The synthesis of bivalent 2β-carbomethoxy-3β-(3,4-dichlorophenyl)-8-heterobicyclo[3.2.1]octanes as probes for proximal binding sites on the dopamine and serotonin transporters

Article abstract of DOI:10.1016/j.bmc.2007.11.009

3-Aryltropanes have been widely explored for potential medications for remediation of cocaine abuse. Research has focused predominantly on 8-azatropanes and it is now well recognized that these compounds can be designed to manifest varied selectivity and potency for inhibition of the dopamine, serotonin, and norepinephrine uptake systems. We had reported that the 8-nitrogen atom present in the 3-aryltropanes is not essential for tropanes to bind to monoamine uptake systems. We demonstrated that compounds in which the amine had been exchanged for an ether or a thioether retained binding potency and selectivity. We have now designed bivalent compounds in which two tropane moieties are linked by an intervening chain. These 8-homo- and 8-heterotropane bivalent compounds allowed a search for adjacent tropane binding sites on the DAT as well as a further exploration of whether the binding sites for 8-azatropanes are the same as those for other 8-heterotropanes. A comparison of these compounds with their progenitor tropanes cast into doubt the existence of proximal binding sites on the DAT, and offered support for the existence of different binding sites for the 8-azatropanes compared with 8-oxa- and 8-thiatropanes. Indeed, 8-aza bivalent tropanes inhibited DAT with potency about 10-fold lower (DAT: IC₅₀ = 31 nM) than their monovalent counterparts. Furthermore, bivalent ligands in which one or both of the tropanes was devoid of an amine suffered a further loss of inhibitory potency. We conclude that it is unlikely that there exist two tropane binding sites in close proximity to one another on either the DAT or SERT.

Full text of DOI:10.1016/j.bmc.2007.11.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1832–1841
The synthesis of bivalent 2b-carbomethoxy-3b-(3,4-dichlorophenyl)-
8-heterobicyclo[3.2.1]octanes as probes for proximal binding sites
on the dopamine and serotonin transporters
Peter C. Meltzer,a,* Olga Kryatova,a Duy-Phong Pham-Huu,a
Patrick Donovana and Aaron Janowskyb
aOrganix Inc., 240 Salem Street, Woburn, MA 01801, USA
bVA Medical Center and Oregon Health and Sciences University, Portland, OR 97239, USA
Received 7 September 2007; revised 23 October 2007; accepted 1 November 2007
Available online 6 November 2007
Abstract—3-Aryltropanes have been widely explored for potential medications for remediation of cocaine abuse. Research has
focused predominantly on 8-azatropanes and it is now well recognized that these compounds can be designed to manifest varied
selectivity and potency for inhibition of the dopamine, serotonin, and norepinephrine uptake systems. We had reported that the
8-nitrogen atom present in the 3-aryltropanes is not essential for tropanes to bind to monoamine uptake systems. We demonstrated
that compounds in which the amine had been exchanged for an ether or a thioether retained binding potency and selectivity. We
have now designed bivalent compounds in which two tropane moieties are linked by an intervening chain. These 8-homo- and 8-
heterotropane bivalent compounds allowed a search for adjacent tropane binding sites on the DAT as well as a further exploration
of whether the binding sites for 8-azatropanes are the same as those for other 8-heterotropanes. A comparison of these compounds
with their progenitor tropanes cast into doubt the existence of proximal binding sites on the DAT, and offered support for the exis-
tence of different binding sites for the 8-azatropanes compared with 8-oxa- and 8-thiatropanes. Indeed, 8-aza bivalent tropanes
inhibited DAT with potency about 10-fold lower (DAT: IC50 = 31 nM) than their monovalent counterparts. Furthermore, bivalent
ligands in which one or both of the tropanes was devoid of an amine suffered a further loss of inhibitory potency. We conclude that
it is unlikely that there exist two tropane binding sites in close proximity to one another on either the DAT or SERT.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
in turn leads to the pharmacological sequelae of cocaine
addiction. The search for potential medications for co-
caine abuse has consequently focused largely on the de-
sign of compounds that target the DAT and SERT, and
a considerable effort has been placed on the search for
DAT and SERT selective molecules.10–16 The DAT, the
predominant mechanism for reuptake of dopamine in
the synapse, has been the focus of particular attention.
Many moderately selective DAT inhibitors have been re-
ported.12,16–20 Far fewer compounds that manifest exclu-
sive DAT inhibitory potency have been discovered.12,21
The class of bicyclo[3.2.1]octanes has been a focus of
much attention in the design of prospective medications
for cocaine abuse.13,14,22,23 8-Azabicyclo[3.2.1]octanes
(8-azatropanes),24 8-oxabicyclo[3.2.1]octanes (8-oxatro-
panes),11 8-thiabicyclo[3.2.1]octanes, (8-thiatropanes),13
and 8-carbabicyclo[3.2.1]octanes, (8-carbatropanes)25
have all been evaluated. These 8-heterotropanes (8-aza,
8-oxa, 8-thia, 8-carba) have provided a broad array of
promising DAT inhibitors.
The search for a pharmacotherapy for the treatment of
cocaine addiction has not yet yielded a compound of pro-
ven clinical utility. Cocaine is a potent stimulant of the
mammalian central nervous system and its stimulatory
activity is a consequence of its interactions with mono-
amine uptake systems located in the striatum.1,2 The
dopamine transporter (DAT) and serotonin transporter
(SERT), located presynaptically on neurons in the stria-
tum, have been specifically implicated in the reinforcing
and stimulatory properties of cocaine.3–9 Cocaine binds
to both the DAT and SERT and inhibits reuptake of syn-
aptic dopamine and serotonin. The excess dopamine in
the synapse then stimulates postsynaptic receptors, which
Keywords: Monoamine transporter ligands; Cocaine medications;
Bivalent ligands.
*
Corresponding author. Tel.: +1 781 932 4142; fax: +1 781 933
6695; e-mail: meltzer@organixinc.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.009

P. C. Meltzer et al. / Bioorg. Med. Chem. 16 (2008) 1832–1841
1833
The reinforcing and addictive properties of cocaine are
thought to be related to its pharmacokinetic profile of
extremely rapid onset and short duration of action.26
Therefore, a biological rationale that has guided much
of the search for pharmacotherapies has been to seek
‘replacement agonist therapies’ that inhibit cocaine
binding but that manifest a slow onset and a long dura-
tion of action. Another approach to such ‘cocaine
replacement therapies’ might utilize the possible exis-
tence of proximal cocaine binding sites on the DAT. If
such proximal binding sites exist, it may be possible to
design highly selective inhibitors of cocaine binding.
Furthermore, if these proximal sites are located at a dif-
ferent distance from one another with respect to dopa-
mine uptake and cocaine binding, it may conceivably
be possible to design a DAT selective, dopamine sparing
partial agonist with prolonged duration of action as well
as slow onset of activity. Therefore the existence of
proximal binding sites could offer a unique window on
the design of cocaine medications.
inhibitory potency than those with shorter interlinking
chains. Furthermore, they observed that the length of
the interlinking spacers influenced the effect on the dis-
crimination ratios (Ki/IC50) between inhibition of radio-
ligand binding to the DAT inhibition and inhibition of
DA reuptake. The longest spacer provided as much as
a 130-fold preference for DAT inhibition over DA reup-
take inhibition. Cashman consequently suggested that
‘certain bivalent aryltropane analogs bind to different
domains on the hDAT’.31
The DAT has been proposed to exist as a dimer or tet-
ramer.33–37 However, the premise that we have explored,
and on which we now report, is the possible existence of
two proximal tropane binding regions on a single dopa-
mine transporter.31 In addition, selected bivalent ligands
have provided an opportunity to further explore
whether 8-azatropanes, 8-oxatropanes, and 8-thiatro-
panes bind at identical domains11 on the DAT.
This bivalent ligand concept has been presented and val-
idated by Portoghese27,28 in his evaluation of molecules
that target opioid receptors. In that work he demon-
strated that potent but non-selective monovalent ligands
that inhibit more than one opioid receptor could be
linked to one another, through a molecular spacer, to
provide bivalent ligands that then proved highly selec-
tive. In this fashion, the linker itself provided a new
‘dimension for the recognition process’27 because each
component of the linked ligands bound to proximal
binding sites on the target receptors, and further, these
sites were localized at different distances from one an-
other on different receptors.
2. Chemistry
2.1. Compound design
The parent compounds selected for this study were
based upon the 2b-carbomethoxy-3b-aryl-8-N,O, or S-
bicyclo[3.2.1]octane skeleton. This skeleton was specifi-
cally selected from among the available stereoisomers
because it generates compounds of comparable nano-
molar potency at DAT and SERT.13 In contrast,
the 2b-carbomethoxy-3a-aryl-8-N,O, or S-bicyclo[3.2.1]
octane and the 2-carbomethoxy-3-aryl-8-N,O, or S-bicy-
clo[3.2.1]oct-2-enes are considerably more potent inhib-
itors of the DAT than the SERT. The 3b-(3,4-
dichlorophenyl)-substitution was selected because this
3,4-dichloro motif has offered the most potent DAT
and SERT inhibitors available.13,38,39 The linking chain
lengths were restricted to a maximum of 10 intervening
methylene groups. Consequently, interaction of each of
the tropane moieties of the bivalent ligands with tropane
binding sites on adjacent DATs of a DAT dimer would
be highly unlikely. The parent C2-ester compounds and
their inhibitory potencies at DAT and SERT are pre-
sented in Table 1.
Tamiz et al.29 later explored the introduction of aspacer in
a class of bivalent 4-arylmethypiperidines in an effort
to obtain selective SERT versus DAT inhibitors. They
reported the striking finding that while the parent mono-
valent methyl 4b-(4-chlorophenyl)-1-methylpiperidine-
3a-carboxylate manifested poor inhibitory potency at
SERT (Ki = 3600 nM), a bivalent analog with a C5 spacer
showed considerable SERT potency (Ki = 1.2 nM). They
concluded that proximal binding sites probably exist on
the SERT.
Christopoulus et al.30 reported the design and evalua-
tion of bivalent muscarinic acetylcholine receptor antag-
onists. They found that the binding potency of the
bivalent compounds was increased by as much as 300-
fold (M1) and 700-fold (M2) over the monovalent parent
compound. Furthermore, M1 and M2 selectivity versus
M3 and M5 receptors could be attained. The implication
of these results was that two proximal binding sites ex-
isted for inhibition of M1 and M2 receptors.
2.2. Synthesis
The starting 2b-carbomethoxy-3b-(3,4-dichlorophenyl)-
8-aza-, 8-oxa-, and 8-thia-bicyclo[3.2.1]octanes (8-
azatropane, 8-oxatropane, and 8-thiatropane) were syn-
thesized (Scheme 1) as described previously. In brief, a
critical ketoester 19 served as the starting point for the
synthesis. The 8-aza-, 8-oxa-, and 8-thia-ketoesters were
resolved10,11,13,40 to provide enantiomerically pure keto-
esters 1R-2. The enoltriflates 3, obtained upon reaction
with N-phenyltriflimide and bis(trimethylsilyl)sodium
imide, were then coupled under Suzuki conditions41 with
3,4-dichlorophenylboronic acid and palladium tetra-
kis(triphenylphosphine) to provide the unsaturated 3-
aryl compounds 4 in good yields. Reduction to provide
the saturated compounds 5 was achieved with samarium
iodide in methanol at low temperature. The desired 3b-
The bivalent approach has also been explored by Fand-
rick et al.31 They reported that while the parent mono-
valent 8-azatropane RTI-3132 inhibited the DAT with
a potency of about Ki = 1 nM, the bivalent RTI-31 li-
gands manifested considerably poorer DAT inhibitory
potency. However, they reported that bivalent com-
pounds with longer intervening linkers showed greater

1834
P. C. Meltzer et al. / Bioorg. Med. Chem. 16 (2008) 1832–1841
Table 1. Monovalent ligands: inhibition of [3H]WIN35, 428 binding to the hDAT and [3H]citalopram binding to the hSERT for 5a, 5b, and 5ca,b
8-Position
Compound
Number
IC50 (nM)
DAT
SERT
8-NCH3
8-O
8-S
5a
5b
5c
O-401
O-1072
O-3516
1.1
3.3
2.0
2.5
4.7
3.0
a Each value is the mean of two or more independent experiments each conducted in different brains and in triplicate. Errors generally do not exceed
10% between replicate experiments.
b Data from Ref. 13.
Scheme 1. Synthesis of 8-hetrobicyclo[3.2.1]octanes. Reagents and conditions: (i) (a) NaN(TMS)2; (b) (1S)-camphanyl chloride; (c) recrystallization
from CH2Cl2 or THF/hexanes, 64%; (d) NaOCH3, MeOH, 90%; (ii) NaN(TMS)2, PhN(Tf)2, 75%; (iii) 3,4-Cl2-PhB(OH)2, Pd(PPh3)4, LiCl, Na2CO3,
74–96%; (iv) Sml2, THF, MeOH, ꢀ60 ꢁC, 20%; (v) LiAlH4, THF, ꢀ78 ꢁC, 85–95%, see Ref. 13 and 11.
aryl (chair) configured compounds 5 were accompanied
by formation of the 3a-aryl configured analogs (gener-
ally, the major products of this reduction) and were sep-
arated from them by column chromatography to
provide the 2b-carbomethoxy-3b-(3,4-dichlorophenyl)
analogs in low yields (ca. 20%). The C2-esters 5 were re-
duced with LiAlH4 in THF at 0 ꢁC to provide the 2b-
hydroxymethyl precursors 6a, b, and c in about 85–
95% yields (Scheme 1).
Thus, the homobivalent ligands 8 (32%) and 16 (93%)
were obtained (Route 1) by reaction of 6a and 6c,
respectively, with succinnic anhydride in the presence
of DMAP, EDCI and HOBT, or succinyl chloride.
The bivalent ligands were obtained (Route 2) upon reac-
tion of the intermediate acids 7b and 7c (prepared from
the primary alcohols, 6b and 6c, upon reaction with suc-
cinnic anhydride in the presence of ethyldimethylamine)
with 6a or 6b in the presence of DMAP and EDCI to
provide the target compounds in yields of 79% (13),
40% (14), and 55% (15). The monovalent 17 (57%) was
prepared by coupling of 6a with heptanoic acid in the
presence of DMAP and EDCI.
In an attempt to obtain all three N,N; N,S; and S,S
(n = 2) bivalent ligands simultaneously, 8-aza 6a and
8-thia 6c were reacted with succinyl chloride in the
presence of DMAP in one pot. However, only the
S,S-16 (n = 2) was obtained, and in excellent yield
(93%). Consequently two routes (Scheme 2) were
adopted for synthesis of the bivalent ligands. The
homobivalent ligands were prepared via Route 1 in
which tandem coupling of the hydroxymethyl precur-
sor 6a (or 6c) was achieved with the appropriate lin-
ker. The heterobivalent ligands were prepared via
Route 2 in which the intermediacy of 7b or 7c avoided
homobivalent side products.
3. Biology
The affinities (IC50 values) for the DAT and SERT
were determined in competition studies with tritium la-
beled ligands. The DAT was labeled with [3H]3b-(4-
fluorophenyl)tropane-2b-carboxylic acid methyl ester
([3H]WIN35, 428 or [3H]CFT (4 nM)) and non-specific
binding was defined as the difference in binding in the

P. C. Meltzer et al. / Bioorg. Med. Chem. 16 (2008) 1832–1841
1835
Scheme 2. Synthesis of bivalent 8-aza-, 8-oxa, and 8-thiatropanes 8–16. Reagents and conditions: (i) heptanoic acid, DMAP, EDCl: (ii) DCC,
DMAP, EtNMe2 or EDCl, DMAP, EtNMe2; (iii) DMAP, EDCl, HOBT, EtNMe2.
presence and absence of (ꢀ)-cocaine (100 lM).
[3H]Citalopram was used to label the SERT and non-
specific binding was measured in the presence of fluoxe-
tine (10 lM). Competition studies were conducted with
a fixed concentration of radioligand and a range of con-
centrations of the test drug. All drugs inhibited
[3H]WIN35, 428 and [3H]citalopram binding in a con-
centration-dependent manner.
linking chain length was important. In order to optimize
the chances of both tropane moieties encountering, and
therefore binding to proximal binding sites, the linker
was designed to be ‘flexible’. Therefore a linker com-
posed of methylene units was selected. Linking chain
˚
lengths were less than 20 A. Consequently, binding to
two sites on different DATs within a DAT dimer34
would not be possible. For ease of synthesis, ester link-
ages were chosen for attachment of the linking chain to
the two tropane moieties.
4. Discussion
The 8-azabicyclo[3.2.1]octane series was utilized to ex-
plore the impact of the length of the spacer with respect
to inhibitory potency of the DAT and SERT. Five com-
pounds (8–12) with linking chains consecutively differing
by two-carbon atoms were evaluated. This provided a lin-
Table 1 presents the parent compounds 5a, 5b, and 5c on
which the bivalent ligands of this study are based. The
data show that these compounds are potent inhibitors
of both DAT (IC50 = 1–4 nM) and SERT (IC50 = 2–
5 nM). Furthermore, these compounds are non-selective
inhibitors, with SERT/DAT ratios of between 1 and 2. It
should be anticipated that bivalent analogs of these par-
ent compounds would display a considerable enhance-
ment of inhibitory potency. In fact, at least a 10- to
100-fold increase in inhibitory potency might be ex-
pected to signify bivalent binding.28,30
˚
ker of between 8 bond lengths (ca. 8.4 A) and 16 bond
˚
lengths (ca. 18.4 A). It is evident from the data in Table
2 that the length of the linking chain had little, if any, sig-
nificant impact upon inhibitory potency at the DAT.
Thus, the two-methylene linked 8 had an IC50 = 12 nM,
the four-methylene linked 9 manifested IC50 = 28 nM,
the six-methylene compound 10 had IC50 = 11 nM, and
the eight-methylene compound 11 manifested IC50
31 nM. The longest linker investigated 12 (10-methylene
=
Since the distance between putative binding sites on the
DAT was unknown, a study of the effect of increasing
˚
units: ca. 18.4 A) had an IC50 = 115 nM. It is therefore

1836
P. C. Meltzer et al. / Bioorg. Med. Chem. 16 (2008) 1832–1841
Table 2. Bivalent ligands: inhibition of [3H]WIN35, 428 binding to the hDAT and [3H]citalopram binding to the hSERT for 8–17a
8X-Position
8Y-Position
n
Compound
Number
IC50 (nM)
DAT
SERT
Homobivalent
8-NCH3
8-NCH3
8-NCH3
8-NCH3
8-NCH3
8-NCH3
8-NCH3
8-NCH3
8-NCH3
8-NCH3
2
8
O-4222
O-4245
O-4623
O-5178
O-4380
12
28
3
106 27
56 16
31 11
4
9
5
3
8
6
10
11
12
11
31
8
10
17
140
1
115
Heterobivalent
8-NCH3
8-NCH3
8-O
8-O
8-S
8-S
8-S
2
2
2
2
13
14
15
16
O-4023
O-3996
O-4022
O-3962
67 11
55 19
2960
405 171
593 188
992
8-S
1540
2200
Monovalent ligand
8-NCH3
6
17
O-5784
8
2
53 27
a Each value is the mean SEM of at least three independent experiments, each conducted in triplicate.
evident that linker length had little bearing upon inhibi-
tory potency at the DAT. Inhibition of the SERT was
somewhat more consistent, in that inhibitory potency in-
creased as the chain linker increased in length from n = 2
(8) to n = 8(11). However, when n = 10 (12)the potency of
inhibition of radioligand binding to the SERT decreased
to a value similar to that obtained when n = 2.
that evidenced by the parent compound 5a (SERT
IC50 = 2.5 nM). Therefore a similar conclusion can be
reached that no second tropane binding site within the
range of the test compounds exists on the SERT. How-
ever, in this case, it is possible that the consistent
improvement in binding as chain length is extended
(n = 2–8) was a consequence of reduced steric encum-
brance at the SERT tropane binding site as the second
tropane extends further away from that actual binding
site.
The most important observation among these com-
pounds was that none of the bivalent 8-azatropanes man-
ifested DAT potency better than, or even equal to, that of
the parent compound 5a (DAT IC50 = 1.1 nM; SERT
IC50 = 2.5 nM). Therefore it can be concluded that the
second tropane in the bivalent ligand did not contribute
to binding. Therefore a second tropane binding site on
the DAT, within the range of the chain lengths explored
in these compounds, does not exist. The reduction (10-
to 30-fold) of binding potency at DAT for 8–12 was most
likely a consequence of steric hindrance offered by the un-
bound second tropane. To confirm the contention that the
second tropane offered only a steric impediment, we pre-
pared the monovalent hexanoyl analogue 17. The binding
potencies observed for 17 (DAT IC50 = 8 nM and SERT
IC50 = 53 nM) were very similar to those manifested by
the homobivalent ligand with six-methylene groups, 10
(DAT IC50 = 11 nM and SERT IC50 = 31 nM). There-
fore, the steric effect presented by the hexanoyl group in
17 was similar to that offered by the second tropane of
homobivalent 10.
In summary, the bivalent 8-azatropanes manifested po-
tent DAT and SERT inhibition thus providing conclu-
sive evidence that these compounds interacted and
bound at the cocaine binding site on both transporters.
However, the fact that these homobivalent compounds
manifested much poorer inhibitory potencies than did
their monovalent progenitors negates the existence of
dual proximal tropane (cocaine) DAT or SERT binding
sites.
We have previously established that 8-oxatropanes and
8-thiatropanes inhibit DAT and SERT with potencies
similar to those of their 8-azatropane counter-
parts.10,11,13 However, the question remained: do the
bivalent-8-heterotropanes interact with the DAT and
SERT similarly? In order to explore this question,
the two-methylene linked hetero-N,O (13), hetero-N,S
(14), hetero-S,O (15), and homo-S,S (16) were com-
pared with the homobivalent two-methylene linked 8-
azatropane, 8. As presented in Table 2, the compounds
that contained an 8-azatropane and an 8-oxatropane
Second, the inhibitory potency of the bivalent ligands at
SERT was also considerably weaker (17–140 nM) than

P. C. Meltzer et al. / Bioorg. Med. Chem. 16 (2008) 1832–1841
1837
(13) or 8-thiatropane (14) retained DAT inhibitory po-
tency (IC50 = 55–67 nM) but lost significant SERT po-
tency (IC50 = 593–405 nM). Those compounds that did
not possess an 8-azatropane (15, 16) lost all potency at
DAT (IC50 = 1.5–3 lM) and SERT (IC50 = 1–2.2 lM).
It is therefore likely that within this class of bivalent li-
gands, an 8-azatropane is required to maintain binding
potency at DAT and SERT, and it can be presumed
that the 8-azatropane binding sites (‘acceptor sites’)11
on both DAT and SERT are not available to either
the 8-oxatropane or 8-thiatropane. Further, it can be
concluded that there is no 8-oxatropane or 8-thiatro-
pane binding site in the proximity of the 8-azatropane
binding site because both 13 and 14 manifested much
weaker potency (DAT: IC50 = ꢁ60 nM; SERT:
IC50 = ꢁ500 nM) than either of their parent com-
pounds (8-oxa: 5b DAT: IC50 = 3.3 nM; SERT:
IC50 = 4.7 nM and 8-thia: 5c DAT: IC50 = 2.0 nM;
SERT: IC50 = 3.0 nM). It is also evident that the accep-
tor site for 8-oxatropanes and 8-thiatropanes is consid-
erably more sensitive to steric factors than is the 8-
azatropane site because the bivalent 8-heterotropanes
15 and 16 were completely inactive at DAT and SERT.
ment with phosphomolybdic acid (PMA). Flash chro-
matography was carried out on Baker Silica Gel
40 lM (silica gel). CombiFlash Chromatography was
conducted on a Teledyne ISCO Sq 16 system. All reac-
tions were conducted under an inert atmosphere of dry
nitrogen. Reagents and solvents were obtained from
commercial suppliers and used as received. Reaction sol-
vents were anhydrous. Yields have not been optimized.
Elemental analyses were performed by Atlantic Micro-
lab, Norcross, GA. HPLC and MS data were obtained
on an Agilent 1100 LC/MSD system. Abbreviations:
HOBT (N-hydroxybenzotriazole), DMAP (dimethyl-
aminopyridine), EDCI (1-ethyl-3-(30-dimethylamino-
propyl)carbodiimide), DCC (dicyclohexylcarbodiim-ide),
brine (saturated aqueous NaCl). A Beckman 1801 Scin-
tillation Counter and Wallac beta-plate and microbeta-
plate readers were used for scintillation spectrometry.
0.1% bovine serum albumin and (ꢀ)-cocaine were pur-
chased from Sigma Chemicals. [3H]WIN35, 428, 2b-car-
bomethoxy-3b-(4-fluorophenyl)-N-[3H]methyltropane
(79.4–87.0 Ci/mmol) and [3H]citalopram (86.8 Ci/mmol)
were purchased from Perkin-Elmer (Boston, MA). (ꢀ)-
Cocaine hydrochloride for the pharmacological studies
was donated by the National Institute on Drug Abuse.
Fluoxetine was purchased from Sigma Chemicals (St.
Louis, MO).
5. Conclusions
8-Homobivalent and 8-heterobivalent tropanes, com-
prised of two tropane moieties linked by intervening
chains of different lengths, were evaluated in a search
for adjacent tropane binding sites on the DAT and
SERT. These bivalent ligands were also used to explore
whether the binding sites on the DAT and the SERT for
8-azatropanes were the same as those for 8-oxatropanes
or 8-thiatropanes. A comparison of these bivalent li-
gands with their progenitor tropanes has cast into doubt
the existence of proximal binding sites on the DAT or
SERT. Indeed, 8-aza-bivalent tropanes inhibited DAT
and SERT with potencies about 10-fold lower at DAT
6.1. (1R)-3b-(3,4-Dichlorophenyl)-2b-hydroxymethyl-8-
methyl-8-azabicyclo[3.2.1]octane (6a)
Compound 5a24 (1.15 g, 5.51 mmol) in dry THF
(39 mL) under a nitrogen atmosphere was cooled to
ꢀ78 ꢁC and LiAlH4 in THF (2 M, 2.1 mL) was added
via syringe. The reaction was then warmed to 0 ꢁC and
stirred for 1 h with complete consumption of starting
material (TLC). The reaction was quenched by slow
addition of a few drops of brine to the cold solution.
When gas evolution ceased, the reaction mixture was di-
luted with aq 10% NaOH (50 mL) and EtOAc (75 mL)
and transferred to a separatory funnel. The aqueous
layer was extracted with EtOAc (3· 75 mL) and the or-
ganic layers combined, dried over MgSO4, filtered, and
evaporated in vacuo to provide a white solid. The solid
was purified by flash chromatography (ratio 32:1; elu-
ent: 48:48:4 EtOAc/hexanes/NEt3) to provide 6a as a
white solid (0.89 g, 84%): mp 99–100 ꢁC; Rf 0.21
(48:48:4 EtOAc/hexanes/NEt3); 1H NMR: d 7.42 (d,
1H), 7.37 (d, 1H), 7.25 (dd, 1H), 7.20–7.10 (bs, OH,
1H), 3.77 (dd, J = 11.3, 1.9 Hz, 1H), 3.45 (d,
J = 2.6 Hz, 1H), 3.35–3.31 (m, 2H), 3.03 (dt, J = 13.2,
6.0 Hz, 1H), 2.47 (td, J = 14.4, 3.1 Hz, 1H), 2.27 (s,
3H), 2.24–2.04 (m, 2H), 1.76–1.57 (m, 3H), 1.50–1.44
(m, 1H); MS (CI, m/z), 300.0 [(M+H)+].
(IC50 = 11–31 nM)
(IC50 = 17–106 nM) at SERT than their monovalent
counterparts (DAT: IC50 = 1.1–3.3 nM; SERT:
and
about
17-fold
lower
IC50 = 2.5–4.7 nM). Furthermore, bivalent ligands in
which one or both of the tropanes was devoid of an
amine suffered a further loss of DAT and SERT inhibi-
tory potency. It is therefore unlikely that there exist two
tropane binding sites in close proximity to one another
on either the DAT or the SERT. It is also likely that dif-
ferent binding sites exist on both the DAT and SERT
for 8-azatropanes compared with 8-oxatropanes or 8-
thiatropanes.
6. Experimental
6.2. (1R)-3b-(3,4-Dichlorophenyl)-2b-(hydroxymethyl)-8-
oxabicyclo[3.2.1]octane (6b)
NMR spectra were recorded on a Jeol 300 NMR spec-
trometer with tetramethylsilane (TMS) as internal stan-
dard and CDCl3 as solvent, unless otherwise mentioned.
Melting points are uncorrected and were measured on a
Mel-Temp melting point apparatus. Room temperature
is 22 1 ꢁC. Thin layer chromatography (TLC) was car-
ried out on Baker Si 250F plates. Visualization was
accomplished with iodine vapor, UV exposure, or treat-
Compound 6b was prepared by LiAlH4 reduction of
methyl ester 5b11 as described above for 6a. Compound
6b was obtained as a thick yellow oil (0.73 g, 96%): Rf
1
0.12 (40:60 EtOAc/hexanes); H NMR: d 7.39 (d, 1H),
7.36 (d, 1H), 7.15 (dd, 1H), 4.68 (d, J = 7.2 Hz, 1H),
4.60 (m, 1H), 3.54 (m, 2H), 3.25 (dt, J = 13.1, 5.6 Hz,

1838
P. C. Meltzer et al. / Bioorg. Med. Chem. 16 (2008) 1832–1841
1H), 2.75 (dd, J = 7.6, 3.2 Hz, 1H), 2.40 (td, J = 13.2,
3.6 Hz, 1H), 2.24–1.51 (m, 5H); MS (CI, m/z), 287.1
[(M+H)+].
dichlorophenyl)-8-methyl-8-azabicyclo[3.2.1]octan-2b-
yl)]methyl dodecanedioate (12: n = 10).
To a solution of (3-(3,4-dichlorophenyl)-8-methyl-8-aza-
bicyclo[3.2.1]octan-2-yl)methanol 6a (0.33 mmol) in
anhydrous CH2Cl2 (4–5 mL) at room temperature was
added Me2NEt (1.7 mmol), followed by addition of suc-
cinic anhydride (0.17 mmol) (compound 8) or the corre-
sponding alkyldioyldichlorides (0.17 mmol) at 0 ꢁC
(compounds 9–12). The reaction mixture was stirred
for 1.5–2 h at room temperature. DMAP (0.17 mmol)
was added, followed by addition of EDCI (0.23 mmol)
(16) and HOBT (0.23 mmol) (compounds 9–12). The
reaction mixture was stirred for 1–3 days at room tem-
perature and monitored by TLC and mass spectrometry.
The reaction mixture was diluted with CH2Cl2, filtered,
washed with aq Na2CO3, brine, dried (Na2SO4), filtered,
and concentrated. Gravity column chromatography
(eluent: acetone) provided compounds 8–12 (19–52%)
(Route 1).
6.3. 3b-(3,4-Dichlorophenyl)-2b-(hydroxymethyl)-8-thia-
bicyclo[3.2.1]octane (6c)
Compound 6c was prepared by LiAlH4 reduction of 5c13
as described for 6a above. Compound 6c was obtained
as a white solid (0.59 g, 95%): Rf 0.62 (50:50 EtOAc/hex-
1
anes); H NMR: d 7.37 (d, 1H), 7.30 (d, 1H), 7.08 (dd,
1H), 3.83 (t, J = 5.0 Hz, 1H), 3.74–3.65 (m, 2H), 3.38
(dt, J = 11.0, 3.7 Hz, 1H), 3.15 (dt, J = 13.1, 5.6 Hz,
1H), 2.41–1.97 (m, 6H), 1.88 (dt, J = 13.5, 4.9 Hz, 1H).
6.4. (1R)-3b-(3,4-Dichlorophenyl)-2b-(3-carboxypropio-
nyloxymethyl)-8-oxabicyclo[3.2.1]octane (7b)
Compound 6b (108 mg, 0.376 mmol) was dissolved in
Ethyldimethylamine
CH2Cl2
(10 mL).
(241 mg,
3.29 mmol) was added via syringe and the reaction
was allowed to stir for 5 min at room temperature. Solid
succinic anhydride (46.3 mg, 0.463 mmol) was then
added and the reaction stirred at room temperature
for 19 h. The reaction was quenched with 1.5 N HCl
(25 mL) and allowed to stir for 5 min. The crude reac-
tion mixture was then diluted with CHCl3 (50 mL) and
aqueous layer was separated and extracted with CHCl3
(3· 50 mL). The combined CHCl3 extracts were washed
with water followed by brine, dried (MgSO4), filtered,
and evaporated in vacuo to obtain 7b as a yellow oil
(190 mg, quant.). This material was used in subsequent
steps without further purification. Rf 0.09 (20:80
MeOH/EtOAc); 1H NMR: d 7.39 (d, 1H), 7.25 (d,
1H), 7.02 (dd, 1H), 4.57–4.55 (m, 1H), 4.50 (d,
J = 6.6 Hz, 1H), 4.18 (t, J = 10.3 Hz, 1H), 3.73 (dd,
J = 10.7, 5.0 Hz, 1H), 3.28 (dt, J = 12.8, 5.1 Hz, 1H),
2.62–2.55 (m, 2H), 2.51–2.43 (m, 2H), 2.23–1.75 (m,
6H), 1.57–1.48 (m, 1H); MS (CI, m/z), 385.1 [(MꢀH)ꢀ].
6.6.1. Bis[(3b-(3,4-dichlorophenyl)-8-methyl-8-azabicy-
clo[3.2.1]octan-2b-yl)]methyl succinate (8: n = 2). White
solid: (32%), mp 148.5–149.5 ꢁC, Rf 0.31 (acetone); H
NMR: d 7.35 (d, 2H), 7.25 (d, 2H), 7.08 (dd, 2H), 4.25
(dd, J = 10.7, 8.8 Hz, 2H), 3.67 (dd, J1 = 10.7, 5.2 Hz,
2H), 3.15–3.30 (m, 4H), 3.00–3.13 (m, 2H), 1.95–2.38
(m, 18H), 1.50–1.70 (m, 6H); 13C NMR: d 24.8, 26.1,
28.9, 33.7, 34.3, 41.9, 45.2, 61.8, 63.6, 63.8, 126.9,
129.6, 130.0, 130.2, 132.3, 142.9, 171.9; MS (CI, m/z),
681.2 [(M+H)+]. Anal. Calcd for C34H40Cl4N2O4.
1
6.6.2. Bis[(3b-(3,4-dichlorophenyl)-8-methyl-8-azabicy-
clo[3.2.1]octan-2b-yl)]methyl adipate (9: n = 4). White so-
lid: (53%), mp 130.7–131.6 ꢁC; Rf 0.33 (acetone); 1H
NMR: d 7.35 (d, 2H), 7.25 (d, 2H), 7.08 (dd, 2H), 4.17
(dd, J = 11, 8 Hz, 2H), 3.70 (dd, J = 11.0, 5.4 Hz, 2H),
3.20–3.30 (m, 4H), 3.05 (dt, J = 13.4, 5.4 Hz, 2H),
1.95–2.23 (m, 18H), 1.50–1.60 (m, 6H), 1.25–1.48 (m,
4H); 13C NMR: d 24.3, 24.8, 26.1, 33.7, 33.8, 34.3,
42.0, 45.3, 61.8, 63.6, 63.9, 126.9, 129.6, 130.0, 130.2,
132.0, 143.0, 173.0; MS (CI, m/z), 709.2 [(M+H)+]. Anal.
Calcd for C36H44Cl4N2O4.
6.5. (1R)-3b-(3,4-Dichlorophenyl)-2b-(3-carboxypropio-
nyloxymethyl)-8-thiabicyclo[3.2.1]octane (7c)
Compound 7c was obtained from 6c as described for 7b
above and used in subsequent steps without purification:
Rf 0.23 (50:50 EtOAc/hexanes); H NMR: d 7.37 (d,
1H), 7.23 (d, 1H), 7.00 (dd, 1H), 4.31 (dd, J = 10.9,
9.2 Hz, 1H), 3.75–3.69 (m, 2H), 3.64–3.57 (m, 1H),
3.18 (dt, J = 13.3, 5.5 Hz, 1H), 2.61–2.53 (m, 2H),
2.47–2.39 (m, 2H), 2.36–2.01 (m, 6H), 1.86 (dt,
J = 13.4, 4.8 Hz, 1H).
6.6.3. Bis[(3b-(3,4-dichlorophenyl)-8-methyl-8-azabicy-
clo[3.2.1]octan-2b-yl)]methyl octanedioate (10: n = 6).
White solid: (19%), mp 136.0–137.0 ꢁC; Rf 0.32 (ace-
tone); 1H NMR: d 7.33–7.40 (m, 2H), 7.20–7.30 (m,
2H), 6.97–7.10 (m, 2H), 4.15–4.26 (m, 2H), 3.65–3.80
(m, 2H), 3.17–3.35 (m, 4H), 3.05–3.15 (m, 2H), 1.95–
2.25 (m, 18H), 1.35–1.72 (m, 10H), 1.15–1.28 (m, 4H);
13C NMR: d 24.8, 24.9, 26.2, 28.9, 33.8, 34.2, 34.4,
42.1, 45.4, 61.9, 63.6, 64.0, 127.0, 129.7, 130.1, 130.3,
143.1, 173.6; MS (CI, m/z), 737.3 [(M+H)+]. Anal. Calcd
for C38H48Cl4N2O4.
1
6.6. General procedure for the synthesis of homobivalent
ligands 8–12 (n = 2, 4, 5, 8, 10)
Bis[(1R)-(3b-(3,4-dichlorophenyl)-8-methyl-8-azabicyclo
[3.2.1]octan-2b-yl)]methyl succinate (8: n = 2), bis[(1R)-
(3b-(3,4-dichlorophenyl)-8-methyl-8-azabicyclo[3.2.1]
octan-2b-yl)]methyl adipate (9: n = 4), bis[(1R)-(3b-(3,4-
dichlorophenyl)-8-methyl-8-azabicyclo[3.2.1]octan-2b-
yl)]methyl octanedioate (10: n = 6), bis[(1R)-(3b-(3,4-
dichlorophenyl)-8-methyl-8-azabicyclo[3.2.1]octan-2b-
yl)]methyl decanedioate (11: n = 8), bis[(1R)-(3b-(3,4-
6.6.4. Bis[(3b-(3,4-dichlorophenyl)-8-methyl-8-azabicy-
clo[3.2.1]octan-2b-yl)]methyl decanedioate (11: n = 8).
Colorless oil: (37%); Rf 0.27 (acetone); 1H NMR: d
7.35 (d, 2H), 7.23 (d, 2H), 7.01 (dd, 2H), 4.18 (dd,
J = 11.0, 8.2 Hz, 2H), 3.71 (dd, J = 11.0, 5.4 Hz, 2H),
3.20–3.30 (m, 4H), 3.05 (dt, J = 13.2, 5.4 Hz, 2H),
1.97–2.25 (m, 18H), 1.40–1.68 (m, 10H), 1.15–1.28 (m,

P. C. Meltzer et al. / Bioorg. Med. Chem. 16 (2008) 1832–1841
1839
8H); 13C NMR: d 24.9, 26.2, 29.2, 33.8, 34.3, 34.4, 42.1,
45.4, 61.9, 63.5, 64.0, 127.0, 129.7, 139.3, 143.1, 173.6;
MS (CI, m/z), 765.3 [(M+H)+]. Anal. Calcd for
C40H52N2O4Cl4.
white solid (200 mg, 79%): mp 117–119 ꢁC; Rf 0.49
1
(10:90 MeOH/CH2Cl2); H NMR: d 7.38 (d, 1H), 7.34
(d, 1H), 7.24 (s, 2H), 7.03–6.98 (m, 2H), 4.58–4.52 (m,
1H), 4.46 (d, J = 7.2 Hz, 1H), 4.26–4.11 (m, 2H), 3.71–
3.64 (m, 2H), 3.31–3.16 (m, 3H), 3.04 (dt, J = 13.5,
5.1 Hz, 1H), 2.42–2.25 (m, 4H), 2.22–1.48 (m, 17H);
MS (CI, m/z), 668.2 [(M+H)+]. Anal. Calcd for
C33H37Cl4NO5.
6.6.5. Bis[(3b-(3,4-dichlorophenyl)-8-methyl-8-azabicy-
clo[3.2.1]octan-2b-yl)]methyl dodecanedioate (12: n = 10).
White solid: (44%); mp 115–116 ꢁC; Rf 0.32 (acetone); 1H
NMR: d 7.35 (d, 2H), 7.26 (m, 2H), 7.02 (dd, 2H), 4.20
(dd, J = 10.7, 8.3 Hz, 2H), 3.71 (dd, J = 11.0, 5.5 Hz,
2H), 3.34–3.22 (m, 4H), 3.11–3.01 (m, 2H), 2.31–1.96
(m, 18H), 1.75–1.14 (m, 22H); 13C NMR: d 24.8, 24.9,
26.2, 29.1, 29.2, 29.4, 33.7, 34.2, 34.3, 42.0, 45.5, 61.8,
63.4, 63.9, 126.9, 129.6, 130.0, 130.2, 143.0, 173.6; MS
(CI, m/z), 793.3 [(M+H)+]. Anal. Calcd for C42H56
N2O4Cl4.
6.9. (3b-(3,4-Dichlorophenyl)-8-oxabicyclo[3.2.1]octan-
2b-yl)methyl, (3b-(3,4-dichlorophenyl)-8-thiabicyclo[3.2.1]
octan-2b-yl)methyl succinate (15)
Compound 15 was prepared from 7c and 6b as described
above for 13, except that DCC was used in place of
EDCI. White solid: (55%); mp 195–196 ꢁC; Rf 0.39
(40% EtOAc/Hex); 1H NMR: d 7.35–7.4 (m, 2H),
7.23–7.27 (m, 2H), 6.96–7.05 ( m, 2H), 4.52–4.60 (m,
1H), 4.45 (d, J = 6.8 Hz, 1H), 4.28 (t, J = 10.1 Hz,
1H), 4.10–4.20 (m, 1H), 3.60–3.78 (m, 4H), 3.22–3.35
(m, 1H), 3.10–3.20 (m, 1H), 1.80–2.40 (m, 17H), 1.47–
1.60 (m, 1H); MS (CI, m/z), 671.3 [(MꢀH)ꢀ]; 704.1
[(M+MeOH)ꢀ]. Anal. Calcd for C32H34SO5Cl.
6.7. ((1R)-3b-(3,4-Dichlorophenyl)-8-methyl-8-azabicyclo
[3.2.1]octan-2b-yl)methyl, (3b-(3,4-dichlorophenyl)-8-thi-
abicyclo[3.2.1]octan-2b-yl)methyl succinate (14)
(Route 2) 3b-(3,4-dichlorophenyl)-2b-(3-carboxy-propi-
onyloxymethyl)-8-thiabicyclo[3.2.1]octane 7c (109 mg,
0.269 mmol) was dissolved in CH2Cl2 (6 mL). To the
resulting solution was added DMAP (107 mg,
0.874 mmol), followed by 6a (82.4 mg, 0.274 mmol),
and finally EDCI (106 mg, 0.555 mmol). The reaction
was stirred at room temperature for 25 h under nitrogen
atmosphere and then diluted with water (25 mL) and
CH2Cl2 (25 mL). The organic layer was separated and
washed with water and brine, dried (MgSO4), filtered,
and evaporated in vacuo to obtain a yellow solid that
contained significant amounts of DMAP and desired
product. The crude 14 was then purified by CombiFlash
chromatography (40 g silica; eluent: CHCl3/MeOH) to
obtain a yellow oil, which was crystallized from
6.10. (3b-(3,4-Dichlorophenyl)-8-thiabicyclo[3.2.1]octan-
2b-yl)methyl, (3b-(3,4-dichlorophenyl)-8-thiabicyclo[3.2.1]
octan-2b-yl)methyl succinate (16)
Succinyl chloride (144 mg, 0.931 mmol) was added
dropwise to a solution of DMAP (284 mg, 2.33 mmol)
in CH2Cl2 (3 mL) at 0 ꢁC. The mixture was stirred at
0 ꢁC for 15 min. A solution of 6a (233 mg, 0.776 mmol)
in CH2Cl2 (3 mL) was added to the DMAP solution and
the reaction was allowed to warm to room temperature
over 2 h. The mixture was then cooled to 0 ꢁC and a
solution of 6c (235 mg, 0.776 mmol) in CH2Cl2 (3 mL)
was added to the reaction mixture which was stirred
overnight at room temperature. A second portion of
succinyl chloride (36 mg, 0.23 mmol) was added and
the reaction mixture was stirred for an additional 2 h.
The reaction was quenched with water (30 mL), fol-
lowed by dropwise addition of aqueous NH4OH until
a pH of 8 was achieved. The crude mixture was ex-
tracted with CHCl3 (3· 30 mL), and the organic layers
combined, dried (Na2SO4), and evaporated under re-
duced pressure to give a product which was purified
by flash chromatography (eluent: 20% ethyl acetate/
80% hexanes). Evaporation of appropriate fractions
provided compound 16 as a white solid (249 mg, 93%):
mp: 188–190 ꢁC; Rf 0.26 (hexanes/ethyl acetate; 20:1);
1H NMR: d 7.36 (d, 2H), 7.22 (d, 2H), 6.98 (dd, 2H),
4.28 (m, 2H), 3.70–3.58 (m, 6H), 3.16 (dt, J = 13.5,
5.4 Hz, 2H), 2.36–2.01 (m, 16H), 1.85 (dt, J = 13.2,
4.7 Hz, 2H). Anal. Calcd for C32H34Cl4O4S2.
EtOAc/heptane to provide 14 as
a white solid
(73.5 mg, 40%): mp 165–166 ꢁC; Rf 0.51 (10:90 MeOH/
1
CH2Cl2); H NMR: d 7.36 (d, 1H), 7.34 (d, 1H), 7.25–
7.20 (m, 2H), 7.02–6.95 (m, 2H), 4.32–4.18 (m, 2H),
3.70–3.58 (m, 4H), 3.27–3.00 (m, 3H), 2.36–1.94 (m,
17H), 1.85 (dt, J = 13.5, 5.0 Hz, 1H), 1.65–1.49 (m,
4H). Anal. Calcd for C33H37Cl4NO4S.
6.8. ((1R)-3b-(3,4-Dichlorophenyl)-8-methyl-8-azabicyclo
[3.2.1]octan-2b-yl)methyl, ((1R)-3b-(3,4-dichlorophenyl)-
8-oxabicyclo[3.2.1]octan-2b-yl)methyl succinate (13)
(Route 2) (1R)-3b-(3,4-dichlorophenyl)-2b-(3-carboxy-
propionyloxymethyl)-8-oxabicyclo[3.2.1]octane 7b (145 mg,
0.376 mmol) was dissolved in CH2Cl2 (9 mL). DMAP
(146 mg, 1.19 mmol) was added to the solution, fol-
lowed by 6a (113 mg, 0.378 mmol) and EDCI (144 mg,
0.753 mmol). The reaction was stirred at room tempera-
ture for 18.5 h, then quenched with water (10 mL), and
extracted with CH2Cl2 (50 mL). The CH2Cl2 layer was
washed with saturated aqueous NaHCO3 (50 mL),
water (50 mL), and brine (50 mL), dried (MgSO4), fil-
tered, and evaporated in vacuo to yield a yellow oil
which was purified by CombiFlash chromatography
(40 g silica; eluent: CH2Cl2/MeOH). Like fractions were
combined and evaporated to provide an oil that crystal-
lized from Et2O/pentane to provide the product 13 as a
6.11. ((1R)-3b-(3,4-Dichlorophenyl)-8-methyl-8-azabicy-
clo[3.2.1]octan-2b-yl)methyl heptanoate (17)
Compound 6a (105 mg, 0.351 mmol) was dissolved in
CH2Cl2 (3 mL). DMAP (134 mg, 1.09 mmol) was added
to the solution, followed by a solution of heptanoic acid
(64.1 mg, 0.492 mmol) in CH2Cl2 (4 mL). EDCI
(134.4 mg, 0.7011 mmol) was then added and the reac-

1840
P. C. Meltzer et al. / Bioorg. Med. Chem. 16 (2008) 1832–1841
tion allowed to be stirred at room temperature for 19 h.
The reaction was quenched with water (20 mL) and ex-
tracted with CH2Cl2 (3· 20 mL). The organic extracts
were combined and washed with saturated aqueous
NaHCO3, water, and brine. The organic layer was dried
(MgSO4), filtered, and evaporated in vacuo to obtain a
solid (211 mg) which was purified by flash chromatogra-
phy (ratio: 30:1; eluent: 2:98 MeOH/CH2Cl2 to 8:92
MeOH/CH2Cl2). Compound 17 was obtained as an am-
ber oil (82.3 mg, 57%): Rf 0.45 (49:49:2 EtOAc/hexanes/
following constituents at a final assay concentration:
[3H]citalopram (1 nM) and membrane preparation.
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 pre-soaked in 0.1%
polyethyleneimine. The filters were washed twice with
5 mL Tris–HCl buffer (50 mM), and radioactivity was
measured as described above. Non-specific binding
was defined as [3H]citalopram bound in the presence
of an excess (10 lM) of fluoxetine.
1
NEt3); H NMR: d 7.35 (d, 1H), 7.25 (d, 1H), 7.02 (dd,
1H), 4.20 (dd, J = 11.0, 8.3 Hz, 1H), 3.71 (dd, J = 11.0,
5.5 Hz, 1H), 3.31–3.20 (m, 2H), 3.05 (dt, J = 13.2,
5.3 Hz, 1H), 2.30–1.97 (m, 9H), 1.75–1.19 (m, 12H),
0.93–0.85 (m, 2H). Anal. Calcd for C22H31Cl2NO2.
6.14. Data analysis
Data were analyzed by the EBDA computer software
programs (Elsevier-Biosoft, UK) or the PRIZM soft-
ware program (San Diego, CA). Baseline values for
the individual drugs were established from the competi-
tion curves and these generally were similar to baseline
values established by 30 lM (ꢀ)-cocaine or 1 lM fluox-
etine. For assays involving brain tissue, the SERT was
assayed in caudate-putamen membranes using condi-
tions similar to those for the dopamine transporter
above.
6.12. Dopamine transporter assay
Assays were conducted by minor modifications of the
methods of Eshleman et al.42 and Pham-Huu et al.13
In assays involving the human transporters, mem-
branes from transfected cells stably expressing the
DAT were labeled with [3H]WIN35, 428 ([3H]CFT,
2b-carbomethoxy-3b-(4-fluorophenyl)-N-[3H]methyltro-
pane, 81–84 Ci/mmol, Perkin-Elmer, Boston, MA). The
affinities of drugs at the [3H]WIN35, 428 binding site
on the DAT were determined by incubating tissue with
a fixed concentration of [3H]WIN35, 428 and a range
of drug concentrations. 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: [3H]WIN35, 428 (4 nM) and cell mem-
brane preparation (0.2 mL). The 1 h incubation at
22 ꢁC was initiated by addition of membranes and ter-
minated by rapid filtration over Whatman GF/B glass
fiber filters pre-soaked in 0.1% polyethyleneimine (Sig-
ma Chem. Co., St. Louis, MO). The filters were
washed twice with 5 mL Tris–HCl buffer (50 mM),
dried, scintillation fluid added to each spot, and radio-
activity was measured on a Wallac beta-plate or
microbeta-plate reader. Non-specific binding was de-
fined as [3H]WIN35, 428 bound in the presence of an
excess (100 lM) of (ꢀ)-cocaine. Stock solutions of
drugs were dissolved in DMSO. The stock solutions
were diluted serially in the assay buffer and added
(0.2 mL) to the assay medium as described above. In
assays involving brain tissue, 1 nM radioligand was
used, and filters were pre-soaked in 0.1% bovine serum
albumin (Sigma Chem. Co.). In addition, non-specific
binding was defined as [3H]WIN35, 428 bound in the
presence of an excess (30 lM) of (ꢀ)-cocaine.
Acknowledgments
This work was supported by the National Institute on
Drug Abuse: DA18825 (P.M.), DA11542 (P.M.),
DAO18165 (A.J.) and by the VA Merit Review and Re-
search Career Scientist Programs (A.J.).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at 10.1016/j.bmc.2007.
11.009.
References and notes
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1991, 14, 299.
3. Kennedy, L. T.; Hanbauer, I. J. Neurochem. 1983, 34,
1137.
4. Schoemaker, H.; Pimoule, C.; Arbilla, S.; Scatton, B.;
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