A second-generation 99mtechnetium single photon emission computed tomography agent that provides in vivo images of the dopamine transporter in primate brain

Article abstract of DOI:10.1021/jm0301484

The dopamine transporter (DAT), located presynaptically on dopamine neurons, provides a marker for Parkinson's disease (Pd) and attention deficit hyperactivity disorder (ADHD). In ADHD, DAT density levels are elevated, while in Pd these levels are deplete

Full text of DOI:10.1021/jm0301484

J . Med. Chem. 2003, 46, 3483-3496
3483
A Secon d -Gen er a tion 99m Tech n etiu m Sin gle P h oton Em ission Com p u ted
Tom ogr a p h y Agen t Th a t P r ovid es in Vivo Im a ges of 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,† Thomas Zona,† Lihua Yang,† Hong Huang,† Ali A. Bonab,‡ Eli Livni,‡
Alan Fischman,‡ and Bertha K. Madras§
Organix Inc., 240 Salem Street, Woburn, Massachusetts 01801, Massachusetts General Hospital, Harvard Medical School,
Boston, Massachusetts, and Department of Psychiatry, Harvard Medical School and New England Regional Primate Research
Center, Southborough, Massachusetts 01772
Received May 1, 2003
The dopamine transporter (DAT), located presynaptically on dopamine neurons, provides a
marker for Parkinson’s disease (Pd) and attention deficit hyperactivity disorder (ADHD). In
ADHD, DAT density levels are elevated, while in Pd these levels are depleted. The depletion
of DAT levels also corresponds with the loss of dopamine. We now describe the design, synthesis,
biology, and SPECT imaging in nonhuman primates of second-generation 99mtechnetium-based
tropane ligands that bind potently and selectively to the DAT. We demonstrate that improved
selectivity and biological stability allows sufficient agent to enter the brain and label the DAT
in vivo to provide a quantitative measure of DAT density in nonhuman primates. We introduce
FLUORATEC (N-[(2-((3′-N′-propyl-(1′′R)-3′′R-(4-fluorophenyl)tropane-2′′â-1-propanoyl)(2-mer-
captoethyl)amino)acetyl)-2-aminoethanethiolato]technetium(V) oxide), a DAT imaging agent
that has emerged from these studies and is now in phase 1 clinical trials in the U.S.
In tr od u ction
in nuclear medicine as those containing the 99mtechne-
tium radionuclide. 99mTechnetium (99mTc; t1/2 ) 6.0 h,
140 keV, γ emission) 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 on site. Consequently, 99mTc-
based compounds are used clinically25,26 as, for example,
blood perfusion agents27 and cardiovascular imaging
agents.28 Recognizing the advantages of 99mTc, we9,29
and others13,30,31 have developed 99mTc-containing DAT
ligands with a view to diagnostic imaging. In 1996,29
we reported technepine, [N-[2-((3′-N′-propyl-3′′â-(4-fluo-
rophenyl)tropane-2′′â-carboxylic acid methyl ester)-
(2-mercaptoethyl)amino)acetyl]-2-aminoethanethio-
lato-99mtechnetium(V) oxide] the first transporter-medi-
ated 99mtechnetium-based in vivo SPECT imaging agent
(Figure 1) that labels the DAT in primates. Although
this ligand provided good SPECT images in primates
under excellent research conditions,9 it proved less
effective in the more demanding clinical setting. This
was thought to be a consequence of inadequate penetra-
tion of the blood-brain barrier (BBB) and therefore
insufficient biological availability at the site of interest,
the DAT. We reasoned that two aspects most probably
dominated this reduced biological availability. First was
the possibility that relatively rapid metabolic inactiva-
tion of the ligand by esterases in the blood would cause
a rapid decrease in the available ligand, and second was
that excessive lipophilicity may cause the ligand to be
retained in the proteinacious material within the blood.
Indeed, the half-life of altropane is approximately 40
min in humans, and this is thought to be primarily a
consequence of C2-ester hydrolysis in vivo.21 Conse-
quently we sought to remove the C2-ester of technepine
(Figure 1) and replace it with a topologically similar
Diagnosis of neurological disorders such as Parkin-
son’s disease (Pd) and attention deficit hyperactivity
disorder (ADHD) currently relies on the clinical skills
of the diagnostician. The severity of Parkinson’s disease
is accompanied by a decrease in dopamine neurons.
Therefore, a measure of the degeneration of dopamine
neurons can provide a window on the quantitative
diagnosis of Pd.1 A biological marker for dopamine
neurons and therefore for Pd has been identified,2,3 and
this marker, the dopamine transporter (DAT), is located
presynaptically on dopamine neurons. ADHD has been
demonstrated to be accompanied by a significant in-
crease in the dopamine transporter.4,5 Consequently,
diagnosis of ADHD should also be possible by quanti-
tation of the DAT on dopamine neurons. Currently there
is no clinically available diagnostic agent that can
enable quantitative diagnosis of these disorders in the
U.S. An iodinated agent, DaTSCAN (ioflupane) is avail-
able outside the U.S.6 The need for a diagnostic tool has
been widely recognized, and many research groups,
including our own, have focused efforts toward the
design of imaging agents that can target the DAT.7-17
A number of agents (Figure 1) designed for single photon
emission computed tomography (SPECT) are currently
in late-phase clinical trials. Among these agents is
altropane,8 an iodinated tropane that binds selectively
to the DAT, and altropane is an effective imaging agent
that can be used to diagnose both Pd and ADHD.18-24
However, iodinated agents are not as widely accepted
* To whom correspondence should be addressed. Phone: 781-932-
4142. Fax: 781-933-6695. E-mail: meltzer@organixinc.com.
† Organix Inc.
‡ Massachusetts General Hospital, Harvard Medical School.
§ Harvard Medical School and New England Regional Primate
Research Center.
10.1021/jm0301484 CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/04/2003

3484 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Meltzer et al.
F igu r e 1. Structures of SPECT imaging agents for the DAT.
group incapable of deactivation by blood esterases. In
addition, we reasoned that if the potency and, more
importantly, the selectivity of the new ligand could be
improved, compared with that of technepine, the en-
hanced BBB penetration would be further comple-
mented by the need for delivery of lesser amounts within
the caudate-putamen. We therefore drew from the body
of SAR data already developed for the parent tro-
panes32,33 to select optimum templates for this new
imaging agent.
We now describe the design, synthesis, biology, and
SPECT imaging in nonhuman primates of a number of
99mtechnetium-based agents. Furthermore, we introduce
FLUORATEC (O-1505T: N-[(2-((3′-N′-propyl-(1′′R)-3′′R-
(4-fluorophenyl)tropane-2′′â-1-propanoyl)(2-mercapto-
ethyl)amino)acetyl)-2-aminoethanethiolato]techne-
tium(V) oxide), a DAT imaging agent that has emerged
from these studies and is now in phase 1 clinical trials
in the U.S.34-36
pounds for further development. The synthetic approach
to the rhenium complexes in Table 1 is outlined in
Schemes 1-3.
The tropanes selected for incorporation in the final
ligands were all prepared along similar lines.32,39 Thus,
the keto ester (1R)-140,41 was converted to the enol
triflate 2 and coupled, via Suzuki coupling, to the
appropriate arylboronic acids. The 2,3-unsaturated tro-
panes 3 thus obtained were utilized to prepare the 2,3-
unsaturated ligands 14 and 15. These 2,3-enes 3 were
also subjected to samarium iodide reduction32,39 to
obtain 4, the boat (2â,3R), and 5, the chair (2â,3â),
configured tropanes. The C2-esters were then converted
to the C2-ketones through the Weinreb amides 7
(Scheme 2).42 Reaction of the Weinreb amides with
ethylmagnesium bromide then provided the ketones 8.
Demethylation with R-chloroethyl chloroformate (ACE-
Cl) then provided the nortropanes 9 as starting materi-
als for incorporation of the chelating ligand, 10.9 The
3â-configured nortropanes and the 2,3-unsaturated
nortropanes 22 and 23 (Scheme 3) were obtained
similarly.
The N2S2 ligand (MAMA′)37,43-45 was attached to
1-chloro 3-propyltriflate in methylene chloride to provide
the chloropropyl-MAMA′ synthon 10,9 which was uti-
lized to alkylate the various nortropane parent com-
pounds (9, 19, 22, 23) in the presence of KI and NaHCO3
to obtain the bistrityl protected compounds 11, 20, 24,
and 25, respectively, in reasonable yields. Rhenium was
then introduced upon reaction of the bistrityl protected
compounds with sodium perrhenate under reductive
conditions (SnCl2) in ethanol to provide rhenium che-
lates 12-15. Compounds 12-15 each exist in two
diastereomeric forms. The chirality of the tropane
moiety is derived from 1 and is therefore fixed. However,
Ch em istr y
The imaging agents described herein are composed
of three parts: (i) a potent 2-substituted 3-aryltropane,
selective for the DAT versus the SERT, (ii) a neutral
chelating unit to which the radionuclide 99mTc is bound,
and (iii) a tether that connects the chelating entity to
the tropane skeleton. Rhenium, an excellent model for
the radioactive 99mTc, has been utilized in place of 99mTc
in studies that do not require the presence of a radio-
label.9,37 It forms stable square-pyramidal N2S2 com-
plexes similar to those formed by 99mTc.38 Also, the
lipophilicity and biological availability of rhenium che-
lates are similar to those of 99mtechnetium chelates.38
Evaluation of the biology of rhenium chelated com-
pounds (Table 1) therefore facilitated selection of com-

99mTechnetium SPECT Agent
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3485
Ta ble 1. Inhibition of [3H]WIN 35,428 Binding to the Dopamine Transporter and [3H]citalopram Binding to the Serotonin
Transporter in Rhesus (Macaca mulatta) or Cynomolgus Monkey (Macaca fascicularis) Caudate-putamena
IC50 (nM)
DATb SERTb SERT
IC50 (nM)
DAT SERT SERT
IC50 (nM)
DAT SERT SERT
DAT/
DAT/
DAT/
Ar
4-FPhc,d
R
compd
compd
compd
15a , O-1135 3500
OMe
26a , O-1186
3.8
1300
342 27a , O-861
27a -1, O-927
27a -2, O-928
242 13a , O-1508
333
2.9
7.4
4.0
5.9
80
67
299
200
27
9
75
34
4000
1.1
4-FPh (1R,12S) OMe
4-FPh (1R,12R) OMe
4-FPhc
Et
12a , O-1505
12a -1, O-2020
12a -2, O-1972
26b, O-1196
12b, O-1561
26c, O-2130
2.05
3
497
1000
14a , O-1768
660
2100
3.2
4-FPh (1R,12R) Et
4-FPh (1R,12S) Et
1.5
10000 6667
3,4-Cl2Phd
3,4-Cl2Ph
2-naphthyld
4-FPh
OMe
10.6
5.3
4
639
337
120
60 27b, O-863
63 13b, O-2131
30 27c, O-1339
37
5
264
400
7.1 15b, O-1136
80
60
1693
2000
964
28
1.7
15.2
Et
14b, O-1769 1200
15c, O-1185 63.4
OMe
NMeOMe
3.64
58.9 16
3260 80
17a , O-1451 40.5
a
b
Each value is the mean of two or more independent experiments each conducted in different brains and in triplicate. DAT ) dopamine
transporter [3H]WIN 35,428; SERT ) serotonin transporter [3H]citalopram. c Mixture of (1R,12S) and (1R,12R) diastereomers. Compounds
26, 27, and 15 have a C2â-COOCH3 (J . Med. Chem. 1997, 40, 1835-1844).
d
the introduction of a chiral rhenium chelate leads to the
formation of these diastereoisomers. These are shown
for 12a in Figure 2: 12a -1 (1R,12R) and 12a -2 (1R,-
12S).
N-[(2-((3′-N′-propyl-3′′R-(4-fluorophenyl)tropane-2′′â-
propionyl)(2-mercaptoethyl)amino)acetyl)-2-amino-
ethanethiolato]rhenium(V) oxide and diastereoisomer
12a -1 is (1R,12R)-N-[(2-((3′-N′-propyl-3′′R-(4-fluorophe-
nyl)tropane-2′′â-propionyl)(2-mercaptoethyl)amino)acetyl)-
2-aminoethanethiolato]rhenium(V) oxide. The absolute
structures of 27a -1 and 27a -2, the C2-ester, 3â-aryl
analogues, have been reported previously.9
Structural assignment of the rhenium chelates was
1
facilitated by H NMR analysis. The presence of dia-
1
stereomeric mixtures was clearly evidenced in the H
NMR spectra of these compounds. In this regard, the
C13 protons are particularly diagnostic for the RedO
complexes. In the RedO chelate 12a , both C13 protons
appear considerably downfield in the region of δ 4.0-
5.2. This downfield shift is due, in part, to the aniso-
tropic effect of the RedO bond and the quaternary
nature of the nitrogen (N12). The diastereotopy of the
C13 protons is evidenced in that the C13â proton
manifests a four-line multiplet [12a -1, δ 4.74 (d, J )
16.5 Hz, 1H, H-13â); 12a -2, δ 5.14 (d, J ) 16.5 Hz, 1H,
H-13â)]. The corresponding 13R proton also shows a
four-line multiplet [12a -1, δ 4.08 (d, J ) 16.5 Hz, 1H,
H-13R); 12a -2, 4.13 (d, J ) 16.5 Hz, 1H, H-13R)] with
an almost 1 ppm upfield shift from the 13â. The effect
of the RedO bond is also apparent on the 16R and â
protons. The 16R proton resonance is a multiplet for
12a -1 at about δ 4.11, and the 16â proton appears as a
multiplet at about δ 4.57. In the diastereomeric mixture
of 12a , the 16â protons overlap, resulting in a multiplet
at about δ 4.60.
The two diastereomers of 12a coelute on TLC in a
variety of solvent systems, and therefore, chromato-
graphic separation is difficult. However, two consecutive
MPLC column chromatographic separations, with a
high ratio of compound to silica gel (171 mg:43 g) and
collection of multiple small fractions, allowed pooling
of early versus late fractions to provide purified samples
of each diastereoisomer. The absolute structure of
diastereoisomer 12a -2 was established by X-ray crystal-
lography and confirmed that the propyl group attached
at N12 bears a syn relationship to the RedO bond
(Figure 3). Therefore, diastereoisomer 12a -2 is (1R,12S)-
Biology
Rhenium is an excellent model for the radioactive
99mTc; therefore, the in vitro binding data were obtained
for the rhenium chelates. Since 99mTc is introduced in a
final preparative step for routine use, a diastereomeric
mixture of the imaging agents will likely be used
clinically. Therefore, the biological activity of the dia-
stereomeric mixture of Re-12a was important; however,
binding constants for each of the diastereoisomers 12a -1
and 12a -2 were also measured.
The affinities (IC50) of the rhenium compounds for the
dopamine and serotonin (SERT) transporters were
determined in competition studies using [3H]-3â-(4-
fluorophenyl)tropane-2â-carboxylic acid methyl ester
([3H]WIN 35,428 or [3H]CFT) to label the dopamine
transporter46 and [3H]citalopram to label the serotonin
transporter.29 Studies were conducted in cynomolgus or
rhesus monkey striata because these compounds are
part of an ongoing investigation of structure-activity
relationships at the dopamine transporter in these
tissues33,41,46 and meaningful comparisons with an
extensive database and in vivo imaging data can be
made. Competition studies were conducted with a fixed
concentration of radioligand and a range of concentra-
tions of the test drug. All drugs inhibited [3H]WIN 35,-
428 and [3H]citalopram binding in a concentration-
dependent manner. Binding constants are presented in
Table 1.
The enhanced selectivity generally manifested by 3R-
aryltropanes32 has been retained for these N-substituted

3486 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Meltzer et al.
Sch em e 1. General Synthesis of Rhenium- and Technetium-Labeled DAT Ligands 12-15a a
a
Reagents and conditions: (i) NaN(TMS)2, THF, -78 °C, PhN(CF3SO2) 2; (ii) LiCl, Pd2dba3, Na2CO3, CH2 (OC2H5) 2, 22 °C, ArylB(OH)
2, ∆; (iii) (a) SmI2, THF, -78 °C, (b) MeOH, TFA (54% 3â and 29% 3R).
rhenium complexes. Indeed, the 3R-(4-fluorophenyl)-C2-
ketone 12a has IC50 ) 2.05 nM (DAT) and IC50 ) 497
nM (SERT). It therefore has a potency similar to that
of technepine 27a (DAT IC50 ) 2.9 nM) and is almost
10-fold more selective (SERT IC50 ) 80 nM). The two
diastereomers of 12a showed similar potency at the DAT
(IC50 ) 1.5, 3.0 nM). Both compounds were extremely
selective; however, 12a -2 was about 20-fold more selec-
tive than 12a -1.
SP ECT Im a ge
Figure 4 shows a representative midstriatal, trans-
axial slice of a high count density SPECT image of a
monkey injected with approximately 20 mCi of 99mTc-
12a . The image was acquired between 15 and 45 min
after injection. There is a relatively intense tracer
accumulation in the striatum. The absence of uptake
in the thalamus and midbrain, regions with high SERT
density, is consistent with the high DAT selectivity of
this compound. Compound 12a has now been used to
provide images in humans.35
Syn th esis of 12a (F LUORATEC:
N-[(2-((3′-N′-P r op yl-(1′′R)-3′′r-(4-flu or op h en yl)-
tr op a n e-2′′â-1-p r op a n oyl)(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 (V) Oxid e)
Discu ssion
The degeneration of dopamine neurons in the stria-
tum can provide a window to the diagnosis of Parkin-
son’s disease,1 and the dopamine transporter has been
identified as a biological marker for these neurons.2,3
This transporter, located presynaptically, also provides
a suitable marker for ADHD because this syndrome is
accompanied by a significant increase in DAT density.4,5
Therefore, the diagnosis of both Pd and ADHD can be
achieved by quantitation of the DAT on dopamine
neurons. SPECT and PET have been utilized to image
Synthesis of 99mTc-12a as a 1:1 diastereomeric mix-
ture was achieved by deprotection of the bistritylated
compound 11 with trifluoroacetic acid under cation
trapping conditions (triethylsilane). The bisthiol ob-
tained was treated with 99mtechnetium glucoheptonate
and the product was purified by HPLC to provide 6 mCi
of the ligand 99mTc-12a for in vivo studies.

99mTechnetium SPECT Agent
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3487
Sch em e 2. General Synthesis of Rhenium- and Technetium-Labeled Ligands 12 and 13a
a
Reagents and conditions: (i) dioxane/H2O, ∆, 24 h; (ii) (a) (COCl) 2, CH2Cl2, DMF, 22 °C, 45 min; (b) (MeO)MeNH, HCl, pyridine, 1
h; (iii) EtMgBr; Et2O, THF, 0-65 °C; (iv) ACE-Cl, ∆; (v) 10, KI, NaHCO3, CH3CN, ∆, 4 h; (vi) SnCl2, EtOH, NaReO4, ∆ or (C2H5)3Si,
Na99mTc pertechnetate; (vii) Me3Al, (MeO)MeNH‚HCl.
Sch em e 3. General Synthesis of Rhenium- and Technetium-Labeled Ligands 14 and 15a
a
Reagents and conditions: (i) EtMgBr; Et2O, Et3N, THF, 0-10 °C; (ii) ACE-Cl, ∆, 5 h; (iii) 10, KI, NaHCO3, CH3CN, ∆, 4 h; (iv) SnCl2,
EtOH, NaReO4, ∆; or (C2H5)3Si, Na99mTc pertechnetate.
the DAT, and radiolabeled imaging agents suitable for
both procedures are now available for research pur-
poses. One such 99mTc-labeled ligand is technepine,
99mTc-27a , reported from these laboratories in 1996.9,29
A shortcoming of technepine was related to the finding
that although it provided excellent images in primates
under ideal research conditions, it did not perform
comparably with altropane,21 our benchmark compound
for a useful clinical SPECT diagnostic agent in a more
demanding clinical setting. The focus of the current
report is the design and synthesis of an improved
second-generation 99mTc-labeled SPECT ligand. To ac-
complish this, we returned to the SAR data for the
8-azabicyclo[3.2.1]octanes32 in which we had established
that the orientation of, and substitution at, the C3
position dictates selectivity for the DAT versus the
SERT. Furthermore, SAR had also shown that varied
functionality could be tolerated at C2,47-52 thus allowing
removal of the hydrolyzable C2-ester present in tech-
nepine. A series of selected rhenium-labeled model
ligands was therefore prepared, and their binding
potencies and selectivities to neurotransmitter uptake

3488 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Meltzer et al.
F igu r e 2. Diastereomers of 12a and 27a .
DAT.9 In summary, the design of these agents hinged
on a tropane moiety that serves as a “guiding ligand”
to which a neutral 99mTc (or Re) chelate could be
attached by a methylene chain (“tether”).
Th e Gu id in g Liga n d . Selection of a potent “guiding
ligand” was aided by structure-activity relationships
of the tropane family of compounds.32 The most potent
tropane DAT inhibitors possess either a C3-(2-naphthyl)
group52 or a C3-(3,4-dichlorophenyl) group.33 However,
such substitution can lead to high lipophilicity and thus
reduce BBB permeation. Furthermore, as bulk is intro-
duced at the N8, steric bulk on the C3 aromatic ring is
less well tolerated.9 Altropane has a C3-(4-fluorophenyl)
group. Therefore, in an initial exploration it was prudent
to utilize all three templates.
We had reported32 that selectivity for the DAT versus
the SERT can be substantially controlled by orientation
of the C3-aryl ring. The 3â-aryl (chair) orientation is
generally least selective for the DAT, while both the
flattened 2,3-ene and the 3R-aryl (boat) substituents
confer considerable selectivity for the DAT.39 Therefore,
we anticipated that either the boat-conformed com-
pounds with the C3R configuration or the 2,3-ene
compounds may provide the optimum guiding ligand.
As discussed above, technepine, with a C2 methyl
ester, manifested insufficient BBB permeability for
routine use, and images obtained in a clinical setting
could not be reliably quantified. A major cause of this
was assumed to be a result of in vivo esterase hydrolysis
of the methyl ester. To eliminate this possibility but
retain overall topology, we explored the introduction of
a C2 ethyl ketone. It has been established that C2-
ketones can lead to extremely potent DAT inhibitors.52
In summary, we based the design of a second-generation
imaging agent on a ligand with enhanced selectivity (3R-
configuration), improved biological half-life (C2-alkyl
ketone), and enhanced potency (C3 substitution: 3,4-
dichlorophenyl, 2-naphthyl, or 4-fluorophenyl).
P oin t of Atta ch m en t. On the basis of the success
with technepine and the fact that it has now been
conclusively proven that sufficient space exists within
the dopamine transporter to accept substantial bulk at
the 8-aza position, we attached the tether and chelator
to the nitrogen of the tropane skeleton.
Ch ela tor . The N2S2 ligand, MAMA′,37,43-45 was se-
lected as the 99mtechnetium chelating moiety for these
ligands. We had already shown that a tropane attached
to a MAMA′ moiety was capable of potent and selective
F igu r e 3. ORTEP diagram of compound 12a -2.
F igu r e 4. Midstriatal, transaxial slice of a SPECT image of
a monkey injected with 12a .
systems (DAT and SERT) were measured (Table 1). Of
the 3R-aryl 2â-ethyl ketones, compounds 12a and 12b
appeared most promising as prospective SPECT ligands.
Compound 12a was particularly potent (IC50 ) 2.05 nM)
and selective (242-fold). Compound 26a , almost equally
potent yet more selective (343-fold) was set aside
because the C2-ester would likely be subject to metabolic
hydrolysis, as had presumably occurred with technepine
(27a ). Thus, 12a was selected for insertion of 99mTc. It
provided suitable SPECT images in monkeys and was
therefore deemed potentially suitable for clinical evalu-
ation. Compound 99mTc-12a , the C3-(4-F-phenyl) ana-
logue, is now in phase 1 clinical trials.
Design of99mTech n etiu m -Labeled Im agin g Agen ts.
We have previously described our approach to the
synthesis of 99mTc-labeled tropanes for imaging of the

99mTechnetium SPECT Agent
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3489
binding to the DAT and, more importantly, the neutral
99mTc-containing ligand was capable of crossing the
blood-brain barrier.9
can be designed to carry the 99mTc radionuclide across
the blood-brain barrier in sufficient quantity and with
sufficient specificity to obtain in vivo images of the
striatum in primates and humans.35
Biology. The binding affinities (IC50) of the rhenium
chelates for the dopamine and serotonin transporters
are presented in Table 1. The most potent and most
selective compound is the 3R-boat-configured 12S dia-
stereomer 12a -2, which has an IC50 of 1.5 nM and a
selectivity of 6667 (DAT/SERT). Of interest is the fact
that the two diastereomers 12a -1 and 12a -2, while
similar in DAT potency (12a -1, IC50 ) 3 nM; 12a -2, IC50
) 1.5 nM), differ markedly in selectivity (333 vs 6667).
Most important, the mixture of diastereomers 12a is
sufficiently potent (IC50 ) 2.05 nM) and selective (DAT/
SERT ) 242) to provide a promising SPECT ligand. It
should be noted that 12a is about equipotent with 27a
(technepine9) at the DAT but is considerably more
selective than 27a . The 3-(3,4-dichlorophenyl) analogue
12b is also potent (DAT IC50 ) 5.3 nM) and manifests
good selectivity (DAT/SERT ) 63). Surprisingly, and
contrary to the SAR previously discovered for the
bicyclo[3.2.1]octanes in general,32 the 2,3-unsaturated
rhenium chelates 14 and 15 manifest poor binding
affinity for the DAT (63-- 3500 nM), and therefore,
none of these compounds were selected for insertion of
99mTc. In a comparison between 2-carbomethoxy com-
pounds and 2-propionyl compounds, it is clear that the
presence of either a ketone or methyl ester does not
affect potency at the DAT significantly.
The 3â-compounds, including technepine 27a , mani-
fested good to excellent binding affinity (2.9-40 nM) for
the DAT; however, selectivity for the DAT versus the
SERT for these chair conformers was, as expected,
weaker than for their boat counterparts 12. This is
because the 3R-boat compounds are far less potent at
the SERT.
A comparison of DAT and SERT inhibition of the
individual diastereomers of 26a and 27a is interesting
(Figure 2). While in the case of 26a , the (1R,12S)-26a -1
is less potent and less selective at the DAT than its (1R,-
12R) diastereomer 26a -2, the reverse is true for the 3R-
configured 2-propionyl analogues, 12a . In this later case,
the (1R,12S)-12a -2 diastereomer proves to be twice as
potent and about 20-fold more selective than its (1R,-
12R)-12a -1 counterpart. During the formation of the
technetium chelates, both diastereomers are produced,
and consequently it is clinically most convenient to
utilize a mixture of diastereoisomers in imaging experi-
ments.
Exp er im en ta l Section
NMR spectra were recorded on a Bruker 100, a Varian XL
400, a Bruker 300, or a J EOL 300 NMR spectrometer with
tetramethylsilane (TMS) as internal standard and CDCl3 as
solvent. Melting points are uncorrected and were measured
on a Gallenkamp melting point apparatus. Optical rotations
were measured at the sodium D line at 21 °C using a J ASCO
DIP 320 polarimeter (1 dcm cell). Thin-layer chromatography
(TLC) was carried out on Baker Si 250F plates. Visualization
was accomplished with iodine vapor, UV exposure, or treat-
ment 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.
All reactions were conducted under an atmosphere of dry
nitrogen. Elemental analyses were performed by Atlantic
Microlab, Atlanta, GA. HPLC analyses were carried out on a
Waters 510 system with detection at 254 nm on a Waters 8
mm, C-18, 10 µm reverse-phase column. A Beckman 1801
scintillation counter was used for scintillation spectrometry.
Pd2dba3 is trisdibenzylideneacetone dipalladium, TFA is tri-
fluoroacetic acid, THF is tetrahydrofuran, and EtOAc is ethyl
acetate. Room temperature is 22 °C. Samples of 0.1% bovine
serum albumin and (-)-cocaine were purchased from Sigma
Chemicals. [3H]WIN 35,428 (2â-carbomethoxy-3â-(4-fluorophe-
nyl)-N-[3H]methyltropane, 79.4-87.0 Ci/mmol) and [3H]citalo-
pram (86.8 Ci/mmol) were purchased from DuPont-New
England Nuclear (Boston, MA). (-)-Cocaine hydrochloride for
the pharmacological studies was donated by the National
Institute on Drug Abuse [NIDA]. Fluoxetine was donated by
E. Lilly & Co.
(1R)-2-Ca r boxym eth oxy-3-[[(tr iflu or om eth yl)su lfon yl]-
oxy]-8-m et h yl-8-a za b icyclo[3.2.1]oct -2-en e (2).53 (1R)-
(-)-2-Carbomethoxy-3-tropinone, 141 (1.0 g, 5.1 mmol), was
dissolved in anhydrous THF (20 mL), and the resulting
solution was cooled to -78 °C. A solution of sodium bistrime-
thylsilylamide (1.0 M, 5.56 mL, 5.56 mmol) was added slowly
to the solution. After 30 min, N-phenyltrifluoromethane sul-
fonamide (1.94 g, 5.43 mmol) was added. The resulting solution
was stirred for a further 45 min at -78 °C and then allowed
to attain room temperature and stirred for a further 2 h. All
solvent was evaporated, and the residue was pumped to
dryness. Flash chromatography (2-16% CH3OH in EtOAc)
gave 2 (1.62 g, 97%) as a yellow oil. Rf ) 0.65 (10% CH3OH in
1
EtOAc). H NMR: δ 3.92 (d, J ) 5 Hz, 1H), 3.80 (s, 3H), 3.42
(t, J ) 6 Hz, 1 H), 2.84 (dd, J ) 18, 4 Hz, 1H), 2.39 (s, 3H),
2.1-2.2 (m, 2H), 1.97 (m, 2H), 1.58 (m, 1H).
(1R)-2-Ca r boxym eth oxy-3-(4-flu or op h en yl)-8-m eth yl-
8-a za bicyclo[3.2.1]oct-2-en e (3a ).53 LiCl (6.45 g, 150.9 mmol)
and an aqueous solution of Na2CO3 (2.0 M, 71 mL) were added
to a solution of 2 (23.23 g, 70.55 mmol), and 4-fluorophenyl-
boronic acid (11.78 g, 84.19 mmol) in diethoxymethane (300
mL) was added at room temperature. The resulting solution
was degassed under N2 for 15 min before the addition, in one
portion, of Pd2dba3 (0.05 equiv) under a strong stream of N2,
and the reaction mixture was heated to reflux for 3 h under
N2. TLC showed the reaction was complete. The mixture was
allowed to cool to room temperature and diluted with Et2O
(300 mL) and filtered through Celite. The Celite was washed
with Et2O (3 × 100 mL) and water (3 × 50 mL). The combined
washes were then basified with NH4OH to pH 11. The layers
were separated, and the Et2O layer was washed with brine.
The aqueous layers were combined and back-extracted with
EtOAc (100 mL). The combined organic layers were dried over
Na2SO4, filtered, and concentrated to dryness to yield a yellow
oil that was further purified by flash chromatography (EtOAc
60%, hexanes 35%, Et3N 5%) to afford 3a as a yellow oil (18.8
g, 89%). Rf ) 0.28 (EtOAc 60%, hexanes 35%, Et3N 5%). 1H
NMR: δ 7.13-7.07 (m, 2H), 7.00 (tt, 2H, J ) 8.7, 2.1 Hz), 3.85
Con clu sion
Rhenium and 99mtechnetium-labeled tropane ligands
have been prepared and evaluated as prospective SPECT
imaging agents for the dopamine transporter. Select
compounds that manifested high potency and selectivity
for the DAT were also shown to cross the blood-brain
barrier and label the dopamine transporter in vivo.
FLUORATEC (N-[(2-((3′-N′-propyl-(1′′R)-3′′R-(4-fluo-
rophenyl)tropane-2′′â-1-propanoyl)(2-mercaptoethyl)-
amino)acetyl)-2-aminoethanethiolato]technetium(V) ox-
ide) has emerged as a second-generation 99mtechnetium-
labeled SPECT imaging agent for the dopamine trans-
porter in striatum and is now in a phase I clinical trial
in the U.S. Thus, we have demonstrated that a molecule

3490 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Meltzer et al.
(d, 1H, J ) 5.5 Hz), 3.50 (s, 3H), 3.35 (t, 1H, J ) 5.6 Hz), 2.74
(dd, 1H, J ) 18.8, 4.8 Hz), 2.44 (s, 3H), 2.28-2.12 (m, 2H),
2.05-1.94 (m, 2H), 1.67-1.60 (m, 1H). Anal. (C16H18O2FN‚1/
6H2O) C, H, N.
3.52 (s, 3H), 3.33 (m, 1H), 2.92 (m, 1H), 2.86 (m, 1H), 2.50 (m,
1H), 2.21 (s, 3H), 2.0-2.1 (m, 2H), 1.6-1.7 (m, 3H). Anal.
(C16H19NO2Cl2) C, H, N, Cl.
(1R)-2â-Ca r boxym eth oxy-3r-(2-n a p h th yl)-8-m eth yl-8-
azabicyclo[3.2.1]octan e (4c) an d (1R)-2â-Car boxym eth oxy-
3â-(2-n a p h th yl)-8-m eth yl-8-a za bicyclo[3.2.1]octa n e (5c).
The procedure described above was followed to provide 4c and
5c as white solids. 4c: (18%). Mp 117-118 °C. Rf ) 0.30
(EtOAc 20%, hexane 75%, Et3N 5%). 1H NMR: δ 7.80-7.74
(m, 3H), 7.66 (s, 1H), 7.47-7.38 (m, 2H), 7.36 (dd, 1H), 3.57
(s, 3H), 3.60-3.48 (m, 1H), 3.40 (d, 1H, d, J ) 6.6 Hz), 3.32 (t,
1H, J ) 7.3 Hz), 2.67 (dd, 1H, J ) 9.5, 1.5 Hz), 2.53 (dt 1H, J
) 13.7, 8.1 Hz), 2.28 (s, 3H), 2.31-2.05 (m, 2H), 1.69-1.49
(m, 3H). Anal. (C20H23NO2) C, H, N. 5c: (35%). Mp 94-95 °C.
Rf ) 0.30 (EtOAc 20%, hexane 75%, Et3N 5%). 1H NMR: δ
7.78-7.73 (m, 3H), 7.68 (s, 1H), 7.45-7.36 (m, 3H), 3.60 (dd,
1H, J ) 6.6, 2.7 Hz), 3.47-3.41 (m, 1H), 3.44 (s, 3H), 3.17 (dt,
1H, J ) 13.2, 5.0 Hz), 3.03 (t, 1H, J ) 3.9 Hz), 2.73 (td, 1H, J
) 12.4, 2.7 Hz), 2.29-2.11 (m, 2H), 2.26 (s, 3H), 1.84-1.75
(m, 2H), 1.71-1.60 (m, 1H). Anal. (C20H23NO2‚1/4H2O) C, H,
N.
(1R)-2-Car boxym eth oxy-3-(3,4-dich lor oph en yl)-8-m eth -
yl-8-a za bicyclo[3.2.1]oct -2-en e (3b ). The procedure de-
scribed above was followed to provide 3b as a yellow oil (512
mg, 83%). Rf ) 0.56 (10% Et3N in EtOAc). 1H NMR: δ 1.61
(m, 1H), 1.9-2.05 (m, 2H), 2.1-2.3 (m, 2H), 2.43 (s, 3H), 2.76
(dd, J ) 19, 4.7 Hz, 1H), 3.36 (t, J ) 4.9 Hz, 1H), 3.52 (s, 3H),
3.86 (d, J ) 5.5 Hz, 1H), 6.96 (dd, J ) 8.3, 1.9 Hz, 1H), 7.2 (d,
J ) 2.2 Hz, 1H), 7.37 (d, J ) 8.2 Hz, 1H). Anal. (C16H17O2-
NCl2) C, H, N.
(1R )-2-Ca r b oxym e t h oxy-3-(2-n a p h t h yl)-8-m e t h yl-8-
a za bicyclo[3.2.1]oct-2-en e (3c). The procedure described
above was followed to provide 3c as a light-yellow solid (94%).
Mp 99-101 °C. Rf ) 0.30 (EtOAc 60%, hexanes 35%, Et3N 5%).
1H NMR: δ 7.83-7.77 (m, 3H), 7.60 (s, 1H), 7.49-7.43 (m,
2H), 7.28-7.24 (m, 1H), 3.91 (d, 1H, d, J ) 5.2 Hz), 3.43 (s,
3H), 3.39 (t, 1H, J ) 5.6 Hz), 2.85 (dd, 1H, J ) 18.8, 4.5 Hz),
2.50 (s, 3H), 2.28-2.00 (m, 4H), 1.75-1.68 (m, 1H). Anal.
(C20H21NO2‚1/5H2O) C, H, N.
(1R)-2â-Ca r boxym eth oxy-3r-(4-flu or op h en yl)-8-m eth -
yl-8-a za b icyclo[3.2.1]oct a n e (4a ) a n d (1R)-2â-Ca r b oxy-
m eth oxy-3â-(4-flu or oph en yl)-8-m eth yl-8-azabicyclo[3.2.1]-
octa n e (5a ). To a solution of SmI2 (2.6 L, 0.1M in THF) was
added a solution of 3a (18.8 g, 68 mmol) in THF (142 mL,
anhydrous) dropwise at -78 °C under N2. The mixture was
then stirred for 45 min. Anhydrous CH3OH (142 mL) was then
added, and the mixture was stirred for a further 2 h. The
reaction was quenched with TFA (73 mL) at -70 °C, and water
(1.5 L) was added. The reaction mixture was allowed to warm
slowly to room temperature. It was then basified (NH4OH to
pH 10) and filtered through Celite, and the Celite was washed
with Et2O and saturated with NaHSO3. The organic layer was
washed with brine, dried (Na2SO4), filtered, and concentrated
to dryness to afford the crude product that was purified by
flash chromatography (EtOAc 20%, hexane 75%, Et3N 5%;
EtOAc 60%, hexanes 35%, Et3N 5%) to yield a white solid
containing 2â,3R- and 2â,3â- (1:1) substituted products (15.93
g, total 91%). The isomers were separated by gravity column
chromatography (3% EtOH/CHCl3; EtOAc 20%, hexane 75%,
Et3N 5%).
F or m a tion of Wein r eb Am id es. (1R)-2â-Ca r boxylic
Acid Meth oxym eth yla m id e-3r-(4-flu or op h en yl)-8-m eth -
yl-8-a za bicyclo[3.2.1]octa n e (7a ). A solution of Al(CH3)3 (28
mL, 2 M in hexane) was added dropwise under N2 at -10 °C
to a solution of N,O-dimethylhydroxylamine hydrochloride
(HN(OCH3)CH3‚HCl) (Weinreb salt) (5.50 g, 55.2 mmol) in
CH2Cl2 (200 mL, anhydrous). The mixture was slowly warmed
to room temperature and stirred for 15 min and then cooled
to -10 °C. A solution of the 2â,3R-substituted ester 4a (5.10
g, 18.4 mmol) in CH2Cl2 (150 mL, anhydrous) was slowly
added. The reaction mixture was stirred at -10 °C for 1 h and
allowed to warm to room temperature. TLC showed completion
of the reaction. It was then quenched with hydrochloric acid
(48 mL, 1 N), diluted with water (500 mL), and basified
(saturated Na2CO3 and NH4OH (concentrated) to pH 9). The
organic layer was separated, and the aqueous layer was
extracted with CH2Cl2 (3 × 150 mL). The combined organic
layers were dried (Na2SO4), filtered, and concentrated to
dryness to afford the crude product, which was purified by
flash chromatography (EtOAc 30%, hexanes 65%, Et3N 5%;
EtOAc 60%, hexanes 35%, Et3N 5%) to afford white crystals
of 7a , the 2â,3R-substituted Weinreb amide (5.42 g, 97%). Mp
142.0-142.5 °C. Rf ) 0.36 (EtOAc 60%, hexanes 35%, Et3N
1
5%). H NMR: δ 7.20-7.15 (m, 2H), 6.92 (tt, 2H), 3.48-3.37
The 2â,3R-substituted ester 4a was obtained as a white solid
(6.22 g, 36%). Rf ) 0.35 (EtOAc 20%, hexane 75%, Et3N 5%).
Mp 48.0-48.9 °C. 1H NMR: δ 7.18-7.14 (m, 2H), 6.94 (tt, 2H),
3.58 (s, 3H), 3.35-3.25 (m, 3H), 2.50-2.39 (m, 2H), 2.25 (s,
3H), 2.30-2.09 (m, 2H), 1.62-1.43 (m, 2H), 1.36-1.27 (ddd, J
) 1.7, 11.0, 13.8 Hz, 1H). Anal. (C16H20F O2N) C, H, N.
The 2â,3â-substituted ester 5a was obtained as a white solid
(6.0 g, 34%).33,54 Rf ) 0.35 (EtOAc 20%, hexane 75%, Et3N 5%).
Mp 90.5-91.5 °C.1H NMR: δ 7.24-7.19 (m, 2H), 6.95 (tt, 2H)
3.56-3.54 (m, 1H), 3.50 (s, 3H), 3.38-3.36 (m, 1H), 2.97 (dt,
1H, J ) 12.7, 5.3 Hz), 2.86 (t, 1H, J ) 3.9 Hz), 2.56 (td, 1H, J
) 12.7, 2.9 Hz), 2.23 (s, 3H), 2.20-2.08 (m, 2H), 1.76-1.61
(m, 3H). Anal. (C16H20F O2N) C, H, N.
(m, 1H), 3.42 (s, 3H), 3.32-3.27 (m, 1H), 3.09 (d, 1H, J ) 5.8
Hz), 3.06 (s, 3H), 2.70 (d, 1H, J ) 11.0 Hz), 2.56-2.46 (m, 1H),
2.32-2.17 (m, 2H), 2.24 (s, 3H), 1.68-1.45 (m, 2H), 1.25 (t,
1H, J ) 12.9 Hz).
(1R)-2â-Ca r boxylic Acid Meth oxym eth yla m id e-3r-(3,4-
d ich lor op h en yl)-8-m eth yl-8-a za bicyclo[3.2.1]octa n e (7b).
The method described for 7a above was followed to provide
7b from 4b. The product 7b was obtained as white crystals
(93%). Rf ) 0.14 (hexanes 40%, EtOAc 55%, Et3N 5%). 1H
NMR: δ 7.27 (d, 1H), 7.25 (d, 1H), 7.05 (dd, 1H), 3.48 (s, 3H),
3.34-3.46 (m, 1H), 3.22-3.32 (m, 1H), 3.08 (brs, 1H), 3.06 (s,
3H), 2.65 (brd, 1H), 2.42-2.54 (m, 1H), 2.21 (s, 3H), 2.1-2.34
(m, 2H), 1.63 (m, 1H), 1.48 (m, 1H), 1.19 (td, 1H).
(1R)-2â-Ca r b oxym et h oxy-3r-(3,4-d ich lor op h en yl)-8-
m eth yl-8-a za bicyclo[3.2.1]octa n e (4b) a n d (1R)-2â-Ca r -
b o x y m e t h o x y -3â-(3,4-d i c h lo r o p h e n y l)-8-m e t h y l-8-
a za bicyclo[3.2.1]octa n e (5b). The procedure described above
was followed to provide 4b and 5b. After column chromatog-
raphy (2.5% EtOH/CHCl3; Et2O 22%, Et3N 3%, hexanes 75%),
compound 4b was isolated as colorless crystals (1.15 g, 29%).
Mp 89-91 °C. Rf ) 0.64 (1% NH4OH/EtOAc). 1H NMR: δ 7.30
(d, 1H), 7.27 (d, 1H), 7.04 (dd, 1H), 3.59 (s, 3H), 3.2-3.47 (m,
3H), 2.35-2.5 (m, 2H), 2.05-2.3 (m, 2H), 1.4-1.6 (m, 2H), 2.23
(s, 3H), 1.28 (ddd, J ) 1.6, 10.4, 14 Hz, 1H). Anal. (C16H19O2-
NCl2‚0.1C6H14) C, H, N.
2â-Ca r boxy-3â-(4-flu or op h en yl)-8-m eth yl-8-a za bicyclo-
[3.2.1]octa n e (16a ). 2â-Carbomethoxy-3â-(4-fluorophenyl)-
tropane 5a (WIN 35,428) (1.25 g, 4.54 mmol) was refluxed for
24 h in a 1:1 dioxane/water (80 mL) solution. The solvent was
removed in vacuo, and the residue was almost completely
dissolved in CHCl3 (275 mL). Remaining undissolved solid was
filtered off, toluene (30 mL) was added, and the volume of the
solution was reduced in vacuo by approximately 75%. After
the resulting white suspension was cooled in a freezer for 2 h,
the precipitated white solid was isolated by filtration and was
washed with cold 1:1 CHCl3/toluene. The solid was pumped
1
dry to yield the product 16a as a white solid (1.11 g, 95%). H
Compound 5b was isolated as a white solid (1.04 g, 26%)
with physical data identical with those reported earlier.33 Mp
82.5-83.5 °C. Rf ) 0.43 (isopropylamine 3%, Et2O 30%,
NMR: δ 7.18-7.24 (m, 2H), 6.8 (m, 2H), 3.5-3.6 (m, 2H), 3.16
(ddd, J ) 13.7 Hz, H-3), 2.62-2.68 (m, 1H), 2.57 (ddd, J )
13.7 Hz, H-3, 1H), 2.24-2.34 (m, 2H), 2.25 (m, 3H), 1.94 (dd,
J ) 9 Hz, 2H), 1.7-1.8 (m, 1H).
1
pentane 67%). H NMR: δ 7.07-7.32 (m, 3H), 3.55 (m, 1H),

99mTechnetium SPECT Agent
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3491
2â-C a r b o x y -3â-(3,4-d i c h lo r o p h e n y l)-8-m e t h y l-8-
a za bicyclo[3.2.1]octa n e (16b). 2â-Carbomethoxy-3â-(3,4-
dichlorophenyl)tropane 5b (1.14 g, 3.47 mmol) was dissolved
in THF/MeOH (1:1; 46 mL) to which was added a solution of
LiOH‚H2O (153 mg) in water (11 mL). The solution was heated
to reflux for 24 h, cooled to 0 °C, and neutralized (pH 7) with
concentrated HCl. Silica gel (1.75 g) was added directly to the
solution, and solvent was removed in vacuo. The material was
purified by flash chromatography (20% MeOH/CHCl3 (900
mL); 30% MeOH/CHCl3 (2 L)). Fractions containing the
product were combined and evaporated in vacuo, and the
residue was dried at high vacuum to provide the product 16b
(3H, s), 2.98-2.91 (1H, m), 2.73 (1H, td, J ) 12.3, 2.7 Hz),
2.28-2.05 (2H, m), 2.22 (3H, s), 1.73-1.54 (3H, m).
F or m a tion of Eth yl Keton es. (1R)-2â-(1-P r op a n oyl)-3r-
(4-flu or op h en yl)-8-m eth yl-8-a za bicyclo[3.2.1]octa n e (8a ).
A solution of EtMgBr (24 mL, 3 M in Et2O) was added dropwise
at 0-5 °C under N2 to a solution of the Weinreb amide 7a
(5.42 g, 17.7 mmol) in THF (500 mL, anhydrous). The reaction
mixture was stirred for 15 min and then slowly warmed to
room temperature and stirred for a further 4 h. The reaction
was quenched with 3 N hydrochloric acid (24 mL), diluted with
water (250 mL) and EtOAc (200 mL), and basified with
(saturated aqueous Na2CO3) to pH 9. The organic layer was
separated, and the aqueous layer was extracted with EtOAc
(2 × 250 mL). The combined organic layers were dried (Na2-
SO4), filtered, and concentrated to dryness to afford the crude
product, which was purified by gradient flash chromatography
(EtOAc 10%, hexanes 88%, Et3N 2%; EtOAc 20%, hexane 75%,
Et3N 5%) to yield 8a as white crystals (3.48 g, 71%). Rf ) 0.39
(EtOAc 20%, hexane 75%, Et3N 5%). Mp 60.5-61.3 °C. 1H
NMR: δ 7.16-7.11 (m, 2H), 6.93 (tt, 2H), 3.32-3.23 (m, 2H,
m), 3.15 (d, 1H, J ) 6.0 Hz), 2.51-2.04 (m, 6H), 2.24 (s, 3H),
1.61-1.44 (m, 2H), 1.32-1.24 (m, 1H), 0.90 (t, 3H, J ) 7.3
Hz). Anal. (C17H22FON) C, H, N.
1
(310 mg, 28%). Rf ) 0.08 (30% MeOH/CHCl3). H NMR (CD3-
OD): δ 7.48 (d, 1H), 7.41 (d, 1H), 7.25 (dd, 1H), 3.93 (m, 2H),
3.3-3.4 (m, 1H), 2.78 (s, 3H), 2.6-2.9 (m, 2H), 2.3-2.5 (m,
2H), 2.1-2.2 (m, 2H), 1.8-1.9 (m, 1H).
2â-Ca r boxylic Acid Meth oxym eth yla m id e-3â-(4-flu o-
r op h en yl)-8-m eth yl-8-a za bicyclo[3.2.1]octa n e (17a ). Ox-
alyl chloride (1 mL, 11.4 mmol) was added dropwise to a stirred
suspension of the acid 16a (1.1 g, 4.2 mmol) in anhydrous CH2-
Cl2 (80 mL) containing DMF (50 mL). The reaction mixture
was stirred for 45 min, during which time the solution became
yellow. The solvent was then removed in vacuo, and the
residue was pumped at high vacuum overnight. CH2Cl2 (80
mL) was added to dissolve the acid chloride, and (MeO)MeNH‚
HCl (450 mg, dried over P2O5) followed immediately by
pyridine (1.1 mL, distilled over CaH2) was added to the
solution. The reaction mixture was stirred for 1 h and
partitioned between CHCl3 (20 mL) and 2 M Na2CO3 (20 mL).
The aqueous layer was extracted CHCl3 (2 × 10 mL), and the
combined organics were dried over Na2SO4, filtered, and
reduced in vacuo to yield 1.09 g of a yellow solid. The crude
product was dissolved in CH2Cl2 and purified by flash chro-
matography (hexanes 20%, EtOAc 75%, Et3N 5%). Product-
containing fractions were combined and concentrated to yield
17a as a light-yellow solid (960 mg, 75%). Mp 120.1-122.5
°C. Rf ) 0.14 (25% hexanes in EtOAc, 5% Et3N). IR (KBr
(1R)-2â-(1-P r op a n oyl)-3r-(3,4-d ich lor op h en yl)-8-m eth -
yl-8-a za bicyclo[3.2.1]octa n e (8b). The procedure described
above for 8a was utilized to convert 7b (105 mg, 0.29 mmol)
to 8b (80 mg, 80%). Rf ) 0.28 (30% EtOAc in hexanes; 5%
1
Et3N). Mp 97.4-98 °C. H NMR: δ 7.29 (d, 1H), 7.24 (d, 1H),
7.10 (dd, 1H), 3.2-3.36 (m, 2H), 3.14 (brd 1H), 3.32-2.52 (m,
3H), 2.21 (s, 3H), 2.06-2.30 (m, 6H), 1.42-1.62 (m, 2H), 1.27
(ddd, 1H), 0.93 (t, J ) 7.4 Hz, 3H). Anal. (C17H21NOCl2) C, H,
N.
2â-(1-P r o p a n o y l)-3â-(4-flu o r o p h e n y l)-8-m e t h y l-8-
a za bicyclo[3.2.1]octa n e (18a ). The procedure described
above for 8a was utilized to convert 17a (823 mg, 2.7 mmol)
to 18a (485 mg, 65%). Rf ) 0.3 (20% EtOAc in hexanes, 5%
1
Et3N). Mp 118-119.5 °C. H NMR: δ 7.17 (m, 2H), 6.69 (m,
1
disk): 2900, 1680, 1500 cm-1. H NMR: δ 7.25 (m, 2H), 6.90
2H), 3.48 (m, 1H), 3.36 (m, 1H), 2.9-3.0 (m, 2H), 2.5-2.6 (m,
1H), 2.23 (m, 3H), 2.0-2.4 (m, 3H), 1.5-1.8 (m, 4H), 0.85 (t,
3H), Anal. (C17H22NOF) C, H, N.
(m, 2H), 3.57 (s, 3H), 3.4 (m, 1H), 3.47 (m, 1H), 3.1 (m, 1H),
3.05 (s, 3H), 3.0 (m, 1H), 2.79 (ddd, 1H), 2.24 (m, 3H, NCH3),
2.0-2.3 (m, 2H), 1.5-1.8 (m, 3H). Anal. (C17H23N2O2F) C, H,
N.
2â-(1-P r op a n oyl)-3â-(3,4-d ich lor op h en yl)-8-m et h yl-8-
a za bicyclo[3.2.1]octa n e (18b). The procedure described
above for 8a was utilized to convert 17b (168 mg, 0.47 mmol)
to 18b (108 mg, 71%). Rf ) 0.22 (EtOAc 10%, hexanes 85%,
2â-Ca r b oxylic Acid Met h oxym et h yla m id e-3â-(3,4-d i-
ch lor op h en yl)-8-m eth yl-8-a za bicyclo[3.2.1]octa n e (17b).
The procedure described above was followed to convert 16b
(300 mg, 9.55 mmol) to 17b (39 mg, 11%). Rf ) 0.10 (50%
EtOAc in hexane, 5% Et3N). 1H NMR: δ 7.31 (d, 1H), 7.13
(dd, 1H), 7.30 (d, 1H), 3.62 (s, 3H), 3.5 (m, 1H), 3.38 (m, 1H),
3.13 (m, 1H), 3.04 (s, 3H), 2.93 (m, 1H), 2.72 (ddd, 1H), 2.21
(m, 3H), 2.0-2.3 (m, 2H), 1.5-1.74 (m, 3H).
1
Et3N 5%). Mp 83-84 °C. H NMR: δ 7.30 (1H, d), 7.28 (1H,
d), 7.08 (1H, dd), 3.54 (1H, d, J ) 4.1 Hz), 3.36-3.35 (1H, m),
3.00 (1H, t, J ) 3.3 Hz), 2.92-2.84 (1H, m), 2.55-2.39 (2H,
m), 2.32-2.05 (2H, m), 2.20 (3H, s), 1.78-1.55 (4H, m), 0.90
(3H, t, J ) 7.3 Hz). Anal. (C17H21NOCl2) C, H, N.
2â-(1-P r o p a n o y l)-3â-(4-flu o r o p h e n y l)-8-m e t h y l-8-
a za bicyclo[3.2.1]oct-2-en e (21a ). Triethylamine (dried over
KOH, 2.5 mL) was added to a solution of 3a (0.22 g, 0.81 mmol)
in THF (5.6 mL). Ethylmagnesium bromide (3 M in Et2O, 1.5
mL) was added dropwise to the above solution. The reaction
mixture was stirred at 5-10 °C for 5 h, and the reaction was
then quenched with 4 N HCl (4.5 mL). The reaction mixture
was then diluted with water (12 mL) and adjusted to pH 8-9.
The resulting mixture was extracted with CH2Cl2 (2 × 25 mL).
The combined extracts were washed with water (15 mL), dried
(K2CO3), filtered, and concentrated to dryness to afford a brown
oil that was purified by column chromatography to provide
21a as a yellow oil (0.104 g, 47%). Rf ) 0.28 (EtOAc 60%,
2â-Ca r b oxylic Acid Met h oxym et h yla m id e-3â-(3,4-d i-
ch lor op h en yl)-8-m eth yl-8-a za bicyclo[3.2.1]octa n e (17b).
Alter n a tive Rou te. To a solution of Weinreb salt (0.53 g, 5.28
mmol) in CH2Cl2 (anhydrous, 20 mL) was added a solution of
Al(CH3)3 (2.7 mL, 2 M in hexane) dropwise at -10 °C under
nitrogen. After the addition, the reaction mixture was slowly
warmed to room temperature, stirred for 15 min, and then
cooled back to -10 °C. A solution of 5b (0.58 g, 1.6 mmol) in
CH2Cl2 (15 mL) was added to the above mixture slowly. The
reaction mixture was stirred at -10 °C for 0.5 h, slowly
warmed to room temperature, and stirred for 24 h. The
reaction was then quenched with potassium sodium tartrate
tetrahydrate (Rochelle’s salt) (saturated aqueous, 21 mL),
diluted with CH2Cl2 (50 mL) and water (20 mL), and basified
with NH4OH to pH 9. The organic layer was separated, and
the water layer was extracted with CH2Cl2 (3 × 50 mL). The
combined organic layers were dried (Na2SO4), filtered, and
concentrated to dryness to afford the crude product, which was
purified by flash chromatography (EtOAc 30%, hexanes 65%,
Et3N 5%; EtOAc 60%, hexanes 35%, Et3N 5%) to provide the
Weinreb amide 17b as a white solid (0.56 g, 88%). Mp 156-
157 °C. Rf ) 0.29 (EtOAc 75%, hexane 20%, Et3N 5%). 1H
NMR: δ 7.31 (1H, d), 7.31 (1H, d), 7.13 (1H, dd), 3.63 (3H, s),
3.54-3.49 (1H, m), 3.41-3.37 (1H, m), 3.16-3.13 (1H, m), 3.05
1
hexanes 35%, Et3N 5%). H NMR: δ 7.16-7.10 (2H, m), 7.03
(2H, tt), 3.74 (1H, d, J ) 5.5 Hz), 3.37 (1H, t, J ) 5.6 Hz), 2.76
(1H, dd, J ) 18.4, 4.7 Hz), 2.42 (3H, s), 2.28-1.86 (6H, m),
1.67-1.59 (1H, m), 0.83 (3H, t, J ) 7.3 Hz).
2â-(1-P r op a n oyl)-3â-(3,4-d ich lor op h en yl)-8-m et h yl-8-
a za bicyclo[3.2.1]oct-2-en e (21b). The procedure described
above was utilized to convert 3b to 21b as a yellow oil (27%).
Rf ) 0.3 (EtOAc 60%, hexanes 35%, Et3N 5%). 1H NMR: δ
7.40 (1H, d), 7.26 (1H, d), 6.99 (1H, dd), 3.72 (1H, d, J ) 5.5
Hz), 3.38 (1H, t, J ) 5.5 Hz), 2.75 (1H, dd, J ) 18.4, 4.9 Hz),
2.41(3H, s), 2.28-1.88 (6H, m), 1.66-1.59 (1H, m), 0.87 (3H,
t, J ) 7.4 Hz).

3492 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Meltzer et al.
N-Dem et h yla t ion . (1R)-2â-(1-P r op a n oyl)-3r-(4-flu o-
r op h en yl)-8-a za bicyclo[3.2.1]octa n e (9a ). Compound 8a
(1.83 g, 6.68 mmol) was suspended in 1-chloroethyl chlorofor-
mate (ACE-Cl; 26 mL, 240 mmol) under N2 and was heated to
reflux at 135 °C for 2 h. The excess ACE-Cl was evaporated,
and CH3OH (55 mL) was added to the residue. The reaction
mixture was heated to reflux for 1 h and then concentrated to
dryness. The residue was diluted with CH2Cl2 (300 mL),
washed with NaHCO3 (saturated), dried (Na2SO4), filtered, and
concentrated to dryness to afford 9a , which was purified by
flash chromatography (EtOAc 75%, hexanes 20%, Et3N 5%)
to provide 9a as a light-yellow solid (1.47 g, 84%). Rf ) 0.20
(EtOAc 75%, hexane 20%, Et3N 5%). Mp 46.6-47.4 °C. 1H
NMR: δ 7.16-7.10 (m, 2H), 6.95 (tt, 2H), 3.63 (t, 1H, J ) 7.4
Hz), 3.45 (d, 1H, J ) 6.6 Hz), 3.13-3.03 (m, 1H), 2.54 (d, 1H,
J ) 10.7 Hz), 2.41-2.22 (m, 2H), 2.11-1.86 (m, 3H), 1.74-
1.54 (m, 3H), 1.33-1.24 (m, 1H), 0.87 (t, 3H, J ) 7.3 Hz). Anal.
(C16H20FNO‚0.2H2O) C, H, N.
mmol), 10 (1.65 g, 2.18 mmol), KI (0.36 g, 2.18 mmol), and
K2CO3 (1.2 g, 8.6 mmol) in anhydrous CH3CN (80 mL) was
heated to reflux under N2 for 20 h. The reaction mixture was
cooled to room temperature and filtered through Celite. The
Celite pad was washed with CH3CN (3 × 20 mL). The organic
layers were combined and concentrated to dryness, and the
crude product was purified by flash chromatography (EtOAc
40%, hexanes 55%, Et3N 5%) to yield 11a as a foam (1.01 g,
60%). Rf ) 0.37 (EtOAc 40%, hexanes 55%, Et3N 5%). Mp 55
1
°C (dec). H NMR: δ 7.51 (t, 1H), 7.40-7.36 (m, 11H), 7.25-
7.09 (m, 20H), 6.93 (t, 2H), 3.28-3.19 (m, 2H), 3.14 (d, 1H, J
) 6.3 Hz), 3.04-2.97 (m, 2H), 2.85 (s, 2H), 2.51-1.89 (m, 16H),
1.53-1.16 (m, 6H), 0.86 (t, 3H, J ) 7.3 Hz). Anal. (C63H66
FN3O2S2) C, H, N.
-
N-[2-(3′-N′-P r op yl-(1′′R)-3′′r-(3,4-d ich lor op h en yl)t r o-
p a n e-2′′â-(1-p r op a n oyl))((2-((tr ip h en ylm eth yl)th io)eth y-
l)a m in o)a cetyl]-S-(tr ip h en yl)-2-a m in oeth a n eth iol (11b).
The procedure described above for 11a was followed to convert
9b (94.5 mg, 0.303 mmol) to 11b obtained as a fluffy white
solid (158 mg, 51%). Rf ) 0.30 (EtOAc/hexanes 3:2). Mp 60 °C
(1R )-2â-(1-P r o p a n o y l)-3r-(3,4-d ic h lo r o p h e n y l)-8-
a za bicyclo[3.2.1]octa n e (9b). The procedure described for
9a was followed to convert 8b (80 mg, 2.45 mmol) to 9b (77
mg, 100%). Rf ) 0.30 (5% Et3N/EtOAc). 1H NMR: δ 0.91 (t,
3H), 1.25 (ddd, 1H), 1.52-1.74 (m, 2H), 1.8-2.56 (m, 7H),
3.06-3.2 (m, 1H), 3.45 (d, 1H), 3.62 (brt, 1H), 7.01 (dd, 1H),
7.25 (d, 1H), 7.31 (d, 1H).
1
(dec). H NMR: δ 6.9-7.6 (m, 34H), 2.6-3.4 (m, 6H), 1.8-2.5
(m, 14H), 1.0-1.7 (m, 8H), 0.89 (t, 3H). Anal. (C63H65
Cl2N3O2S2) C, H, N.
-
N-[2-(3′-N′-P r op yl-(1′′R)-3′′â-(4-flu or op h en yl)tr op a n e-
2′′â-(1-p r op a n oyl))((2-((tr ip h en ylm eth yl)th io)eth yl)a m i-
n o)a cet yl]-S-(t r ip h en yl)-2-a m in oet h a n et h iol (20a ). The
procedure described above for 11a was followed to convert 19a
(27 mg, 0.09 mmol) to 20a as a foam (47 mg, 46%). Rf ) 0.09
(60% EtOAc in hexanes, 1% Et3N). 1H NMR: δ 7.1-7.6 (m,
32H), 6.9-7.0 (m, 2H), 3.3-3.5 (m, 2H), 1.2-3.1 (m, 29H), 0.77
(t, 3H). Anal. (C63H66FN3O2S2‚2/3CHCl3) C, H, N.
N-[2-(3′-N′-P r op yl-(1′′R)-3′′â-(3,4-d ich lor op h en yl)t r o-
p a n e-2′′â-(1-p r op a n oyl))((2-((t r ip h en ylm et h yl)t h io)et h -
yl)a m in o)a cetyl]-S-(tr ip h en yl)-2-a m in oeth a n eth iol (20b).
The procedure described above for 11a was followed to convert
19b (30 mg, 0.096 mmol) to yield the title compound 20b as a
foam (41 mg, 41%). Rf ) 0.14 (60% EtOAc in hexanes, 1%
Et3N). 1H NMR: δ 7.0-7.6 (m, 34H), 3.52 (m, 1H), 3.34 (m,
1H), 1.2-3.1 (m, 29H), 0.81 (t, 3H).
N-[2-(3′-N′-P r op yl-(1′′R)-3′′-(4-flu or op h en yl)tr op -2-en e-
2′′-(1-pr opan oyl))((2-((tr iph en ylm eth yl)th io)eth yl)am in o)-
a cetyl]-S-(tr ip h en yl)-2-a m in oeth a n eth iol (24a ). The pro-
cedure described above for 11a was followed to convert 22a
(63 mg, 0.24 mmol) to 24a (132 mg, 0.135 mmol) as a white
solid (56%). Rf ) 0.38 (EtOAc 60%, hexanes 35%, Et3N 5%).
1H NMR: δ 7.49 (t, 1H), 7.39-7.34 (m, 13H), 7.26-7.14 (m,
16H), 7.10-7.05 (m, 2H), 6.99 (t, 2H, J ) 8.6 Hz), 3.75 (d, 1H,
J ) 5.5 Hz), 3.34 (t, 1H, J ) 5.8 Hz), 3.00 (q, 2H, J ) 6.4 Hz),
2.86 (2H, s), 2.57 (dd, 1H, J ) 18.4, 4.7 Hz), 2.43-2.32 (m,
9H), 2.25 (t, 2H, J ) 6.3 Hz), 2.12-1.78 (m, 7H), 1.61-1.50
(m, 2H), 0.81 (t, 3H, J ) 7.3 Hz). IR (film): 3339 (br, m), 3057
(m), 2936 (s), 1672 (vs), 1599 (m), 1508 (vs), 1491 (s), 1444 (s),
1358 (m), 1225 (m), 1185 (m), 1158 (m), 1034 (m), 911 (m),
847 (w) cm-1. Anal. (C63H64FN3O2S2‚1/2H2O) C, H, N.
2â-(1-P r opan oyl)-3â-(4-flu or oph en yl)-8-azabicyclo[3.2.1]-
octa n e (19a ). The procedure described above for 9a was
followed to convert 18a (335 mg, 1.22 mmol) to 19a (104 mg,
1
33%). Rf ) 0.36 (10% Et3N/EtOAc). H NMR: δ 7.10 (m, 2H),
6.94 (m, 2H), 3.70 (m, 1H), 3.56 (m, 1H), 3.18 (m, 1H), 2.88
(m, 1H), 2.4 (m, 1H), 1.9-2.3 (m, 3H), 1.4-1.8 (m, 5H), 0.70
(t, 3H).
2â-(1-P r op a n oyl)-3â-(3,4-d ich lor op h en yl)-8-a za bicyclo-
[3.2.1]octa n e (19b). The procedure described above for 9a was
followed to convert 18b (0.27 g, 0.84 mmol) to 19b as a light-
yellow solid (69 mg, 26%). Rf ) 0.12 (EtOAc 75%, hexanes 20%,
Et3N 5%). 1H NMR: δ 7.34 (1H, d), 7.26 (1H, d), 7.02 (1H,
dd), 3.79-3.77 (1H, m), 3.67 (1H, d, J ) 5.5 Hz), 3.47 (1H, s),
3.16 (1H, dt, J ) 12.9, 5.2 Hz), 2.95 (1H, d, J ) 5.5 Hz), 2.47-
1.97 (4H, m), 1.84-1.55 (4H, m), 0.77 (3H, t, J ) 7.3 Hz).
2-(1-P r op a n oyl)-3-(4-flu or op h en yl)-8-a za bicyclo[3.2.1]-
oct-2-en e (22a ). The procedure described above for 9a was
followed to convert 21a to obtain 22a as a yellow oil (66%). Rf
1
) 0.30 (EtOAc 90%, MeOH 6%, Et3N 4%). H NMR: δ 7.13-
6.99(4H, m), 4.12 (1H, d, J ) 5.2 Hz), 3.82 (1H, t, J ) 5.9 Hz),
2.80 (1H, dd, J ) 18.3, 3.4 Hz), 2.20 (1H, dd, J ) 18.3, 1.0
Hz), 2.12-1.84 (6H, m), 1.72-1.63 (1H, m), 0.83 (3H, t, J )
7.2 Hz).
2-(1-P r op a n oyl)-3-(3,4-d ich lor op h en yl)-8-a za b icyclo-
[3.2.1]oct-2-en e (22b). The procedure described above for 9a
was followed to convert 21b (106 mg, 0.325 mmol) to obtain
22b (83 mg, 0.266 mmol) as a yellow oil (82%). Rf ) 0.15
(EtOAc 75%, hexane 20%, Et3N 5%). 1H NMR: δ 7.40 (1H, d),
7.23 (1H, d), 6.96 (1H, dd), 4.10 (1H, d, J ) 5.5 Hz), 3.82 (1H,
t, J ) 5.8 Hz), 2.79 (1H, dd, J ) 18.4, 4.9 Hz), 2.20-1.89 (7H,
m), 1.72-1.63 (1H, m), 0.87 (3H, t, J ) 7.4 Hz).
N-[2-(3′-N′-P r op yl-(1′′R)-3′′-(3,4-d ich lor op h en yl)tr op -2-
en e-2′′-(1-p r op a n oyl))((2-((tr ip h en ylm eth yl)th io)eth yl)-
a m in o)a cet yl]-S-(t r ip h en yl)-2-a m in oet h a n et h iol (24b ).
The procedure described above for 11a was followed to convert
22b (44 mg, 0.14 mmol) to 24b (112 mg, 0.109 mmol), which
was isolated as a light-yellow solid (76%). Rf ) 0.43 (EtOAc
(1 R )-2 -M e t h o x y c a r b o n y l -3 -(4 -fl u o r o p h e n y l )-8 -
a za bicyclo[3.2.1]oct-2-en e (23a ). The procedure described
above for 9a was followed to convert 8a (362 mg, 1.3 mmol) to
obtain 23a as a yellow solid (234 mg, 68%). Rf ) 0.70 (10%
1
1
MeOH in hexanes, 0.5% NH4OH). Mp 67-68 °C. H NMR: δ
60%, hexanes 35%, Et3N 5%). H NMR: δ 7.50 (t, 1H), 7.38-
6.8-7.2 (m, 4H), 4.2 (m, 1H), 3.8 (m, 1H), 1.50-3.0 (m, 6H).
Anal. (C15H16NO2F) C, H, N.
7.33 (m, 13H), 7.26-7.14 (m, 18H), 6.92 (dd, 1H), 3.72 (d, 1H,
J ) 5.2 Hz), 3.36-3.32 (m, 1H), 3.00 (q, 2H, J ) 6.6 Hz), 2.86
(s, 2H), 2.58-2.32 (m, 8H), 2.26 (t, 2H, J ) 6.3 Hz), 2.10-1.95
(m, 4H), 1.86-1.78 (m, 2H), 1.57 (m, 5H), 0.86 (t, 3H, J ) 7.3
Hz). IR (film): 3341 (br, m), 3055 (w), 2937 (m), 1673 (s), 1514
(m), 1491 (m), 1467 (m), 1444 (s), 1357 (w), 1184 (w), 1135
(w), 1031 (m), 911 (m) cm-1. Anal. (C63H63Cl2N3O2S2‚2/3H2O)
C, H, N.
(1R )-2-Me t h oxyca r b on yl-3-(3,4-d ich lor op h e n yl)-8-
a za bicyclo[3.2.1]oct-2-en e (23b). The procedure described
for 9a was followed to convert 3b (200 mg, 0.61 mmol) to 23b
as a yellow oil. Rf ) 0.30 (10% Et3N in EtOAc). 1H NMR: δ
7.40 (d, 1H), 7.2 (dd, 1H), 6.9 (dd, 1H), 4.2 (m, 1H), 3.8 (m,
1H), 3.53 (s, 3H), 2.5-2.9 (m, 1H), 1.50-2.3 (m, 5H). Anal.
(C15H16NO2Cl2) C, H, N.
Atta ch m en t of MAMA′ Syn th on . N-[2-(3′-N′-P r op yl-
(1′′R)-3′′r-(4-flu or op h en yl)tr op a n e-2′′â-(1-p r op a n oyl))((2-
((tr iph en ylm eth yl)th io)eth yl)am in o)acetyl]-S-(tr iph en yl)-
2-a m in oeth a n eth iol (11a ). A suspension of 9a (0.45 g, 1.72
N-[2-(3′-N′-P r op yl-(1′′R)-3′′-(4-flu or op h en yl)tr op -2-en e-
2′′-(m eth oxyca r bon yl))((2-((tr ip h en ylm eth yl)th io)eth yl)-
a m in o)a cet yl]-S-(t r ip h en yl)-2-a m in oet h a n et h iol (25a ).
The procedure described above for 11a was followed to convert
23a (134 mg, 0.50 mmol) to 25a as a light-yellow foam (148

99mTechnetium SPECT Agent
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3493
1
mg, 29%). Rf ) 0.18 (10% Et3N in hexane). H NMR: δ 6.8-
7.8 (m, 34H), 3.8-3.9 (m, 1H), 3.5 (s, 3H), 3.2-3.4 (m, 1H),
1.3-3.15 (m, 22H).
FN3O4ReS2; 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 temper-
ature factors, distances, and angles are available as Supporting
Information.
N-[(2-((3′-N′-P r op yl-(1′′R)-3′′r-(3,4-d ich lor op h en yl)tr o-
p a n e-2′′â-1-p r op a n oyl)(2-m er ca p toeth yl)a m in o)a cetyl)-
2-a m in oeth a n eth iola to]r h en iu m (V) Oxid e (12b: O-1561).
The procedure described above for the preparation of 12a was
followed to convert 11b (24 mg, 0.024 mmol) to the solid 12b
(4.6 mg, 27%). 1H NMR: δ 7.34 (d, 1H), 7.3 (d, 1H), 7.0 (dd,
1H), 4.70, 5.06 (2d, J ) 16.7 Hz), 4.4-4.65 (m, 1H), 3.9-4.2
(m, 3H), 3.6-3.8 (m, 1H), 3.0-3.5 (m, 7H), 2.8-3.0 (m, 1H),
1.1-2.5 (m, 14H), 0.8-1.0 (2t, 3H). Anal. (C25H34Cl2N3O3ReS2‚
1/5C6H14) C, H, N.
Alter n a tive Rou te to 12b. To a solution of 9b (20 mg,
0.064 mmol) in anhydrous CH3CN (3 mL) was added the
rhenium chelate of 10 9 (30 mg, 0.064 mmol, 1 equiv), KI (10.6
mg, 0.064 mol, 1.0 equiv), and K2CO3 (10.6 mg, 0.064 mmol).
The resulting mixture was brought to reflux for 30 h and then
loaded onto SiO2 (1 g) and evaporated to dryness. The silica-
adsorbed material was purified by column chromatography
(10% Et3N/EtOAC) to yield 12b as a maroon solid (26 mg,
55%).
N-[2-(3′-N′-P r op yl-(1′′R)-3′′-(3,4-d ich lor op h en yl)t r op -
2-en e-2′′-(m eth oxyca r bon yl))((2-((tr ip h en ylm eth yl)th io)-
et h yl)a m in o)a cet yl]-S-(t r ip h en yl)-2-a m in oet h a n et h iol
(25b). The procedure described above for 11a was followed to
convert 23b (107 mg, 0.34 mmol) to 25b as a foam (111 mg,
31%). Rf ) 0.18 (5% Et3N in hexane). H NMR: δ 6.8-7.6 (m,
33H), 3.8-3.9 (m, 1H), 3.5 (s, 3H), 3.2-3.4 (m, 1H), 1.4-3.2
1
(m, 22H),
In ser tion of Rh en iu m . N-[(2-((3′-N′-P r op yl-(1′′R)-3′′r-
(4-flu or op h en yl)t r op a n e-2′′â-1-p r op a n oyl)(2-m er ca p t o-
eth yl)a m in o)a cetyl)-2-a m in oeth a n eth iola to]r h en iu m (V)
Oxid e (12a : O-1505). A solution of 11a (1.00 g, 1.02 mmol)
in EtOH (50 mL) was brought to reflux under N2. A solution
of SnCl2 (0.26 g, 1.33 mmol, in 6.3 mL of 0.05 N HCl) was
added to the above solution and followed immediately by the
addition of NaReO4 (0.36 g, 1.33 mmol, in 6.3 mL of 0.05 N
HCl). The reaction mixture was maintained at reflux for 26
h. It was then diluted with CH3CN (50 mL) and filtered
through Celite. The Celite was washed with hot CH3CN (3 ×
25 mL). The combined organic washes were concentrated to
dryness to afford the crude Re complex 12a (1.48 g). Gradient
flash chromatography (EtOAc 40%, hexanes 56%, Et3N 4%;
EtOAc 75%, hexane 20%, Et3N 5%; EtOAc 90%, hexanes 6%,
Et3N 4%) provided a 1:1 mixture of diastereoisomers of 12a
as a pink solid (0.34 g, 47%). Rf ) 0.24 (EtOAc 75%, hexane
20%, Et3N 5%). Mp 104 °C (dec). Anal. (C25H35FN3O3ReS2‚1/2-
EtOAc) C, H, N.
Sep a r a tion of th e d ia ster eoisom er s of th e Re Com p lex
12a . A mixture of the Re diastereoisomers 12a (0.4 g) was
separated by gravity chromatography. The silica gel was
pretreated with 1% NH4OH/EtOAc. After the column was
packed, 80% EtOAc/hexane was used to wash away the excess
base. The 12a to silica gel ratio was 1:500-650. The eluent
was 80% EtOAc/hexane; EtOAc. The two isomers were ob-
tained: 12a -2 (110 mg, 27%), mp 191.5-192.8 °C, and 12a -1
(30 mg, 7.5%), mp 96 °C (dec).
(1R,12R)-N-[(2-((3′-N′-p r op yl-3′′r-(4-flu or op h en yl)t r o-
p a n e-2′′â-p r op ion yl)(2-m er ca p t oet h yl)a m in o)a cet yl)-2-
a m in oeth a n eth iola to]r h en iu m (V) Oxid e (12a -1: O-2020).
HRMS (FAB) [M + H]: calcd 696.1740; found 696.1751. 1H
NMR: δ 7.15-7.10 (m, 2H), 6.97 (t, 2H, J ) 8.7 Hz), 4.74 (d,
1H, J ) 16.5 Hz), 4.57 (m, 1H), 4.20-4.05 (m, 2H), 4.08 (d,
1H, J ) 16.5 Hz), 3.78-3.67 (m, 2H), 3.43 (dd, 1H, J ) 12.1,
3.0 Hz), 3.34-3.07 (m, 5H), 2.96 (dd, 1H, J ) 13.5, 4.1 Hz),
2.52-2.78 (m, 3H), 2.32-1.40 (m, 10H), 1.34-1.20 (m, 1H),
0.82 (t, 3H, J ) 7.3 Hz). 13C NMR (75 MHz, CDCl3): δ 213.3,
187.4, 163.1, 159.9, 139.4, 139.4, 129.3, 129.2, 115.5, 115.2,
66.7, 65.6, 63.6, 62.5, 59.7, 59.7, 59.0, 50.1, 47.7, 40.2, 39.3,
37.5, 36.9, 30.5, 29.5, 24.2, 7.5.
(1R,12S)-N-[(2-((3′-N′-P r op yl-3′′r-(4-flu or op h en yl)t r o-
p a n e-2′′â-p r op ion yl)(2-m er ca p t oet h yl)a m in o)a cet yl)-2-
a m in oeth a n eth iola to]r h en iu m (V) Oxid e (12a -2: O-1972).
HRMS (FAB) [M + H]: calcd 696.1740; found 696.1768. 1H
NMR: δ 7.14-7.10 (m, 2H), 6.95 (t, 2H, J ) 8.5 Hz), 5.14 (d,
1H, J ) 16.5 Hz), 4.61 (m, 1H), 4.16-3.93 (m, 3H), 4.13 (d,
1H, J ) 16.5 Hz), 3.43 (td, 1H, J ) 13.3, 3.6 Hz), 3.34-3.08
(m, 6H), 2.87 (dd, 1H, J ) 13.3, 4.3 Hz), 2.53-1.80 (m, 10H),
1.70-1.49 (m, 3H), 1.29-1.20 (m, 1H), 0.84 (t, 3H, J ) 7.3
Hz). 13C NMR (75 MHz, CDCl3) δ 213.3, 187.7, 163.1, 159.9,
139.4, 139.4, 129.3, 129.2, 115.5, 115.2, 67.0, 64.6, 63.5, 62.28,
59.9, 59.3, 58.9, 49.8, 47.5, 40.2, 38.8, 37.3, 36.9, 30.5, 29.5,
23.4, 7.5.
N-[(2-((3′-N′-P r op yl-(1′′R)-3′′â-(4-flu or op h en yl)tr op a n e-
2′′â-1-p r op a n oyl)(2-m er ca p toeth yl)a m in o)a cetyl)-2-a m i-
n oeth a n eth iola to]r h en iu m (V) Oxid e (13a :O-1508). The
procedure described above for 12a was followed to convert 20a
to 13a as a maroon foam (6.7 mg, 44%). Rf ) 0.07 (60% EtOAc
in hexanes, NH4OH 0.5%). HRMS (FAB) (C25H35FN3O3ReS2):
1
calcd 695.172; found 695.162. H NMR: δ 7.14 (m, 2H), 6.93
(m, 2H), 4.87 (d, 0.5H, J ) 16.5 Hz), 4.73 (d, 0.5H, J ) 16.5
Hz), 4.5-4.6 (m, 1H), 1.4-4.1 (m, 26H), 0.7-0.9 (2t, 2H).
N-[(2-((3′-N′-P r op yl-(1′′R)-3′′â-(3,4-d ich lor op h en yl)tr o-
p a n e-2′′â-1-p r op a n oyl)(2-m er ca p toeth yl)a m in o)a cetyl)-
2-a m in oeth a n eth iola to]r h en iu m (V) Oxid e (13b: O-2131).
The procedure described above for 12a was followed to provide
13b as a maroon solid (71%). Mp 131 °C (dec). Rf ) 0.35 (EtOAc
75%, hexanes 20%, Et3N 5%). HRMS [M + H]+: calcd
746.1054; found 746.1027. 1H NMR: δ 7.33-7.27 (m, 2H),
7.10-7.05 (m, 1H), 4.79 (d, 0.5H, J ) 16.5 Hz), 4.71 (d, 0.5H,
J ) 16.8 Hz), 4.60-4.52 (m, 1H), 4.13-3.97 (m, 2H), 3.90-
3.35 (m, 5H), 3.31-3.14 (m, 3H), 3.02 (s, 1H), 2.91-2.83 (m,
2H), 2.62-2.32 (m, 3H), 2.22-2.05 (m, 4H), 1.84-1.66 (m, 6H),
0.90 (t, 1.5H, J ) 7.4 Hz), 0.84 (t, 1.5H, J ) 7.4 Hz). 13C NMR
(75 MHz, CDCl3) δ 209.1, 187.2, 143.4, 143.4, 131.9, 129.9,
129.6, 129.3, 129.2, 126.6, 126.5, 66.7, 66.7, 65.8, 64.9, 63.8,
62.1, 61.8, 60.4, 60.4, 59.7, 59.7, 58.5, 58.5, 51.2, 51.0, 47.6,
39.1, 38.8, 35.5, 35.4, 34.1, 34.0, 33.9, 27.1, 27.0, 25.4, 24.4,
24.2, 7.8. IR (film): 2939 (s), 1713 (s), 1660 (vs), 1474 (s), 1351
(s), 1311 (s), 1028 (w), 968 (vs), 910 (s), 731 (vs). Anal. (C25H34
-
Cl2N3O3ReS2‚0.2C6H14) C, H, N.
N-[(2-((3′-N′-P r op yl-(1′′R)-3′′-(4-flu or op h en yl)t r op -2′′-
en e-2′′-1-p r op a n oyl)(2-m er ca p t oet h yl)a m in o)a cet yl)-2-
a m in oeth a n eth iola to]r h en iu m (V) Oxid e (14a : O-1768).
The procedure described above for 12a was followed to provide
14a as a red-brown foam (52%). Rf ) 0.29 (EtOAc 95%, Et3N
5%). 1H NMR: δ 7.14-7.02 (m, 4H), 4.73 (d, 0.5H, J ) 16.2
Hz), 4.69 (d, 0.5H, J ) 16.2 Hz), 4.62-4.56 (m, 1H), 4.19-
4.01 (m, 3H), 3.88 (d, 1H, J ) 5.8 Hz), 3.74-3.62 (m, 1H),
3.52-3.13 (m, 5H), 2.89 (dd, 0.5H, J ) 13.2, 4.7 Hz), 2.87 (dd,
0.5H, J ) 13.1, 5.1 Hz), 2.64-2.58 (m, 3H), 2.27-1.58 (m, 9H),
1.25-1.12 (m, 1H), 0.84 (t, 3H, J ) 7.3 Hz). 13C NMR (75 MHz,
CDCl3): δ 208.9, 187.2, 141.1, 140.9, 137.4, 137.1, 136.0, 129.2,
129.1, 115.8, 115.6, 66.9, 64.2, 64.1, 62.2, 59.8, 58.2, 57.9, 56.5,
47.8, 45.1, 38.8, 36.6, 35.6, 34.1, 34.1, 30.3, 22.9, 22.8, 8.5. IR
(film): 2937 (m), 1661 (s), 1508 (m), 1461 (w), 1350 (m), 1313
(m), 1223 (m), 967 (s), 730 (m) cm-1 . HRMS (FAB) [M + H]
(C25H33FN3O3ReS2 (Na): calcd 716.1404; found 716.1405.
Sin gle-Cr ysta l X-r a y An a lysis of Dia ster eoisom er 12a -
2. Orthorhombic crystals of 12a -2 were obtained by slow
growth at the interface of CHCl3/anhydrous ethanol main-
tained 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 are as follows: crystal size, 0.40 mm × 0.38 mm
× 0.14 mm; cell dimensions, a ) 7.4147(5) Å, b ) 15.9123(11)
N-[(2-((3′-N′-P r op yl-(1′′R)-3′′-(3,4-d ich lor op h en yl)tr op -
2′′-en e-2′′-1-p r op a n oyl)(2-m er ca p toeth yl)a m in o)a cetyl)-
Å, c ) 22.2645(14) Å, R ) 90°, â ) 90°, γ ) 90°; formula, C24H33
-

3494 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Meltzer et al.
2-a m in oeth a n eth iola to]r h en iu m (V) Oxid e (14b: O-1769).
The procedure described above for 12a was followed to provide
14b as a brown foam (51%). Rf ) 0.27 (EtOAc 95%, Et3N 5%).
1H NMR: δ 7.42 (d, 1H, J ) 8.3 Hz), 7.25 (d, 1H, J ) 2.0 Hz),
6.98 (dd, 0.5H, J ) 8.3, 2.0 Hz), 6.97 (dd, 0.5H, J ) 8.3, 2.0
Hz), 4.73 (d, 0.5H, J ) 16.5 Hz), 4.69 (d, 0.5H, J ) 16.5 Hz),
4.62-4.56 (m, 1H), 4.17-3.99 (m, 3H), 3.85 (d, 1H, J ) 5.8
Hz), 3.75-3.59 (m, 1H), 3.49-3.13 (m, 5H), 2.89 (dd, 0.5H, J
) 13.3, 4.5 Hz), 2.88 (dd, 0.5H, J ) 13.3, 4.5 Hz), 2.63-2.55
(m, 3H), 2.30-1.96 (m, 7H), 1.88 (t, 1H, J ) 10.4 Hz), 1.69-
1.62 (m, 1H), 1.25-1.10 (m, 1H), 0.88 (t, 3H, J ) 7.3 Hz). 13C
NMR (75 MHz, CDCl3): δ 208.3, 187.2, 142.2, 139.8, 135.5,
133.0, 132.6, 130.6, 129.2, 126.8, 66.9, 64.3, 64.1, 62.1, 59.8,
58.4, 58.1, 56.4, 47.8, 45.1, 38.8, 36.7, 35.2, 34.0, 30.3, 22.9,
22.8, 8.4. IR (film): 2937 (m), 1660 (s), 1468 (m), 1350 (m),
Rhesus monkeys weighing approximately 7 kg were anes-
thetized with ketamine/xylazine (15.0 and 1.5 mg/kg) and
positioned prone on the imaging bed of the SPECT camera.
Before the start of imaging, a venous catheter was inserted in
a peripheral vein for radiopharmaceutical administration. The
heads of the animals were immobilized with
a custom-
fabricated head holder. Approximately 20-25 mCi of 99mTc
labeled compound was injected intravenously over 60 s.
Dynamic SPECT imaging was initiated at the end of the
infusion and consisted of 2 min acquisitions during the first
hour and 5 min acquisitions thereafter.
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.
1313 (m), 967 (s), 731 (m) cm-1. HRMS (FAB) [M + H] (C25H32
-
Cl2N3O3ReS2 (Na): calcd 766.0718; found 766.0720.
N-[(2-((3′-N′-P r op yl-(1′′R)-3′′b-(4-flu or op h en yl)tr op -2′′-
en e-2′′-ca r bom et h oxy)(2-m er ca p t oet h yl)a m in o)a cet yl)-
2-a m in oeth a n eth iola to]r h en iu m (V) Oxid e (15a :O-1135).
The procedure described above for 12a was followed to provide
15a as a foam (34 mg, 37%). Rf ) 0.09 (10% Et3N in EtOAc).
1H NMR: δ 6.9-7.2 (m, 4H), 4.5-4.9 (m, 2H), 3.5 (s, 3H), 1.4-
4.3 (m, 23H). HRMS (FAB) [M + H]: calcd 696.1348; found
696.1405. Anal. (C24H31FN3O4S2Re) C, H, N.
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 fascicularis)
was stored at -85 °C in the primate brain bank at the New
England Regional Primate Research Center. The caudate-
putamen was dissected from coronal slices and yielded 1.4 (
0.4 g of tissue. Membranes were prepared as described
previously. Briefly, the caudate-putamen was homogenized 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 no. 5) for 15 s. All
experiments were conducted in triplicate, and each experiment
was repeated in each of two to three preparations from
individual brains.
N-[(2-((3′-N′-P r op yl-(1′′R)-3′′-(3,4-d ich lor op h en yl)tr op -
2′′-en e-2′′-car bom eth oxy)(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 (15b: O-1136).
The procedure described above for 12a was followed to provide
15b as a tan foam (56 mg, 78%). Rf ) 0.09 (10% Et3N in
EtOAc). 1H NMR: δ 7.4 (d, 1H), 7.2 (d, 1H), 6.95 (dd, 1H),
4.4-4.9 (m, 2H), 3.55 (s, 3H), 1.4-4.2 (m, 23H). HRMS(FAB):
calcd 746.0663; found 746.0689. Anal. (C24H30Cl2N3O4S2Re) C,
H, N.
99m Tc La belin g. Compounds 12a ,b, 13a ,b, 17a , 26a -c,
27a -c were prepared by identical methods.
Dep r otection . Twenty microliter aliquots of Et3N, CH2CI2,
and (C2H5)3Si were added to 5.0 mg of each trityl-protected
precursor to cleave the thiol protecting group. After a 20 min
incubation, 200 µL of 1.0 M HCl/ether was added to protonate
the thiols. The solvent was evaporated, followed by successive
washes with hexanes for removal. The deprotected compound
was then dissolved in DMSO to produce a stock solution at a
concentration of 1 mg/mL.
Dop a m in e Tr a n sp or ter Assa y. The dopamine transporter
was labeled with [3H]WIN35,428 ([3H]CFT, 2â-carbomethoxy-
3â-(4-fluorophenyl)-N-[3H]methyltropane, 81-84 Ci/mmol, Du-
Pont-NEN). 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 concentrations of unlabeled WIN 35,428. 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 concentra-
tion: WIN 35,428, 0.2 mL (1 pM to 100 or 300 nM), [3H]WIN
35,428 (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 terminated by rapid
filtration over Whatman GF/B glass fiber filters presoaked in
0.1% bovine serum albumin (Sigma Chem. Co.). The filters
were washed twice with 5 mL of Tris-HCl buffer (50 mM),
incubated overnight at 0-4 °C in scintillation fluor (Beckman
Ready-Value, 5 mL), and radioactivity was measured by liquid
scintillation spectrometry (Beckman 1801). The cpm values
were converted to dpm following determination of counting
efficiency (> 45%) of each vial by external standardization.
Total binding was defined as [3H]WIN 35,428 bound in the
presence of ineffective concentrations of unlabeled WIN 35,428
(1 or 10 pM). Nonspecific binding was defined as [3H]WIN
35,428 bound in the presence of an excess (30 µM) of (-)-
cocaine. Specific binding was the difference between the two
values. Competition experiments to determine the affinities
of other drugs at [3H]WIN 35,428 binding sites were conducted
using procedures similar to those outlined above. Stock solu-
tions 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.
Ra d iola belin g. Approximately 200 mCi of sodium 99mTc
pertechnetate was added to a Glucoheptonate kit (Dupont
Pharmaceuticals, Billerica, MA) and allowed to incubate at
room temperature for 15 min. The 150 mCi of the resulting
99mTc-Glucoheptonate was added to an equal volume of 50 mM
acetate buffer, pH 5.2 (approximately 2 mL) and 50 µL of
deprotected precursor stock solution (1 mg/mL in DMSO, 50
µg). This solution was incubated at room temperature for 20
min. The course of the radiolabeling was monitored with a
Rainin HPLC system using a reverse-phase Vydac C8 column
(4.6 mm × 250 mm, 5 µm). The column was eluted with a 0.1
M ammonium acetate/acetonitrile mobile phase at 1.5 mL/min
flow rate and with a gradient of 5-100% acetonitrile over 15
min. Radioactive detection was achieved with a Frisk-Tech
rate meter (Bicron Corp.). The final radiolabeled product was
purified using a C18 Sep-Pak (Waters Inc.) eluted with
ethanol. Labeling yields and radiochemical purities were
greater than 85% and 98%, respectively. Each product was
diluted with sterile saline to yield a <10% ethanol solution
followed by filtration through a 0.22 µm filter prior to injection.
SP ECT Im a gin g. SPECT images were acquired with a
MultiSPECT 2 γ camera (Siemens, Hoffman Estates, IL)
equipped with fan-beam collimators and peaked to the 140 keV
photopeak of 99mTc (15% window). This camera has an intrinsic
resolution of 4.6 mm (fwhm) and a sensitivity of ∼240 cps/
mCi. Images were acquired over 360° (60 projections/head, 128
× 128 matrix) in the continuous mode. Image reconstruction
was performed using a conventional filtered back-projection
algorithm to an in-plane resolution of 10 mm fwhm and
attenuation correction via the Chang method.
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

99mTechnetium SPECT Agent
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3495
[3H]citalopram (specific activity of 82 Ci/mmol, DuPont-NEN)
for the serotonin transporter was determined in experiments
by incubating tissue with a fixed concentration of [3H]citalo-
pram 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% polyethyleneimine. 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). The cpm values
were converted 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]citalo-
pram bound in the presence of an excess (10 mM) of fluoxetine.
Specific binding was the difference between the two values.
Competition experiments to determine the affinities of other
drugs at [3H]citalopram binding sites were conducted using
procedures similar to those outlined above.
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Da ta An a lysis. Data were analyzed by the EBDA computer
software programs (Elsevier-Biosoft, U.K.). Final estimates 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.
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Ack n ow led gm en t. This work was supported by
Boston Life Sciences Inc. and the National Institute on
Drug Abuse (Grants NIDA4-8309 (P.C.M), NIDA06303
(B.K.M.), NIDA09462 (B.K.M.), RR00168 (B.K.M.)). The
authors thank Dr. Clifford George (Naval Research
Laboratory, Washington, D.C.) for conducting the X-ray
structural analysis. The authors thank Drs. Alan J ones
and Ashfaq Mahmood (Brigham and Women’s Hospital,
Harvard Medical School, Boston, MA) for their contri-
butions to an earlier part of this program.
(18) Fischman, A. J .; Babich, J . W.; Elmaleh, D. R.; Meltzer, P. C.;
Barrow, S. A.; Madras, B. K. SPECT imaging of dopamine
transporter sites in normal and MPTP treated Rhesus monkeys.
J . Nucl. Chem. 1995, 36, 87.
(19) Fischman, A. J .; Babich, J . W.; Elmaleh, D. R.; Barrow, S. A.;
Meltzer, P. C.; Hanson, R. N.; Madras, B. K. SPECT imaging of
dopamine transporter sites in normal and MPTP-treated rhesus
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(20) 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. Neurosci.-Net 1997, 1, 00010.
Su p p or tin g In for m a tion Ava ila ble: Crystal data and
refinement parameters, coordinates, anisotropic temperature
factors, distances, and angles for 12b-2. This material is
available free of charge via the Internet at http://pubs.acs.org.
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