3
3.0
3.4
3.0
3.7
3.4
2.8
hippocampus
rest of brain
Ta ble 4. Brain Distribution of Activity in Sprague Dawley
Rats 60 min after iv Injection of [123I]ZIET in the Presence of
Monoamine Transporter Ligandsa
tissue
no blocker citalopram
RTI-113
reboxetine
blood
0.22 ( 0.03 0.21 ( 0.01 0.24 ( 0.01 0.26 ( 0.01
F igu r e 1. Brain distribution of activity in Sprague-Dawley
rats after i.v. injection of [123I]ZIET. (a) Values are reported as
the mean percent dose per gram of tissue ( standard error.
The data for the 60 and 120 min groups were analyzed
separately using one-way ANOVAs with repeated measures,
and p-values were calculated using Tukey’s posthoc test for
pairwise multiple comparisons. (b) Blood %ID/g at 60 min was
less than hypothalamus (p < 0.001), brainstem (p < 0.05),
frontal cortex (p < 0.001), occipital cortex (p < 0.001), striatum
(p <0.001), hippocampus (p <0.001), and the rest of the brain
(p < 0.05) at the same time point. Blood %ID/g at 60 min was
greater than the cerebellum (p <0.001) at the same time point.
(c) Blood %ID/g at 120 min was less than hypothalamus (p <
0.001) and striatum (p < 0.01) at the same time point. Blood
%ID/g at 120 min was greater than cerebellum (p < 0.001) at
the same time point. (d) Hypothalamus %ID/g at 60 min was
greater than brainstem (p <0.01) and the rest of the brain
(p < 0.001) at the same time point. (e) Hypothalamus %ID/g
at 120 min was greater than brainstem (p < 0.001), frontal
cortex (p < 0.05), occipital cortex (p < 0.001), hippocampus
(p < 0.05), and the rest of the brain (p < 0.001) at the same
time point. (f) Cerebellum %ID/g at 60 and 120 min was less
than all other brain regions (p <0.001 for all) at the same time
points. (g) Frontal cortex %ID/g at 60 min was greater than
the rest of the brain (p < 0.001) at the same time point. (h)
Striatum %ID/g at 60 min was greater than the brainstem and
the rest of the brain (p < 0.001 for both) at the same time
point. (i) Striatum %ID/g at 120 min was greater than
brainstem (p < 0.05) and the rest of the brain (p < 0.01) at
the same time point.
hypothalamus 0.42 ( 0.11 0.20 ( 0.03 0.55 ( 0.07 0.46 ( 0.02
brainstem
cerebellum
frontal cortex
occipital cortex 0.46 ( 0.13 0.20 ( 0.04 0.42 ( 0.04 0.42 ( 0.02
striatum
hippocampus
rest of brain
thyroid
0.33 ( 0.09 0.14 ( 0.02 0.35 ( 0.03 0.36 ( 0.02
0.15 ( 0.03 0.10 ( 0.02 0.13 ( 0.02 0.15 ( 0.01
0.53 ( 0.13 0.21 ( 0.03 0.49 ( 0.06 0.47 ( 0.02
0.42 ( 0.11 0.26 ( 0.04 0.45 ( 0.02 0.45 ( 0.01
0.40 ( 0.11 0.17 ( 0.02 0.42 ( 0.04 0.41 ( 0.02
0.37 ( 0.09 0.18 ( 0.02 0.36 ( 0.04 0.34 ( 0.01
0.54 ( 0.11 0.93 ( 0.09 1.01 ( 0.03 1.12 ( 0.15
a
Values are reported as the mean percent dose per gram of
tissue ( standard error. n ) 3 for the control group, n ) 4 for the
RTI-113 and citalopram group, and n ) 5 for the reboxetine group.
The monoamine transporter ligands were given 15 min prior to
[
123I]ZIET at doses of 4 mg/kg.
The ratios of the uptake of radioactivity in the brain
regions examined over time versus the cerebellum, a
region with low density of serotonergic innervation, are
shown in Table 3. At 120 min the highest ratios relative
to the cerebellum were seen in the hypothalamus
(4.3:1) and in the striatum (3.7:1). The brainstem,
frontal cortex, occipital cortex, and hippocampus all
demonstrated ratios greater than 3:1 at 120 min, while
the remainder of the brain had a ratio of 2.8:1 at this
time. The high uptake of [123I]ZIET in the SERT-rich
regions and the 13-27% washout in the hypothalamus,
brainstem, striatum, and hippocampus from 60 to 120
min demonstrate that [123I]ZIET has potentially favor-
able kinetic properties for in vivo quantification and
imaging of the SERT by SPECT.
brain. The same results were seen within both the RTI-
113 and reboxetine groups (p < 0.003). In contrast, no
significant differences were detected between any of the
brain regions including the cerebellum in the citalopram
group. In light of the results obtained with PET imaging
using [11C]ZIET discussed in the following section, the
uptake of radioactivity in the striatum warrants further
discussion. In the blocking study, pretreatment with
citalopram decreased striatal uptake of activity relative
to the RTI-113, reboxetine, and no blocker groups (p )
0.002, p < 0.001, and p ) 0.02, respectively). This result
indicates that a large component of striatal uptake of
To assess the in vivo selectivity of [123I]ZIET for the
SERT versus the DAT and the NET, blocking studies
were performed using citalopram as a brain available
SERT-selective ligand, RTI-113 as a DAT-selective
ligand and reboxetine as a NET-selective ligand (Scheme
1, Table 1). In these studies, rats received the mono-
amine transporter ligand (4 mg/kg) 15 min prior to
receiving [123I]ZIET and were compared to a group that
received only [123I]ZIET (no blocker group). The rats
were sacrified 60 min after injection of [123I]ZIET. After
dissection, the uptakes of radioactivity in different parts
of the brain were determined. The data (Table 4, Figure
1) were analyzed via a two-way ANOVA, and p values
were calculated using Tukey’s test for all pairwise
multiple comparisons. No significant differences were
found between the no blocker, RTI-113, and reboxetine
groups (p > 0.93), while the citalopram group was
significantly different than these three groups (p <
0.001). Within the no blocker group, the %ID/g in the
cerebellum was significantly lower than in the other
brain regions studied (p < 0.001) which is in agreement
with the expected regional distribution of SERT in rat
[
123I]ZIET is due to SERT binding. However, within the
citalopram group, there was a trend toward a signifi-
cantly higher %ID/g in the striatum relative to the
cerebellum (p ) 0.058) not observed in other brain
regions. This observation suggests that another, non-
SERT component of striatal uptake might be present.
Importantly, the %ID/g values observed in the striatum
were virtually identical between the RTI-113, reboxe-
tine, and no blocker groups, suggesting that [123I]ZIET
did not have appreciable DAT binding in the rat
striatum.

Radioligands for in Vivo Imaging
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1127
F igu r e 2. Transverse microPET images acquired using [11C]ZIET.
In Vivo Non h u m a n P r im a te Im a gin g. MicroPET
imaging studies were performed with [11C]ZIET and
[11C]ZBrET to assess the regional distribution, imaging
properties and in vivo selectivity of these radioligands
for the SERT in rhesus and cynomolgus monkeys. [11C]-
9 was also evaluated in monkey and demonstrated a
high nonspecific binding and a high uptake in the
regions rich in DAT correlating its binding affinities
(Table 1). This ligand did not warrant further investiga-
tions.
cerebellum.39,15 To assess the specificity of [11C]ZIET
and [11C]ZBrET uptake, displacement studies were
performed using the SERT ligand citalopram. A dose
of citalopram (1.5 mg/kg) administered 40 min after
injection of [11C]ZIET and [11C]ZBrET resulted in a
significant reduction of radioactivity in the thalamus,
midbrain, and pons in both cases (Figures 6 and 7) but
no displacement from the putamen and the caudate
suggesting that these tracers may bind to the DAT given
the high DAT density of these regions. To assess the
potential binding of [11C]ZIET and [11C]ZBrET to the
DAT, chase studies were performed using 0.3 mg/kg of
the DAT ligand RTI-113 administered 40 min after
injection of radioactive tracer. In the study with [11C]-
ZBrET and RTI-113 acquired for a period of time of 85
min, no displacement of radioactivity from any part of
the brain was observed (Figure 9). A slight displacement
of [11C]ZIET from the striatum was observed when RTI-
113 was administered, with a percentage of washout of
4 and 5% from the caudate and putamen, respectively
(Figure 8). However, if this slight decrease represents
[11C]ZIET binding to DAT sites, the administration of
RTI-113 would be expected to displace the radioactivity
uptake in the striatum because administration of citalo-
pram did not chase any activity from this region of the
brain. Similarly, RTI-113 has no effect on the [11C]-
ZBrET uptake, and yet ZBrET has a higher affinity for
the DAT and less selectivity for the SERT than ZIET.
Therefore, the inability of RTI-113 to significantly
displace the radioactivity from the striatum suggests
that [11C]ZIET and [11C]ZBrET bindings in the striatum
is not due to reversible DAT binding. To confirm that
the uptake of [11C]ZIET or [11C]ZBrET in the striatum
was due to specific binding, a chase study using phar-
macological doses of nonradioactive ZIET or ZBrET was
performed. The administration of 0.3 mg/kg of ZIET or
ZBrET 40 min after injection of the corresponding 11C-
labeled tropane demonstrated a significant reduction of
The microPET images acquired from 0 to 85 min
using [11C]ZIET and [11C]ZBrET showed specific ac-
cumulation in the putamen, midbrain, thalamus, pons,
medulla, and caudate as seen in Figures 2 and 3,
respectively, with the lowest uptake occurring in the
cerebellum. The corresponding time-activity curves
describing the kinetics of [11C]ZIET and [11C]ZBrET
binding in a rhesus monkey are presented in Figure 4
and Figure 5, respectively. The highest ratios relative
to cerebellum occurred at the end of the study and were
2.3, 2.1, 2.1, 2.0, and 1.7 for the putamen, midbrain,
pons, thalamus and medulla, respectively, when [11C]-
ZIET was injected and 2.3, 1.9, 1.8, 1.7, 1.6, and 1.3 for
the putamen, midbrain, caudate, thalamus, pons, and
medulla, respectively, when [11C]ZBrET was injected.
The higher ratios obtained with the more lipophilic [11C]-
ZIET compare to [11C]ZBrET can be explained by its
higher selectivity and slightly faster kinetics. The
regional brain distribution of [11C]ZIET and [11C]ZBrET
reflected the known distribution of SERT-rich sites in
the brain. However, the uptake of radioactivity in the
putamen, especially with [11C]ZBrET, was slightly
higher than in the midbrain and the thalamus. This
distribution has not been observed with previously
reported selective SERT imaging agents that have
shown the highest levels of SERT in the midbrain and
thalamus followed by intermediate level in the striatum,
low levels in the cortexes and negligible levels in the

1128 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Plisson et al.
F igu r e 3. Transverse, coronal, and sagittal microPET images acquired using [11C]ZBrET.
F igu r e 4. Time-activity curves for brain regions for [11C]-
ZIET in a rhesus monkey. Images were acquired for a total
time of 85 min.
F igu r e 5. Time-activity curves for brain regions for [11C]-
ZBrET in a rhesus monkey. Images were acquired for a total
time of 85 min.
radioactivity in the midbrain, putamen, caudate, thala-
mus, pons, and medulla. The percentages of washout
of [11C]ZIET were 35, 32, 30, 30, and 29% from the
putamen, midbrain, thalamus, pons, and medulla,
respectively, at 85 postinjection, and the percentages
of washout of [11C]ZBrET were 55, 49, 49, 49, 40, and
36% from the putamen, midbrain, thalamus, caudate,
pons, and medulla, respectively, at 85 min postinjections
(Figures 10 and 11). These studies indicate that radio-
activity uptake in the striatum is primarily due to
specific binding rather than nonspecific binding. To
define the nature of [11C]ZIET and [11C]ZBrET uptake
in the striatum, we performed a study in which the
monkey was pretreated with 0.3 mg/kg of RTI-113, 30
min before injection of radioligand, followed, 40 min
later by administration of 1.5 mg/kg of citalopram. The
time-activity curves (Figures 12 and 13) demonstrate
that the specific DAT ligand RTI-113 did not prevent
the accumulation of radioactivity in the striatum, but
unlike the previous citalopram studies, significant
displacement from the putamen, caudate, thalamus,
midbrain, pons, and medulla occurred. As seen in
Figures 14 and 15, the same results were obtained when
the monkeys were pretreated with citalopram (30 min
prior to the injection of radioligand) and RTI-113 was
administered 40 min postinjection. The accumulation
of radioactivity in the striatum was not blocked by the
presence of citalopram, but the radioactivity was dis-
placed from this region by RTI-113. The logical next
study was to inject simultaneously RTI-113 and citalo-

Radioligands for in Vivo Imaging
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1129
F igu r e 9. Time-activity curves for brain regions for [11C]-
ZBrET in a rhesus monkey with RTI-113 administered at
40 min. Images were acquired for a total time of 85 min.
F igu r e 6. Time-activity curves for brain regions for [11C]-
ZIET in a cynomolgus monkey with citalopram administered
at 40 min. Images were acquired for a total time of 85 min.
F igu r e 10. Time-activity curves for brain regions for [11C]-
ZIET in a rhesus monkey with ZIET administered at 40 min.
Images were acquired for a total time of 85 min.
F igu r e 7. Time-activity curves for brain regions for [11C]-
ZBrET in a rhesus monkey with citalopram administered at
40 min. Images were acquired for a total time of 115 min.
F igu r e 11. Time-activity curves for brain regions for [11C]-
ZBrET in a rhesus monkey with ZBrET administered at
40 min. Images were acquired for a total time of 85 min.
F igu r e 8. Time-activity curves for brain regions for [11C]-
ZIET in a cynomolgus monkey with RTI-113 administered at
40 min. Images were acquired for a total time of 85 min.
pram. These experiments carried out by injecting a mix
consisting of 0.3 mg/kg of RTI-113 and 1.5 mg/kg of
citalopram 80 min after the injection of [11C]ZIET and
[11C]ZBrET showed a dramatic reduction of radioactivity
from the SERT-rich regions including the striatum
(Figures 16 and 17). These dual-administration studies
indicate that uptake of radioactivity can be displaced
from the striatum when both SERT and DAT sites are
occupied.
1.5 and 1.3, respectively. The initial brain uptake of
CNS radioligands has been reported to be optimum
when log P7.4 ) 2.4-2.8.41 Our radiotracer’s values were
below the optimal range; nonetheless, both [11C]ZIET
and [11C]ZBrET achieved brain penetrance and good
specific (midbrain) to nonspecific binding (cerebellum)
ratios (2.1:1 and 1.9:1, respectively) at 85 min postin-
jection.
Meta bolite An a lysis in Mon k eys. Arterial plasma
samples from rhesus monkeys following antecubital vein
injection of [11C]ZIET and [11C]ZBrET were analyzed
for nonpolar, potentially brain permeable, metabolites
Lip op h ilicity. The lipophilicities of ZIET and ZBrET
were measured using the procedure of Wilson et al.40
The measured log P7.4 values for ZIET and ZBrET were

1130 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Plisson et al.
F igu r e 15. Time-activity curves for brain regions for [11C]-
ZBrET in a rhesus monkey with citalopram administered
30 min prior injection of [11C]ZBrET and RTI-113 administered
at 40 min. Images were acquired for a total time of 85 min.
F igu r e 12. Time-activity curves for brain regions for [11C]-
ZIET in a rhesus monkey with RTI-113 administered 100 min
prior injection of [11C]ZIET and citalopram administered at
40 min. Images were acquired for a total time of 85 min.
F igu r e 16. Time-activity curves for brain regions for [11C]-
ZIET in a rhesus monkey with a mix of citalopram and RTI-
113 administered at 80 min. Images were acquired for a total
time of 115 min.
F igu r e 13. Time-activity curves for brain regions for [11C]-
ZBrET in
a rhesus monkey with RTI-113 administered
100 min prior injection of [11C]ZBrET and citalopram admin-
istered at 40 min. Images were acquired for a total time of
85 min.
F igu r e 17. Time-activity curves for brain regions for [11C]-
ZBrET in a cynomolgus monkey with a mix of citalopram and
RTI-113 administered at 80 min. Images were acquired for a
total time of 115 min.
F igu r e 14. Time-activity curves for brain regions for [11C]-
ZIET in a rhesus monkey with citalopram administered
30 min prior injection of [11C]ZIET and RTI-113 administered
at 40 min. Images were acquired for a total time of 85 min.
tabolite found in arterial plasma using HPLC separation
and gamma-counter detection was in both cases a more
polar component. The abundance of the polar metabolite
increased to 91% of the plasma activity by 80 min for
[11C]ZIET and 89% by 80 min for [11C]ZBrET. The
identity of the polar metabolites was not elucidated
because they were not expected to enter the brain. In
both cases, the metabolites were eluted immediately
after the void volume and are probably the correspond-
ing free acid. This hypothesis is based on our experience
by a HPLC method. For both radiotracers, the major
radioactive component appearing in the initial arterial
plasma samples displayed a major peak on HPLC
spectra corresponding to unmetabolized authentic [11C]-
ZIET and [11C]ZBrET. The fraction of plasma radioac-
tivity corresponding to unmetabolized [11C]ZIET or
[11C]ZBrET rapidly decreased from 69% at 5 min to 12%
at 40 min for [11C]ZIET and from 63% at 5 min to 14.5%
at 40 min for [11C]ZBrET. The major radioactive me-

Radioligands for in Vivo Imaging
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1131
oil obtained was purified by flash chromatography (hexane:
ethyl acetate:triethylamine 75:21:4) to give a white solid
(155 mg, 41%). Spectroscopic and physical data were identical
to those previously reported.29
with other tropanes and a plasma metabolite study
reported in the literature42 showing that the major
metabolite obtained from [N-methyl-11C]â-CIT was the
[N-methyl-11C]â-CIT acid which did not enter the brain
in high quantity. The absence of labeled lipophilic
metabolites in the arterial plasma that could reenter
the brain permits uncorrected calculation of [11C]ZIET
and [11C]ZBrET input functions and subsequent quan-
tification of binding sites by tracer kinetic modeling
without correcting for lipophilic metabolites.
2â-Ca r bom eth oxy-3â-[4′-((Z)-2-tr im eth ylsta n n yleth en -
yl)p h en yl]tr op a n e (3). To a solution of 2â-carbomethoxy-3â-
(4′-bromophenyl)tropane (2) (214 mg, 0.63 mmol) in toluene
(10 mL) were added tetrakis(triphenylphosphine)palladium (0)
(44 mg, 0.04 mmol) and (Z)-1,2-trimethylstannylethylene (4)
(0.3 g, 0.78 mmol). The reaction mixture was stirred at 90 °C
for 3 h. The toluene was removed under reduced pressure, and
the black residue was purified by flash chromatography
(hexane:ethyl acetate:triethylamine 80:18.5:1.5) to afford a
yellow solid (160 mg, 57%). 1H NMR (CDCl3, 400 MHz) δ
-0.01-0.01 (m. 9H), 1.60-1.76 (m, 3H), 2.08-2.14 (m, 1H),
2.17-2.24 (m, 1H), 2.24 (s, 3H), 2.56-2.63 (m, 1H), 2.90-2.92
(m, 1H), 2.99-3.05 (m, 1H), 3.37-3.41 (m, 1H), 3.46 (s, 3H),
3.53-3.57 (m, 1H), 6.13 (d, J ) 13.6 Hz, 1H), 7.14-7.20 (m,
4H), 7.52 (d, J ) 13.6 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ
-7.87 (3C), 25.29, 26.19, 33.62, 34.09, 42.11, 51.37, 53.13,
62.47, 65.48, 127.10 (2C), 127.25 (2C), 132.98, 138.82, 142.51,
147.35, 172.35. HRMS (FAB) calcd for C21H32NO2Sn (120Sn)
[MH+]: 450.1455, found 450.1460.
(Z)-1,2-Tr im eth ylsta n n yleth ylen e (4). To a solution of
hexamethylditin (0.49 g, 1.37 mmol) in 5 mL of 1,4-dioxane
was added tetrakis(triphenylphosphine)palladium(0) (80 mg,
0.07 mmol). The reaction mixture was warmed at 60-65 °C,
and purified acetylene (passed successively through the fol-
lowing traps: cold trap at -78 °C, concentrated sulfuric acid,
solid potassium hydroxide, and Drierite) was bubbled through
the reaction mixture. After a few minutes, the reaction mixture
turned black. After 2 h the reaction was complete, and the
volatiles were evaporated under reduce pressure. Purification
by flash chromatography (hexane + 0.2% triethylamine) gave
a yellow oil (260 mg, 49%) which was used without any further
purification.
Con clu sion
Two new SERT ligands have been synthesized, ra-
diolabeled, and evaluated in rats and nonhuman pri-
mates. Competition binding assays in cells stably ex-
pressing the human monoamine transporters demon-
strated that ZIET and ZBrET have high affinities for
the SERT. ZIET showed significantly higher selectivity
for SERT over DAT than ZBrET, with ratios of 200 to
1, and 38 to 1, respectively. Biodistribution studies in
rats showed that [123I]ZIET uptake occurred specifically
in regions rich in SERT. In vivo microPET imaging
studies in monkeys demonstrated that [11C]ZIET and
[11C]ZBrET exhibited high uptake in the striatum,
diencephalon, and brainstem. However, a component of
striatal uptake not displaceable by SERT or DAT
ligands alone was observed. A series of a combination
of blocking and displacement studies with RTI-113 and
citalopram demonstrated that the component of striatal
uptake of [11C]ZIET was primarily SERT. These results
support the candidacy of [11C]ZIET and [123I]ZIET for
further study as radioligands for in vivo quantitation
of SERT sites by PET and SPECT, respectively.
2â-Ca r bom eth oxy-3â-[4′-((Z)-2-iod oeth en yl)p h en yl]tr o-
p a n e (ZIET). To a solution of 2â-carbomethoxy-3â-[4′-((Z)-2-
trimethylstannylethenyl)phenyl]tropane (3) (0.390 g, 0.87
mmol) in dichloromethane (15 mL) at 25 °C was added
dropwise iodine monochloride (1 M in dichloromethane, 0.85
mL). The reaction mixture was stirred for 10 min and then
quenched with an aqueous solution of sodium metabisulfite
(5% w/w, 10 mL). The organic layer was separated, washed
with water, and evaporated under reduced pressure. Purifica-
tion by flash chromatography (hexane:ethyl acetate:triethyl-
amine 80:18.5:1.5) afforded a yellow oil (0.261 g, 73%).
Additional purification by HPLC was performed to obtain the
pure standard for labeling (Waters, X-terra RP18 7.8 × 100
mm, 35% H2O in methanol + 0.1% Et3N, 3 mL/min, tR ) 10.2
Exp er im en ta l Section
All reagents used were obtained from commercially avail-
able sources. Solvents used in reactions were purchased from
Aldrich Chemicals (Milwaukee, WI) while solvents for chro-
matography were obtained from VWR (West Chester, PA).
Melting points are uncorrected and were determined in
capillary tubes using a Mel-Temp II apparatus obtained from
Laboratory Devices, Inc. (Holliston, MA). 1H NMR spectra were
recorded on a Varian spectrometer at 400 MHz or 300 MHz
and referenced to the NMR solvent (chemical shifts in ppm
values, J values in Hz). Mass spectra were determined on a
VG 70-S double focusing mass spectrometer using high-
resolution electron ionization (EI) or a J EOL J MS-SX102
double focusing mass spectrometer using fast atomic bombard-
ment (FAB). Silica gel column chromatography was performed
using Merck silica gel 60 (40-63 µm particle size). Thin-layer
chromatography (TLC) was performed using 250 µm layers of
F-254 silica on aluminum plates obtained from Whatman
(Clifton, NJ ).
Ch em istr y. 2â-Ca r bom eth oxy-3â-(4′-br om op h en yl)tr o-
p a n e (2). Magnesium chips (96 mg, 4 mmol) were stirred
vigorously for 2 h under dry argon flow. Ether (0.6 mL) was
added as well as a crystal of iodine. A solution of 1,4-
dibromobenzene (1 g, 4 mmol) in ether (3.5 mL) was added
dropwise in order to control and maintain the reflux. After
completion of the addition, the reaction mixture was refluxed
for 1 h then cooled to -40 °C. A solution of ecognine methyl
ester (0.2 g, 1.1 mmol) in ether/dichloromethane (5 mL/5 mL)
was added dropwise. The reaction mixture was stirred at -40
°C for 3 h then cooled to -70 °C and quenched with a solution
of trifluoroacetic acid (0.45 mL, 5.8 mmol) in dichloromethane
(2 mL). The reaction mixture was allowed to warm to -10 °C,
and 5N HCl was added. The aqueous layer was retained,
washed with ether, basified with (30% w/w) ammonium
hydroxide, and extracted with dichloromethane. The yellow
1
min). H NMR (CDCl3, 400 MHz) δ 1.58-1.77 (m, 3H), 2.10-
2.22 (m, 2H), 2.21 (s, 3H), 2.59-2.71 (m, 1H), 2.94-3.09 (m,
2H), 3.41-3.46 (m, 1H), 3.51 (s, 3H), 3.59-3.63 (m, 1H), 6.48
(d, J ) 8.4 Hz, 1H), 7.25-7.28 (m, 3H), 7.57 (d, J ) 8.0 Hz,
2H). 13C NMR (100 MHz) δ 25.36, 26.09, 33.86, 34.10, 42.16,
51.38, 52.84, 62.40, 65.49, 78.15, 127.23 (2C), 128.24 (2C),
134.21, 138.51, 143.82, 172.30. HRMS (EI) calcd for C18H22
-
NIO2: 411.0695. Found: 411.0688. HPLC Waters Nova-Pak
C18 column 3.9 × 150 mm, absorbance wavelength: 254 nM,
eluent A: 30% H2O in MeOH + 0.1% NEt3, 1 mL/min, tR
)
12.27 min, eluent B: 30% H2O in CH3CN + 0.1% NEt3, 1 mL/
min, tR ) 4.02 min.
2â-Ca r bom eth oxy-3â-[4′-((Z)-2-br om oeth en yl)p h en yl]-
tr op a n e (ZBr ET). To a solution of 2â-carbomethoxy-3â-[4′-
((Z)-2-trimethylstannylethenyl)phenyl]tropane (3) (0.110 g,
0.245 mmol) in dichloromethane (7.5 mL) at 25 °C was added
N-bromosuccinimide (49 mg, 0.272 mmol). The reaction mix-
ture was stirred for 10 min and then quenched with an
aqueous solution of sodium metabisulfite (5% w/w, 10 mL).
The organic layer was separated, washed with water, and
evaporated under reduced pressure. Purification by flash
chromatography (hexane:ethyl acetate:triethylamine 80:18.5:
1.5) afforded a yellow oil (0.063 g, 70%). Additional purification
by HPLC was performed to obtain the pure standard for

1132 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Plisson et al.
labeling (Waters, X-terra RP18 7.8 × 100 mm, 35% H2O in
methanol + 0.1% Et3N, 3 mL/min, tR ) 8.2 min). 1H NMR
(CDCl3, 300 MHz) δ 1.57-1.77 (m, 3H), 1.97-2.26 (m, 2H),
2.24 (s, 3H), 2.55-2.65 (m, 1H), 2.91-3.04 (m, 2H), 3.38-3.40
(m, 1H), 3.50 (s, 3H), 3.56-3.59 (m, 1H), 6.35 (d, J ) 8.1 Hz,
1H), 7.00 (d, J ) 8.1 Hz, 1H), 7.26 (d, J ) 8.4 Hz, 2H), 7.61 (d,
J ) 8.4 Hz, 2H). 13C NMR (CD3OD, 100 MHz) δ 25.69, 26.65,
34.62, 34.76, 42.00, 51.80, 53.51, 63.88, 66.56, 106.28, 128.29,
129.92, 133.42, 134.29, 144.54, 174.12. HRMS (EI) calcd for
The C18 SepPak was washed with water (40 mL) and the
tropane was eluted with ethanol (3 mL). After evaporation of
ethanol, the residue (5 mg) was purified by HPLC (Waters,
X-terra RP18 7.8 × 100 mm, 33% H2O in methanol + 0.1%
1
Et3N, 4 mL/min, tR ) 7.3 min) to give an oil (3 mg, 48%). H
NMR (CDCl3, 300 MHz) δ 1.54-1.75 (m, 3H), 2.06-2.34 (m,
2H), 2.27 (s, 3H), 2.55-2.68 (m, 1H), 2.87-3.45 (m, 2H), 3.38-
3.50 (m, 1H), 3.50 (s, 3H), 3.56-3.65 (m, 1H), 7.25 (m, 1H),
7.42 (s, 1H), 7.46 (d, J ) 8.1 Hz, 2H).
C
18H22BrNO2 (79Br): 363.0834. Found: 363.0837 (Int: 95%),
2â-car bom eth oxy-3â-[4′-((Z)-2-tr im eth ylstan n yleth en yl)-
p h en yl]n or tr op a n e (11). To a solution of 2â-carbomethoxy-
3â-(4′-bromophenyl)nortropane (10) (270 mg, 0.83 mmol) in
toluene (13 mL) were added tetrakis(triphenylphosphine)-
palladium(0) (58 mg, 0.05 mmol) and (Z)-1,2-trimethylstan-
nylethylene (4) (0.45 g, 1.27 mmol). The reaction mixture was
stirred at 90°C for 3 h. The toluene was removed under
reduced pressure, and the black residue was purified by flash
chromatography (hexane:ethyl acetate:triethylamine 65:30:5)
to afford a yellow oil (188 mg, 52%). 1H NMR (CDCl3, 300 MHz)
δ -0.04-0.14 (m, 9H), 1.60-2.19 (m, 6H), 2.38-2.48 (m, 1H),
2.74-2.77 (m, 1H), 3.21-3.29 (m, 1H), 3.37 (s, 3H), 3.68-3.75
(m, 2H), 6.16 (d, J ) 8.8 Hz, 1H), 7.14 (d, J ) 8.7 Hz, 2H),
7.18 (d, J ) 8.7 Hz, 2H), 7.53 (d, J ) 8.8 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ -7.89 (3C), 27.76, 29.22, 33.75, 35.59,
51.30, 51.38, 53.82, 56.47, 127.37 (4C), 133.52, 139.52, 141.67,
147.14, 174.09. HRMS (EI) calcd for C20H29NO2Sn (120Sn):
435.1220, found 435.1219.
calcd for C18H22BrNO2 (81Br): 365.0813. Found: 365.0829
(Int: 100%). HPLC Waters Nova-Pak C18 column 3.9 × 150
mm, absorbance wavelength: 254 nM, eluent A: 30% H2O in
MeOH + 0.1% NEt3, 1 mL/min, tR ) 12.21 min, eluent B: 30%
H2O in CH3CN + 0.1% NEt3, 1 mL/min, tR ) 4.47 min.
N-(ter t-Bu toxyca r bon yl)-2â-ca r bom eth oxy-3â-[4′-for m -
ylp h en yl]n or tr op a n e (5). Protocol and spectroscopic and
physical data were identical to those previously reported.28
N-(ter t-Bu toxyca r bon yl)-2â-ca r bom eth oxy-3â-[4′-(2,2′-
d ibr om o)eth en ylp h en yl]n or tr op a n e (6). To a solution of
carbon tetrachloride (94 mg, 0.28 mmol) in dichloromethane
(1.5 mL) at -5 °C was added dropwise a solution of triphen-
ylphosphine (149 mg, 0.57 mmol) in dichloromethane (1.5 mL).
The resulting yellow solution was stirred for 10 min at -5 °C,
then the aldehyde 5 (53 mg, 0.14 mmol) dissolved in dichlo-
romethane (1.5 mL) was added dropwise. The reaction mixture
was stirred for 25 min at 25 °C. The reaction mixture was
diluted with dichloromethane and washed with a solution of
ammonium hydroxide (5% w/w). The organic layer was dried
over anhydrous magnesium sulfate and evaporated. Purifica-
tion by flash chromatography (hexane:ethyl acetate 75:25) gave
2â-ca r bom eth oxy-3â-[4′-((Z)-2-br om oeth en yl)p h en yl]-
n or tr op a n e (ZBr ENT). To a solution of 2â-carbomethoxy-
3â-[4′-((Z)-2-trimethylstannylethenyl)phenyl]nortropane (11)
(0.188 g, 0.43 mmol) in dichloromethane (20 mL) at 0 °C was
added dropwise a solution of bromine (0.5 M in carbon
tetrachloride, 1.0 mL). The reaction mixture was stirred at this
temperature for 5 min, and then quenched with an aqueous
solution of sodium metabisulfite (5% w/w, 10 mL). The organic
layer was separated, washed with water, and evaporated
under reduced pressure. Purification by flash chromatography
(dichloromethane:methanol 95:5) afforded a yellow oil (0.11
g, 73%). The precursor for labeling was purified again by HPLC
(Waters, X-terra RP18 7.8 × 100 mm, 35% H2O in methanol +
0.1% Et3N, 3 mL/min, tR ) 7.1 min). 1H NMR (CDCl3, 400
MHz) δ 1.66-1.84 (m, 3H), 2.06-2.25 (m, 2H), 2.42-2.49 (m,
1H), 2.77-2.78 (m, 1H), 2.87 (br s, 1H), 3.24-3.30 (m, 1H),
3.38 (s, 3H), 3.78-3.85 (m, 2H), 6.39 (d, J ) 8.0 Hz, 1H), 7.02
(d, J ) 8.0 Hz, 1H), 7.19 (d, J ) 8.4 Hz, 2H), 7.62 (d, J ) 8.4
Hz, 2H). 13C NMR (CDCl3, 100 MHz) δ 27.31, 28.62, 33.15,
35.49, 50.57, 51.39, 53.71, 56.25, 106.13, 127.13 (2C), 128.99
(2C), 131.92, 133.36, 141.89, 173.83. HRMS (EI) calcd for
1
a yellow oil (63 mg, 84%). H NMR (CDCl3, 400 MHz) δ 1.42
and 1.46 (s, 9H), 1.65-1.85 (m, 3H), 2.0-2.18 (m, 2H), 2.74-
2.77 (m, 1H), 2.87-2.92 (m, 1H) 3.2-3.27 (m, 1H), 3.44 (s, 3H),
4.42-4.68 (m, 2H), 7.20-7.26 (m, 2H), 7.41 (s, 1H), 7.46 (d,
J ) 8.4 Hz).
N-(ter t-Bu toxyca r bon yl)-2â-ca r bom eth oxy-3â-[4′-((Z)-
2-br om oeth en yl)p h en yl]n or tr op a n e (7). To a degassed
solution of N-(tert-Butoxycarbonyl)-2â-carbomethoxy-3â-[4′-
(2,2′-dibromo)ethenylphenyl]nortropane (6) (62 mg, 0.117 mmol)
in dry toluene (2.5 mL) were added tetrakis(triphenylphos-
phine)palladium (0) (6 mg, 0.005 mmol) then a solution of
tributyltin hydride (35 mg, 0.13 mmol) in toluene (0.5 mL).
The reaction mixture was stirred for 1 h at 25 °C, then diluted
with dichloromethane and washed with water. Purification by
flash chromatography (hexane:ethyl acetate 75:25) gave an oil
1
(50 mg). H NMR (CDCl3) showed that the product obtained
contains 5% of dehalogenated compound.
2â-ca r bom eth oxy-3â-[4′-(2,2′-d ibr om o)eth en ylp h en yl]-
n or tr op a n e (8). To a solution of N-(tert-butoxycarbonyl)-2â-
carbomethoxy-3â-[4′-(2,2′-dibromo)ethenylphenyl]nortropane
(6) (23 mg, 0.043 mmol) in dichloromethane (1.5 mL) at -5
°C was added dropwise trifluoroacetic acid (0.15 mL). The
reaction mixture was stirred for 45 min at 25 °C. The solvent
and reagent were evaporated under reduced pressure. The
residue was dissolved in dichloromethane and washed with
ammonium hydroxide (5% w/w). Purification by HPLC (Wa-
ters, Novapak C18 semiprep 25 × 100 mm, 20% H2O in
methanol + 0.1% Et3N, 6 mL/min, tR ) 31.0 min) gave a thick
oil (13 mg, 70%).1H NMR (CDCl3, 400 MHz) δ 1.61-1.80 (m,
3H), 1.98-2.18 (m, 2H), 2.30-2.65 (br s, 1H), 2.38-2.45 (m,
1H), 2.74-2.79 (m, 1H), 3.20-3.27 (m, 1H), 3.73 (s, 3H), 3.71-
3.76 (m, 2H), 7.19 (d, J ) 8.0 Hz, 2H), 7.43 (s, 1H), 7.46 (d,
J ) 8.0 Hz, 2H). HRMS (FAB) calcd for C17H20O2NBr2 (2 79Br)
[MH+]: 427.9861. Found: 427.9852 (Int: 52.6%), calcd for
C
17H20BrNO2 (79Br): 349.0677. Found: 349.0678 (Int: 100%),
calcd for C17H20BrNO2 (81Br): 351.0656. Found: 351.0633
(Int: 93%).
2â-Car bom eth oxy-3â-[4′-((Z)-2-iodoeth en yl)ph en yl]n or -
tr op a n e (ZIENT). To a solution of 2â-carbomethoxy-3â-[4′-
((Z)-2-trimethylstannylethenyl)phenyl]nortropane (11) (0.1 g,
0.23 mmol) in dichloromethane (10 mL) at 0 °C was added
dropwise iodine monochloride (1 M in dichloromethane, 0.28
mL). The reaction mixture was stirred at this temperature for
7 min and then quenched with an aqueous solution of sodium
metabisulfite (5% w/w, 10 mL). The organic layer was sepa-
rated, washed with water, and evaporated under reduced
pressure. Purification by flash chromatography (dichlo-
romethane:methanol 95:5) afforded a yellow oil which was
purified by HPLC (Waters, X-terra RP18 7.8 × 100 mm, 35%
H2O in methanol + 0.1% Et3N, 3 mL/min, tR ) 8.15 min) to
give a thick oil (60 mg, 66%) that slowly crystallizes. 1H NMR
(CDCl3, 400 MHz) δ 1.64-1.81 (m, 3H) 2.00-2.17 (m, 2H),
2.39-2.47 (m, 1H), 2.76-2.77 (m, 1H), 2.87 (br s, 1H), 3.23-
3.29 (m, 1H), 3.38 (s, 3H), 3.72-3.79 (m, 2H), 6.52 (d, J )
8.4 Hz, 1H), 7.20 (d, J ) 8.4 Hz, 2H), 7.27 (d, J ) 8.4 Hz, 1H),
7.56 (d, J ) 8.4 Hz, 2H). 13C NMR (CDCl3, 100 MHz) δ 27.84,
29.27, 33.73, 35.80, 51.15, 51.36, 53.79, 56.49, 78.97, 127.29
(2C), 128.50 (2C), 135.05, 138.42, 142.82, 173.99. HRMS (EI)
calcd for C17H20INO2: 397.0539, Found 397.0528.
C
17H20O2NBr2 (1 79Br + 1 81Br) [MH+]: 429.9840. Found:
429.9836 (Int: 100.0%), calcd for C17H20O2NBr2 (2 81Br) [MH+]:
431.9820. Found: 431.9822 (Int: 49.2%).
2â-Ca r bom eth oxy-3â-[4′-(2,2′-d ibr om o)eth en ylp h en yl]-
tr op a n e (9). To a solution of 2â-carbomethoxy-3â-[4′-(2,2′-
dibromo)ethenylphenyl]nortropane (6 mg, 0.014 mmol) in N,N-
dimethylformamide (0.4 mL) was added 2 drops of methyl
iodide. The reaction mixture was heated at 100 °C for 8 min
then diluted with water (4 mL) and applied onto a C18 SepPak.

Radioligands for in Vivo Imaging
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1133
Ra d iola belin g. No-carrier-added [11C]CO2 was produced
through the bombardment of 14N2 gas containing 1% 16O2 by a
Siemens 11 MeV RDS 112 cyclotron at Emory University
Hospital through the 14N[p,R]11C reaction. Radiosyntheses
employing [11C]CH3I utilized a GE MicroLab methyl iodide
system for the conversion of [11C]CO2 to [11C]CH3I. The
syntheses of [11C]ZIET and [11C]ZBrET were performed using
the nortropane precursors, ZIENT and ZBrENT. The precursor
(∼ 0.5 mg) dissolved in N,N-dimethylformamide (250 µL) was
placed in a sealed v-vial (1.0 mL). The [11C]CH3I was delivered
as a gas to the reaction vial and bubbled through the solution
containing the precursor with ice bath cooling. After delivery
of [11C]CH3I, the sealed vessel was heated at 105 °C for 5 min.
The reaction mix was diluted with HPLC mobile phase
(500 µL) prior to injection onto the semiprep HPLC column.
HPLC purification was performed using a reverse phase C18
column (Waters, NovaPak C18, 25 × 100 mm) at a 9 mL/min
flow rate with a buffered mobile phase consisting of 32% water
in acetonitrile and containing 1.4 g of ammonium acetate per
liter of solvent. At a flow rate of 9 mL/min, the retention time
of [11C]ZIET and [11C]ZBrET were approximately 14 and
12 min, respectively, and fractions eluting at this time
containing activity were purified using a modified solid-phase
extraction (SPE) procedure based upon Lemaire’s method.43
The desired fractions were combined with an equal volume of
a 6 mg/mL aqueous sodium carbonate solution. After stirring,
the homogeneous solution was applied to a Waters C18 SepPak
(previously activated with ethanol (10 mL) and water (10 mL))
by transferring under vacuum through a cook line. The eluate
was directed to the waste.
combined and subject to SPE formulation as described previ-
ously. A sample of the final product from each production was
taken to determine the pH, the radiochemical purity and the
specific activity of the final dose. The same analytical HPLC
conditions used to assess doses of [11C]ZIET were used for [123I]-
ZIET.
Lip op h ilicity Mea su r em en ts. Measurement of distribu-
tion coefficients of radiolabeled compounds were performed
based on the method described by Wilson et al. using 0.02 M
sodium phosphate buffer at pH 7.4 and 1-octanol.40
Each log
P7.4 determination was performed in quadruplicate for each
radiotracer. A ∼20 µCi portion of the radiotracer was added
to 5 mL of the phosphate buffer and shaken in a separatory
funnel with 10 mL of octanol for 2 min to remove any polar
radiolabeled impurities accompanying the radiotracer. The
aqueous phase was discarded, the octanol phase was retained,
and four 2 mL aliquots of the octanol phase were pipetted into
four test tubes each containing 2 mL of the phosphate buffer.
The test tubes were capped and vigorously shaken for 10 min.
After shaking, the test tubes were centrifuged at 300g for
5 min to thoroughly separate the two phases. For each of the
four test tubes, a 0.5 mL portion of the upper octanol phase
was pipetted into a tube for counting. The remainder of the
octanol phase was carefully removed, the pipet tip was
changed, and a 0.5 mL portion of the buffer phase was pipetted
into a tube for counting. The radioactivity in the octanol and
the phosphate buffers for each replicate were determined with
a
Packard Cobra II auto-gamma counter (Perkin-Elmer,
Downers Grove, IL) and decay corrected. The log P7.4 values
were calculated for each replicate with the following equation:
log P7.4 ) log [counts in octanol phase/counts in buffer
phase]. The log P7.4 measurements from each replication were
averaged to give the log P7.4 value for the radiotracer.
The C18 SepPak was successively washed with saline (0.9%
NaCl, 40 mL) and ethanol (0.5 mL) (all directed to the waste).
The radiolabeled tropane was eluted with ethanol (1.5 mL)
and driven to a sterile empty vial containing 3.5 mL of saline.
The resulting solution was transferred under argon pressure
through a Millipore filter (pore size 1.0 µm) followed by a
smaller one (pore size 0.2 µm), to a 30 mL sterile vial
containing 10 mL of saline. The total synthesis time (including
purification and formulation) of [11C]ZIET and [11C]ZBrET
labeling was approximately 45 min from end of bombardment.
Radiochemical purity greater than 99% and specific activity
of 0.4-0.6 Ci/µmol were demonstrated using reverse-phase
analytical HPLC (NovaPak, 3.9 × 150 mm) at 1.0 mL/min flow
rate with a mobile phase consisting of 70:30:0.1 methanol:
water:triethylamine.
Rod en t Biod istr ibu tion Stu d ies. Tissue distribution
studies were performed in male Sprague-Dawley rats (200-
250 g) after intravenous injection of [123I]ZIET in 0.1 to 0.4
mL of 10% ethanol/saline. The animals were allowed food and
water ad libitum before the experiments. The animals were
anesthetized with an intramuscular injection of 0.1 mL/100 g
of a 1/1 ketamine (500 mg/mL)/xylazine (20 mg/mL) solution,
and cannulas were placed in the tail veins. The animals were
killed at the appropriate time points, their tissues were
dissected, and selected tissues were weighed and counted along
with dose standards in a Packard Cobra II auto-gamma
counter. The raw counts were decay-corrected to a standard
time, and the counts were normalized as the percent of total
injected dose per gram of tissue (%ID/g). The statistical
analyses of the rodent biodistribution studies were performed
with GraphPad Prism software package for one-way analysis
of variances (ANOVAs) and with SigmaStat software package
(SPSS, Inc., Chicago, IL) for two-way ANOVAs. The reported
p-values were calculated using Tukey’s posthoc test for all
pairwise comparisons.
The synthesis of [123I]ZIET was performed using no-carrier-
added Na[123I]I purchased from MDS Nordion, Inc. (Ottawa,
ON Canada). The trimethyltin precursor 3 (100 µg) dissolved
in ethanol (0.3 mL) was added to a sealed vial containing Na-
[
123I]I (∼5 mCi) in 0.1 N sodium hydroxide (∼10-30 µL). In
rapid succession, an excess of 0.4 N aqueous hydrochloric acid
(100 µL) was added followed by 3% hydrogen peroxide (50 µL).
The reaction vial was inverted several times to mix the
reagents, and the reaction was allowed to proceed at room
temperature for 25 min. The reaction was quenched by the
addition of aqueous sodium hydrogensulfate (0.25 g/mL,
50 µL), and the reaction mixture was drawn into a syringe
containing an aqueous solution of saturated sodium bicarbon-
ate (0.5 mL). The reaction mix was passed through a C18
SepPak (preconditioned with methanol (10 mL)), and the
SepPak was eluted sequentially with water (3, 10, and 5 mL).
The radiolabeled product was then eluted from the C18 SepPak
with 1 mL portions of methanol which were collected as
individual fractions. The methanol fractions containing sig-
nificant activity (∼five total fractions) were combined and
evaporated at 65 °C with argon flow over the course of
approximately 30 min. The residue was diluted with 0.4 mL
of water prior to HPLC. Purification via HPLC was performed
using an X-Terra column (Waters, Prep RP18 7.8 × 100 mm,
5 µm particle size) at a 3.0 mL/min flow rate with a mobile
phase consisting of 38% water in methanol + 0.1% of trieth-
ylamine. Under these conditions, the [123I]ZIET product had
a retention time of approximately 15 min. Fractions containing
radioactivity eluting at the expected retention time were
Tissue distribution studies were performed in male
Sprague-Dawley rats (200-250 g) after intravenous injection
of ∼20 µCi of [123I]ZIET in 0.2 mL of 10% ethanol/saline. The
rats were killed in groups of five at 5, 30, 60, and 120 min
after injection of [123I]ZIET. The brains of the rats were
dissected, and the amount of radioactivity in the various brain
regions was measured. In the blocking experiments, the rats
were divided into four groups, and three of the groups were
given a competing monoamine transporter ligand 15 min prior
to the injection of [123I]ZIET. The monoamine transporter
ligands used in this study were RTI-113, reboxetine, and
citalopram at doses of 4 mg/kg for each drug. The rats in each
of the four groups were killed 60 min after the injection of
[
123I]ZIET.
In Vitr o Com p etition Assa ys. Competition assays were
performed based on methods reported previously.44,45 Cell
membranes from human embryonic kidney cell line (HEK-293)
stably expressing hNET or hSERT (gift from Dr. Randy
Blakely, Vanderbilt University) and dog kidney cell line
(MDCK) stably expressing hDAT (gift from Dr. Gary Rudnick,
Yale University) were used in these assays. Cells were grown

1134 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Plisson et al.
to confluency in DMEM containing 10% fetal bovine serum
and geneticin sulfate and then harvested using pH 7.4
phosphate-buffered saline (PBS) containing 0.53 mM ethyl-
enediaminetetraacetic acid (EDTA) at 37 °C. Cell pellets were
prepared through centrifugation at 2000g for 10 min, the
supernatant was decanted, and the pellets were homogenized
with a Polytron PT3000 (Brinkman, Littau, Switzerland) at
11 000 rpm for 12 s in 30 volumes of PBS. The resulting cell
membrane suspensions were centrifuged at 43 000 × g for 10
min, the supernatants were decanted, and the resulting pellets
were stored at -70 °C until used in assays. Competition assays
were performed in 13 × 100 mm polystyrene tubes in a 2.0
mL final volume consisting of 1.7 mL of assay buffer, 100 µL
of competing ligand in assay buffer, 100 µL of radioligand in
assay buffer, and 100 µL of cell membrane suspension in assay
buffer. Cell membrane pellets were characterized prior to
competition assays to determine membrane concentrations
that gave optimal signal while not significantly affecting the
concentration of free radioligand. The cell membrane pellets
were resuspended in the appropriate volume of assay buffer
through brief homogenization using a Polytron PT3000 just
prior to their use in assays. Competing ligands were assayed
in triplicate at twelve concentrations ranging from 10-13 to 10-5
M. To ensure solubility, the competing ligands were dissolved
in 1/1 ethanol/5 mM aqueous hydrochloric acid and then
serially diluted in 5 mM hydrochloric acid. For SERT assays,
the assay buffer consisted of 53 mM Tris buffer, 126 mM
sodium chloride, and 5.3 mM potassium chloride (pH 7.9 at
room temperature). The equilibrium incubation time was 2 h
at room temperature. The radioligand used for SERT assays
was [3H]citalopram obtained from Dupont NEN (Boston, MA;
3100 GBq/mmol), and the measured Kd used for Ki calculation
was 1.8 nM. For NET assays, the assay buffer consisted of 53
mM Tris buffer, 320 mM sodium chloride, and 5.3 mM
potassium chloride (pH 7.4 at 4 °C). The equilibrium incuba-
tion time was 4 h at 4 °C. The radioligand used for NET assays
was [3H]nisoxetine obtained from Dupont NEN (3000 GBq/
mmol), and the measured Kd used for Ki calculation was 0.71
nM. For DAT assays, the assay buffer consisted of 42 mM
sodium phosphate buffer and 320 mM sucrose (pH 7.4 at room
temperature). The equilibrium incubation time was 1 h at
room temperature. The radioligand used for DAT assays was
throughout the imaging session. Respiration was maintained
through a mechanical ventilator with measurement of expira-
tory oxygen and carbon dioxide levels to ensure physiological
levels of respiration. The radiolabeled compounds were injected
via the antecubital vein over the course of 5 min. Quantitative
brain imaging studies employing [11C]ZIET and [11C]ZBrET
were performed using a Concorde MicroPET P4 system
(Knoxville, TN). In each study, the animal was positioned in
the scanner with its head immobilized with a thermoplastic
face mask (Tru Scan, Annapolis, MD). A transmission scan
was obtained with a germanium-68 source prior to the PET
study for attenuation correction of the emission data. Emission
data acquired were subject to iterative reconstruction (OSEM,
two iterations, 40 subsets) with no pre- or postfiltering to
provide images with an isotropic resolution of 3 mm fwhm.
For generation of time-activity curves, regions of interest
(ROIs) were drawn manually based on the anatomical land-
marks visible in reconstructed PET images using ASIPro
software (Concorde, Knoxville, TN). Several imaging studies
were performed using [11C]ZIET and [11C]ZBrET in rhesus and
cynomologus monkeys with and without injection of pharma-
cological doses of monoamine transporter ligands. Emission
data were collected in the MicroPET imaging studies continu-
ously for 85 or 115 min after injection of [11C]tropane and then
binned for analysis.
Meta bolite An a lysis in Mon k eys. Arterial plasma analy-
ses of [11C]ZIET and [11C]ZBrET metabolism were performed
in rhesus and cynomolgus monkeys, respectively. [11C]tropanes
were injected as described above and arterial blood samples
(4 mL) were collected at approximately 5, 40, 60, 80 min after
tracer injection. The blood was centrifuged and the plasma
separated. Acetonitrile (0.7 mL) was added to 0.5 mL of plasma
sample, and the resulting suspension was vortexed then
centrifuged at 10000 rpm for 3 min. The supernatant was
removed and filtered through a 0.45 µm PTFE membrane. A
0.5 mL amount of the filtrate was injected and analyzed by
HPLC with a UV detector (254 nm) and a flow-through
radioactivity detector. Chromatographic separation was per-
formed with a Waters, X-terra RP18 column (3.5 × 8 mm), a
mobile phase of methanol:water:triethylamine 80:20:0.1, and
a flow rate of 1.5 mL/min. Low activity samples (i.e. 40, 60,
and 80 min) were also fractionated (0.5 min collections) and
measured for radioactivity using a Packard Cobra automatic
gamma-counter. All activity measurements were half-life
decay-corrected.
[
123I]RTI-55 obtained from DuPont NEN (81.4 TBq/mmol), and
the measured Kd used for Ki calculation was 0.43 nM. The
radioligand concentrations used in the assays were approxi-
mately 0.3 to 0.4 nM for both [3H]citalopram and [3H]-
nisoxetine and 0.01 to 0.02 nM for [125I]RTI-55. All assays were
initiated by the addition of the cell membrane suspension. At
the end of the incubation, the assays were terminated by the
addition of ∼5 mL of assay buffer at 4 °C followed by rapid
vacuum filtration with 3 × 5 mL washes with assay buffer at
4 °C through GF/B filters (Whatman, Inc., Clifton, NJ )
presoaked in assay buffer containing 6 g of polyethylenimine
per liter. In the case of [3H]nisoxetine and [3H]citalopram, the
dried filters were placed in scintillation vials with 6 mL of
liquid scintillation cocktail (Ultima Gold, Packard Bioscience,
Merden, CT) and shaken. In the case of [125I]RTI-55, the dried
filters were placed in 12 × 60 mm glass test tubes and counted
directly. The amount of radioactivity present on the filters was
determined with a liquid scintillation counter (LKB 1209
Rackbeta, Perkin-Elmer Wallac, Inc., Gaithersburg, MD) in
the case of the tritiated ligands or in a gamma-counter (LKB-
Wallac RiaGamma 1274, Perkin-Elmer Wallac, Inc.) in the
case of [125I]RTI-55. The data from the competition curves were
analyzed and Ki values calculated using GraphPad Prism
software (GraphPad Software, San Diego CA). The Ki values
were reported as the geometric mean of at least three separate
assays for each compound unless otherwise stated.
Ack n ow led gm en t. This research was sponsored by
the Office of Health and Environmental Research, U.S.
Department of Energy under Grant No. DE-FG02-
97ER62637 and the Emory Conte Center for the
Neuroscience of Mental Disorders.The authors would
like to thank Mary David for assistance in preparation
of this manuscript. We also acknowledge the use of
Shared Instrumentation provided by grants from the
NIH and NSF.
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