3-Aryl-2-carbomethoxybicyclo[3.2.1]oct-2-enes inhibit WIN 35,428 binding potently and selectively at the dopamine transporter

Article abstract of DOI:10.1016/S0968-0896(99)00322-3

The search for medications for cocaine abuse has focused upon the design of potential cocaine antagonists or cocaine substitutes which interact at the dopamine transporter of mammalian systems. This manuscript describes the synthesis and biological evalua

Full text of DOI:10.1016/S0968-0896(99)00322-3

Bioorganic & Medicinal Chemistry 8 (2000) 581±590
3-Aryl-2-carbomethoxybicyclo[3.2.1]oct-2-enes Inhibit WIN
35,428 Binding Potently and Selectively at the Dopamine
Transporter
Peter C. Meltzer, a,* Paul Blundell, a Hong Huang, a Shanghao Liu, a
Yaw F. Yong a and Bertha K. Madras b
aOrganix Inc., 240 Salem Street, Woburn, MA 01801, USA
bDepartment of Psychiatry, Harvard Medical School and New England Regional Primate Center, Southborough, MA 01772, USA
Received 26 August 1999; accepted 28 October 1999
AbstractÐThe search for medications for cocaine abuse has focused upon the design of potential cocaine antagonists or cocaine
substitutes which interact at the dopamine transporter of mammalian systems. This manuscript describes the synthesis and biolo-
gical evaluation of 8-substituted 2-carbomethoxy-3-arylbicyclo[3.2.1]oct-2-enes. These compounds prove potent and selective inhi-
bitors of the dopamine transporter. Their selectivity results primarily from a reduced inhibitory potency toward the serotonin
transporter. This work supports the notion that the orientation of the 3-aryl ring in the bicyclo[3.2.1]octane system a€ects the
interaction of these molecules with the serotonin transporter far more markedly than it a€ects the interaction with the dopamine
transporter. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
studies have concentrated on the development of certain
bicyclo[3.2.1]octanes, congeners of the prototypical
cocaine-like tropane, WIN 35,428.19,27±33 During the
course of these studies we have learned that the speci®c
presence of nitrogen at the 8-position of these tropanes
is not a prerequisite for binding to the DAT and that
replacement with either an oxygen30 or even a carbon19
can lead to potent compounds.31 Furthermore, we27 and
others,18,20 have learned that substitution on the aro-
matic ring of such compounds plays a considerable role
in the binding potency of these compounds to the DAT
and can a€ect their relative potency with respect to
inhibition of the SERT.20,34 Finally, studies have also
shown that the orientation of the 3-aryl ring in either
the a- or b-con®guration can a€ect selectivity of these
compounds for the DAT versus the SERT.19,35 This
latter observation prompted us to explore whether the
orientation of the aromatic ring in a more planar rela-
tionship to the tropane skeleton could further enhance
biological selectivity. This approach was particularly
attractive since these unsaturated compounds are read-
ily available on route to the more classical saturated
bicyclo[3.2.1]octanes.
The dopamine transporter (DAT), present in ®bers of
mammalian neurons, has been the focus of considerable
research. It is at this site that cocaine is understood to
exert its pharmacological activity by inhibition of reup-
take of endogenous dopamine.1±7 The DAT also pro-
vides a measure of certain neurological diseases. For
example, in normal human aging the concentration of
this transporter decreases about 5±7% for each decade
of life.8,9 In Parkinson's disease the transporter is con-
siderably depleted and consequently a measure of the
DAT concentration can provide a diagnostic assessment
of the progression of the disease.10,11 Although no
medication currently exists which can reverse this dis-
ease, quanti®cation of the DAT will provide an excel-
lent marker for the success of such agents as they are
developed in the future.12±17
In the search for medications for cocaine abuse, the
DAT has provided a focus for the design of potential
cocaine antagonists or cocaine substitutes.7,18±26 Our
In this work we present the synthesis and binding
constants (DAT and SERT) for three series of
*Corresponding author. Tel.: +1-781-932-4142; fax: +1-781-933-
6695; e-mail: meltzer@organixinc.com
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00322-3

582
P. C. Meltzer et al. / Bioorg. Med. Chem. 8 (2000) 581±590
bicyclo[3.2.1]oct-2-enes (Fig. 1). We report that a num-
ber of these 2,3-enes manifest substantial binding
potency at the DAT and relatively poor potency at
the SERT. This results in extremely selective DAT
inhibitors.
We have previously reported the syntheses of the three
critical intermediate keto esters, 6,30 728 and 8.19 In
summary, 6 had previously been prepared by the reac-
tion of 2,5-dimethoxytetrahydrofuran (1) with 1,3-bis-
(trimethylsiloxy)-1-methoxybuta-1,3-diene (2).36,37 Com-
pound 7 was readily obtained from tropinone 3 via
introduction of the C2-methyl ester followed by enan-
tiomeric resolution of the tartrate salts to obtain both
the 7-1R and 7-1S enantiomers.28,38,39 Compound 8 was
obtained from 3-chlorobicyclo[3.2.1]oct-2-ene (4) upon
treatment with sulfuric acid40 and subsequent introduc-
tion of the 2-carbomethoxy group by reaction with
methyl cyanoformate in the presence of lithium diiso-
propyl amide.
Results
Chemistry
The compounds of this study are 2,3-disubstituted-
bicyclo[3.2.1]oct-2-enes. Their synthesis is presented in
Scheme 1.
Figure 1.
Scheme 1. Synthesis of 3-aryl-8-heterobicyclo[3.2.1]oct-2-enes. Reagents: (i) TiCl4, (ii) LDA, CNCO2CH3, (iii) H2SO4, (iv) NaN(TMS)2, PhNTf2, (v)
(S)-( )-camphanic chloride,(vi) ArB(OH)2, Pd2(dba)3, (vii) (COCl)2, RHNR.

P. C. Meltzer et al. / Bioorg. Med. Chem. 8 (2000) 581±590
583
Enantiomeric purity of the 1R and 1S isomers of 6 and
7 was con®rmed by formation of their enol campha-
nates upon reaction of the enolates with (S)-( )-cam-
phanic chloride. The methyls of the resultant
camphanate diastereomers had suciently di€erent
similar throughout these compounds. Calculation of the
dihedral angles by means of the Karplus equation indi-
cates that H5±C5±C4±H4a is about 85±90ꢀ and H5±C5±
C4±H4b is about 40±45ꢀ. This implies that the ®ve car-
bon centers at C1±C2±C3±C4±C5 are almost coplanar,
thus resulting in a ¯attened ring system.
1
chemical shifts to enable their use in H NMR spectro-
scopy to quantify enantiomeric excess.30
Biology
The ketoesters 6, 7 and 8 were treated with N-phenyl-
bis(tri¯uoromethanesulfonyl)amine and sodium bis(tri-
methylsilyl) amide in tetrahydrofuran30 to obtain the
enol tri¯ates 9, 10, 11 in good yield. The enol tri¯ates
were then coupled41 with the appropriate arylboronic
acids in diethoxymethane in the presence of lithium
chloride, sodium carbonate and tris(dibenzylideneace-
tone) dipalladium(0)30,35 to provide the desired aryl
octenes 12 (Table 2), 13 (Table 3) and 14 (Table 4).
Compounds 15 (Table 2) were obtained upon hydrolysis
of 12, formation of the acid chloride, and subsequent
reaction with the appropriate amines. It is interesting
that while hydrolysis of saturated 8-azatropanes can
generally be accomplished by simple re¯ux in dioxane
water mixtures, this is not a sucient means for these
unsaturated enes (12, 13 and 14). Indeed, LiOH hydro-
lysis was required in these cases.
The anities of a series of bicyclo[3.2.1]oct-2-ene ana-
logues for the dopamine and serotonin transporters
were determined in competition studies using [3H]3b-(4-
¯uorophenyl)tropane-2b-carboxylic acid methyl ester
([3H]WIN 35,428 or [3H]CFT) to label the dopamine
transporter5 and [3H]citalopram to label the serotonin
transporter.31 Each compound was tested 2±5 times and
each assay conducted in a di€erent brain. Binding data
(IC50) for the 8-oxabicyclo[3.2.1]oct-2-enes are pre-
sented in Table 2. Data for the 8-azabicyclo[3.2.1]oct-2-
enes are presented in Table 3, and data for the bicy-
clo[3.2.1]oct-2-enes are shown in Table 4. Studies were
conducted in monkey striatum because these com-
pounds are part of an ongoing investigation of
structure±activity relationships at the DAT in this tis-
sue.5,7,27,28 Hence, meaningful comparisons with an
extensive data base can be made. IC50 values are repor-
ted as the assay concentration of [3H]WIN 35,428 and
[3H]citalopram were below Kd values. Competition stu-
dies were conducted with a ®xed concentration of radi-
oligand and a range of concentrations of the test drug.
All drugs inhibited [3H]WIN 35,428 and [3H]citalopram
binding in a concentration-dependent manner. The 8-
azabicyclo[3.2.1]octenes (Table 3) are all enantiomeri-
cally pure. The 8-oxabicyclo[3.2.1]octenes, with the
exception of O-1059 and O-1108 (Table 2), and the
bicyclo[3.2.1]octenes (Table 4) are racemic. Conse-
quently, comparison of binding data has to be made
with care since the DAT is a stereoselective uptake
mechanism and consequently one enantiomer (generally
the 1R) is considerably more potent than the other.
Structural characterization of these compounds is readily
1
accomplished by H and 13C NMR spectroscopy. The
1H NMR is particularly diagnostic as exempli®ed for
the series of 3-(3,4-dichlorophenyl)bicyclo[3.2.1]-octenes
and enol tri¯ates in Table 1. Thus the H4a and H4b
protons consistently appear at about d 2.0±2.1 as a gem
coupled doublet (J=17.9±18.7 Hz), and d 2.6±3.0 as a
double doublet showing both the gem coupling (J=
17.9±18.9 Hz) as well as the coupling to the H5 bridge-
head proton (J=4.4±4.9 Hz), respectively. Only in the
case of 11 is the H4a to H5 coupling seen (J=1.9 Hz).
It is interesting to note that the coupling constants
measured between H5 and each of H4a and H4b are
Table 1. 1H NMR data for representative enol tri¯ates 9, 10, 11 and 2,3-enes 12g, 13d, 14ca
X
Compound
H1
H5
H4b
H4a
COOCH3
O
NCH3
CH2
12g
13d
14c
5.00 (d)b
3.86 (d)
3.00 (t)c
4.64 (t)
3.36 (t)
2.45 (m)d
2.91 (J=4.9, 18.7)e
2.76 (J=4.7, 18.9)
2.63 (J=4.7, 18.7)
2.06 (J=18.7)
1.97 (J=18.4)
2.14 (J=18.7)
3.55
3.52
3.49
O
NCH3
CH2
9
10
11
5.04 (d)
3.93 (d)
3.10 (t)
4.69 (t)
3.42 (t)
2.51 (m)
3.00 (J=4.7, 17.9)
2.84 (J=4.4, 18.7)
2.70 (J=4.7, 18.4)
2.13 (J=17.9)
1.97 (J=18.4)
2.17 (J=1.9, 18.1)
3.82
3.81
3.81
aResonance positions in ppm from TMS.
bd=Doublet.
ct=Triplet.
dm=Multiplet.
eCoupling (J) in Hz.

584
P. C. Meltzer et al. / Bioorg. Med. Chem. 8 (2000) 581±590
Table 2. Inhibition of [3H]WIN 35,428 binding to the dopamine transporter and [3H]citalopram binding to the serotonin transporter by 8-oxabi-
cyclo[3.2.1]octenes in cynomolgus monkey caudate-putamen
Ar
R2
No.
Compound
Dopamine transporter
[3H]WIN 35,428
Serotonin transporter
[3H]citalopram
Selectivity
SERT/DAT
C6H5
4-FC6H4
4-ClC6H4
4-BrC6H4
4-IC6H4
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
4-Cl-3-FC6H3
3-ClC6H4
1-Naphthyl
2-Naphthyl
2-Anthracenyl
Benzofuran
3,4-(OCH2O)C6H3
4-CHOC6H4
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
(1R/S)
O-1141
O-1132
O-1134
O-1155
O-1165
O-1014
O-1059
O-1108
O-1143
O-1614
O-1170
O-1140
O-1537
O-1147
O-1611
O-1497
>10,000
2730
238
>10,000
>50,000
>60,000
>30,000
4,820
1960
2120
>10,000
5300
>10,000
>40,000
704
>10,000
>20,000
>2000
>10,000
Ð
19
278
632
71
159
461
803
74
8
25
36
0.2
63
Ð
Ð
(1R/S)
(1R/S)
(1R/S)
(1R/S)
(1R/S)
(1R)
62
67.9
12.3
4.6
58.2
72
1280
1720
19.7
(1S)
(1R/S)
(1R/S)
(1R/S)
(1R/S)
(1R/S)
(1R/S)
(1R/S)
(1R/S)
>40,000
413
>4000
>10,000
a
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
cC4H7
cC4H8
15a
15b
15c
(1R/S)
(1R/S)
(1R/S)
O-1169
O-1178
O-1179
2890
436
324
>10,000
>10,000
>50,000
3
42
154
b
cC4H8Oc
aCyclobutyl.
bPiperidinyl.
cMorpholinyl.
Table 3. Inhibition of [3H]WIN 35,428 binding to the dopamine transporter and [3H]citalopram binding to the serotonin transporter by 8-azabi-
cyclo[3.2.1]octenes in cynomolgus monkey caudate-putamen
Ar
No.
Compound 13
Dopamine transporter
[3H]WIN 35,428
Serotonin transporter
[3H]citalopram
Selectivity
SERT/DAT
C6H5
4-FC6H4
4-FC6H4
3,4-Cl2C6H3
3,4-Cl2C6H3
2-Naphthyl
3,4-Cl2C6H3; NH
4-FC6H4; NH
a
b
c
d
e
f
(1R)
O-1449
O-1104
O-1119
O-1109
O-1120
O-1173
O-1130
O-1131
2590
408
1590
1.16
490
2.9
0.99
740
>20,000
7990
>20,000
867
1920
11
20
18
747
4
38
54
1
(1R)
(1S)
(1R)
(1S)
(1R)
(1R)
(1R)
109
53.6
829
g
h
Discussion
Structure±activity relationships (SAR)
classes of 3-(substituted-aryl) compounds at the DAT is
similar and can be summarized as 3,4-Cl2>3-(2-naph-
thyl)>F>H. Within the 8-oxabicyclo[3.2.1]octenes, in
which a larger series was made, the rank order can be
extended as follows: 3,4-Cl2>3-(2-naphthyl)>I=
Br>Cl>F>H. Also, the 8-oxa-3-(2-naphthyl) (12l) is
considerably more potent than the 8-oxa-3-(1-naphthyl)
(12k) (Table 2).19 Davies20 has reported substantial
potency for an 8-aza-3-(2-naphthyl)-2-ethylketo tropane
The SAR in the three 2,3-ene families is surprisingly
similar. Thus, the 3-unsubstituted phenyl compounds
are all poor inhibitors of both the DAT and SERT. In
contrast, the 3-(3,4-dichlorophenyl) analogues (12g,
13d, 14c) are all potent inhibitors of the DAT
(IC50=1.1±7.1 nM). The order of potency for the three

P. C. Meltzer et al. / Bioorg. Med. Chem. 8 (2000) 581±590
585
Table 4. Inhibition of [3H]WIN 35,428 binding to the dopamine transporter and [3H]citalopram binding to the serotonin transporter by bicyclo-
[3.2.1]octenes in cynomolgus monkey caudate-putamen
Ar
No.
Compound 14
Dopamine transporter
[3H]WIN 35,428
Serotonin transporter
[3H]citalopram
Selectivity
SERT/DAT
C6H5
a
b
c
(1R/S)
O-1443
O-1436
O-1231
O-1482
3820
390
7.1
>10,000
>30,000
5160
1
77
726
119
4-FC6H4
3,4-Cl2C6H3
2-Naphthyl
(1R/S)
(1R/S)
(1R/S)
d
11.0
1310
and this is now con®rmed for the 8-oxa-2-carbomethoxy
tropane 12l, the 8-aza-2-carbomethoxy tropane 13f
(Table 3) and for the 8-carbacycle 14d (Table 4). Most
signi®cantly, these 2,3-unsaturated compounds are quite
selective for the DAT compared with the SERT. For all
three series, the 3-(3,4-dichlorophenyl) analogues were
highly selective (12g, 13d, 14c) (460- to 1000-fold selectiv-
ity). Within the oxabicyclooctenes, the 4-bromophenyl
(12d; 632-fold) is next most selective followed by the 4-
chlorophenyl (12c; 278-fold) and 4-iodo (12e; 71-fold).
The 3-(2-naphthyl) compounds, (12l, 13f, 14e) while
potent, are less selective than their 3,4-dichlorophenyl
counterparts. This is because the 3,4-dichlorophenyl
analogues manifest considerably reduced anity at
the SERT compared with the 2-naphthyl analogues.
The 3-(2-benzofuranyl) 12n has IC50=413 nM which
lies between that of the 3-(1-naphthyl) analogue 12k
(IC50=1720 nM) and the 3-(2-naphthyl) analogue 12l
(IC50=19.7 nM).
C1). However, Carroll has recently reported com-
pounds42 in which two C3 aromatic rings, linked by an
intervening carbon chain, manifest substantial DAT
inhibitory potency.
The 8-oxa enes prove intolerant of amide functionality
at position C2. Thus 15a, 15b and 15c are of reduced
potency and so di€er from their nitrogen counterparts
which show a marked tolerance for amide functionality
in the C2 position.43
Within the 8-aza series, the N-methyl, 13d, was 14 times
less potent at the SERT than the N-unsubstituted com-
pound 13g (Table 3). This result parallels data observed
for the saturated tropanes.27,44
The dopamine transporter
The dopamine transporter provides a channel through
which dopamine is passed, presumably subsequent to
`recognition' by the macromolecule. There are diverse
DAT inhibitors which are substantially di€erent in
molecular shape, size and functionality and which bind
potently to the DAT. Therefore, these ligands are
`recognized' by the transporter. However, most are not
substrates and, therefore, the action of recognition and
transport must require di€erent processes. The attri-
butes that govern transport are unknown but may
involve conformational changes induced by one ligand
but not by another. With respect to binding, however,
there is increasing evidence that these diverse ligands
bind at di€erent sites on the DAT.29,45,46 We refer to the
actual site on the DAT at which a speci®c array of
amino acids is involved in intermolecular binding
between the ligand and the protein as the ligand accep-
tor site. We postulate that there are a number of such
ligand acceptor sites within the DAT and that binding
at any one of these acceptor sites can cause inhibition of
dopamine reuptake.30,47
In stark contrast to the marked stereoselective binding
of the 8-azatropenes to the DAT, stereoselective binding
of the 8-oxatropenes is much less pronounced. Thus,
while the 1R-8-aza-compound 13d has an IC50=
1.16 nM, and its 1S-enantiomer 13e manifests only
490 nM, anity of the 1R-8-oxa compound 12g is
4.6 nM whereas the anity of its 1S enantiomer 12h is
58.2 nM. Surprisingly, this 1S enantiomer 12h is almost
twice as potent as ( )-cocaine, notwithstanding the fact
that it is of opposite stereochemistry. Biostereo-
selectivity is yet to be determined for the carbacycles 14.
The in¯uence of aromatic bulk at the C3 position is also
evident. As shown in the 8-oxa series (Table 2), the a-
nity of 3-(1-naphthyl) is 1720 nM (12k) whereas the 3-
(2-naphthyl) (12l) is 100 times more potent. Although
aromatic bulk in the 3-(2)-position can increase anity,
limitations to this principal were observed with 3-(2-
anthracenyl) (12m) which displayed an IC50=>40,000.
Therefore, it is clear that excess steric bulk (2-anthrace-
nyl), and bulk that lies transverse to the tropane skele-
ton as in the 3-(1-naphthyl), is deleterious to binding.
This might imply that the cavity on the DAT that
accepts the aromatic ring is less than about 7.3 A in
depth (`length' of an anthracenyl system), and less than
about 4.2 A in width (`width' of a naphthyl connected at
In the three examples presented in this manuscript
(X=N, O and C), although the relative order of
potency of the various aryl substituted compounds
remains similar, the potencies di€er quite markedly,
especially for the weaker compounds. This may imply
that these families occupy di€erent ligand acceptor sites

586
P. C. Meltzer et al. / Bioorg. Med. Chem. 8 (2000) 581±590
on the DAT. If these acceptor sites are at di€erent
depths within the channel formed by the membrane-
surrounded DAT, then it is conceivable that, presuming
no conformational change has been a€ected, those that
bind deeper within the channel will not necessarily
inhibit the binding of those that bind more shallowly.
This implies that all ligands that bind within the
DAT will inhibit reuptake of dopamine but may show
di€erences in their binding constants with respect to
comparison with other non-substrate DAT ligands.
This concept is currently under investigation in our
laboratories.
England Nuclear (Boston, MA). A Beckman 1801 scin-
tillation counter was used for scintillation spectrometry.
0.1% Bovine serum albumin was purchased from Sigma
Chemicals. (R)-( )-Cocaine hydrochloride for the
pharmacological studies was donated by the National
Institute on Drug Abuse [NIDA]. Fluoxetine was
donated by E. Lilly & Co.
General synthetic procedures
General procedure for the synthesis of enol tri¯ates 9,
10, 11 (1R, 1S)-2-carbomethoxy-3-{[(tri¯uoromethyl)sul-
fonyl]oxy}-8-oxabicyclo[3.2.1]-2-octene (9). Sodium bis-
(trimethylsilyl)amide (1.0 M solution in THF, 45 mL)
was added dropwise to a solution of 2-carbomethoxy-8-
oxabicyclo[3.2.1]octanone, 6 (7.12 g, 38.65 mmol) in
THF (100 mL) at 78 ꢀC under nitrogen. After stirring
for 30min, N-phenyltri¯uoromethanesulfonimide (15.2 g,
42.5 mmol) was added as a solid at 78 ꢀC. The reaction
was allowed to warm to room temperature and was then
stirred overnight. The volatile compounds were then
removed on a rotary evaporator. The residue was dis-
solved in CH2Cl2 (200 mL) and washed with H2O
(100 mL) and brine (100 mL). The dried (MgSO4)
CH2Cl2 layer was concentrated to dryness on a rotary
evaporator. The residue was puri®ed by ¯ash chroma-
tography (eluent: 5±10% EtOAc in hexanes) to a€ord
9.62 g of 9. Pale yellow oil; yield: 79%; 1H NMR
(CDCl3, 300 MHz): d 1.74 (m, 1H), 2.0±2.3 (m, 4H),
3.00 (dd, J=4.65, 17.85 Hz, 3H), 3.82 (s, 3H), 4.69 (m,
1H), 5.04 (d, J=4.95 Hz, 1H).
The selectivity exhibited by the unsaturated compounds
has not been presented prior to this report, however, it
is extremely important from a number of perspectives.
In the search for potential medications from this class of
compounds, these unsaturated compounds are the pre-
cursors of the classical 3-aryltropanes and are, there-
fore, synthetically more readily accessible. Further, they
cannot undergo epimerization in the C2-position as can
the 2b,3b tropanes, nor is there the substantial yield loss
which results from reduction of these 2,3-enes to pro-
vide the classical 2b, 3b tropanes. Finally, their biologi-
cal half life may be anticipated to be increased
compared with that of the saturated analogues as a
consequence of the relatively more stable conjugated
methyl ester. Consequently these unsaturated com-
pounds o€er considerable advantages for the develop-
ment of clinically useful compounds.
(1R)-N-Methyl-2-carbomethoxy-3-{[(tri¯uoromethyl)sulf-
onyl]oxy}-8-azabicyclo[3.2.1]-2-octene (10). Pale yellow
oil; yield: 78%; H NMR (CDCl3, 300 MHz): d 1.58
(m, 1H), 1.97 (m, 2H), 2.1±2.2 (m, 2H), 2.39 (s, 3H,
NCH3), 2.84 (dd, J=4, 19 Hz, 1H, H-4b), 3.42 (t,
J=6 Hz, 1H, H-5), 3.8 (s, 3H, CO2CH3), 3.93 (d,
J=5 Hz, 1H, H-1).
Conclusion
1
In this work it has been demonstrated that appropriate
orientation of the 3-aryl ring in the bicyclo[3.2.1]octane
system is critical for potent interaction with the SERT.
Thus, the topology of the ligand dictates its ability to
occupy the acceptor site on the SERT.30 The striking
selectivity for the DAT manifested by the bicyclo-
[3.2.1]oct-2,3-enes suggests that the DAT is con-
siderably more ¯exible with respect to binding than is
the SERT. It is possible that there are a number of tro-
pane ligand acceptor sites30 on the DAT and very few,
or even only a single tropane acceptor site on the SERT.
(1R,1S)-2-Carbomethoxy-3-{[(tri¯uoromethyl)sulfonyl]-
oxy}bicyclo[3.2.1]-2-octene (11). Clear oil; yield: 69%;
1H NMR (CDCl3, 300 MHz): d 1.41±1.7 (m, 3H), 1.8±
2.05 (m, 3H), 2.17 (dd, J=1.9 and 18 Hz, 1H, H-4a),
2.51 (m, 1H, H-5), 2.70 (dd, J=4.7, 18 Hz, 1H, H-4b),
3.10 (t, J=3.8 Hz, 1H, H-1), 3.81 (s, 3H, CO2CH3).
General procedure for the synthesis of the 2-octenes, 12, 13,
14 (1R,1S)-2-carbomethoxy-3-phenyl-8-oxabicyclo[3.2.1]-
2-octene (12a). 2-Carbomethoxy-3-{[(tri¯uoromethyl)-
Experimental
1H NMR spectra were recorded on a JEOL 300 NMR
spectrometer. TMS was used as an internal standard.
Melting points are uncorrected and were measured on a
Gallenkamp melting point apparatus. Thin layer chro-
matography (TLC) was carried out on Baker Si250F
plates. Visualization was accomplished with either UV
exposure or treatment with phosphomolybdic acid
(PMA). Flash chromatography was carried out on
Baker Silica Gel 40 mM. Elemental analyses were per-
formed by Atlantic Microlab, Atlanta, GA. All reac-
tions were conducted under an inert (N2) atmosphere.
[3H]WIN 35,428 (2b-carbomethoxy-3a-(4-¯uorophenyl)-
N-[3H]methyltropane, 79.4±87.0 Ci/mmol) and [3H]citalo-
pram (86.8 Ci/mmol) were purchased from DuPont-New
sulfonyl]oxy}-8-oxabicyclo[3.2.1]-2-octene,
9
(2.0 g,
6.3 mmol), phenyl boronic acid (1.02 g, 8.36 mmol), di-
ethoxymethane (20 mL), LiCl (578 mg, 13.6 mmol), tris-
(dibenzylideneacetone)dipalladium(0) (247 mg, 0.25 mmol)
and Na2CO3 (2 M solution, 6.1 mL) were combined and
heated at re¯ux for 1 h. The mixture was cooled to room
temperature, ®ltered through Celite and washed with
ether (100 mL). The mixture was made basic with
NH4OH and washed with brine. The dried (MgSO4)
ether layer was concentrated to dryness. The residue
was then puri®ed by ¯ash chromatography (eluent: 10%
EtOAc in hexanes). This a€orded 1.28 g (82%) of a light
brown viscous oil (12a): Rf 0.26 (20% EtOAc in hexanes);

P. C. Meltzer et al. / Bioorg. Med. Chem. 8 (2000) 581±590
587
1H NMR (CDCl3, 100 MHz): d 7.1±7.5 (m, 5H), 4.95±
5.1 (m, 1H), 4.55±4.75 (m, 1H), 3.52 (s, 3H), 2.95 (dd,
1H, J=5, 18 Hz), 1.7±2.2 (m, 5H). Anal. (C15H16O3) C,
H.
(m, 2H), 7.69 (m, 2H), 7.98 (m, 2H), 8.29 (s, 1H), 8.38
(s, 1H), 8.42 (s, 1H).
(1R,1S)-2-Carbomethoxy-3-(4-formylphenyl)-8-oxabicy-
clo[3.2.1]-2-octene (12p). Compound 12p was prepared
from 9 with 4-formylphenylboronic acid as described
above. A white solid was obtained (25%): Rf 0.22 (30%
EtOAc in hexanes); mp 93.8±94.8 ꢀC; 1H NMR (CDCl3,
300 MHz): d 1.8 (m, 1H), 2.0±2.3 (m, 4H), 2.95 (dd,
J=5.2, 18 Hz, 1H, H-4b), 3.50 (s, 3H, OCH3), 4.66 (m,
1H, H-5), 5.0 (d, J=5.2 Hz, 1H, H-1), 7.27 (d,
J=4.7 Hz, 2H) 7.86 (d, J=4.7 Hz, 2H). Anal.
(C16H16O4) C, H.
(1R,1S)-2-Carbomethoxy-3-(4-¯uorophenyl)-8-oxabicyclo-
[3.2.1]-2-octene (12b). Compound 12b was prepared
from 9 with 4-¯uorophenylboronic acid as described
above. A light brown viscous oil was obtained (88%): Rf
0.19 (20% EtOAc in hexanes); 1H NMR (CDCl3,
100 MHz): d 7.0±7.2 (m, 4H), 4.95±5.05 (m, 1H), 4.55±
4.75 (m, 1H), 3.52 (s, 3H), 2.95 (dd, 1H, J=5, 18 Hz),
1.7±2.3 (m, 5H). Anal. (C15H15O3F) C, H.
(1R,1S)-2-Carbomethoxy-3-(3-chloro-4-¯uorophenyl)-8-
oxabicyclo[3.2.1]-2-octene (12i). Compound 12i was
prepared from 9 with 3-chloro-4-¯uorophenylboronic
acid as described above. An o€-white solid was obtained
(96%): Rf 0.25 (EtOAc in hexanes); mp 75±76 C; H
NMR (CDCl3, 100 MHz): d 1.5±2.4 (m, 5H), 2.9 (dd,
J=5, 20 Hz, 1H, H-4b), 3.56 (s, 3H, OCH3), 4.55±4.80
(m, 1H, H-5), 5.0 (m, 1H, H-1), 6.9±7.4 (m, 3H). Anal.
(C15H14O3FCl) C, H, Cl.
(1R)-N-Methyl-2-carbomethoxy-3-phenyl-8-azabicyclo-
[3.2.1]-2-octene (13a). Compound 13a was prepared
from 10(1R) with phenylboronic acid as described
above. A yellow oil was obtained (25%). Rf 0.47 (5%
ꢀ
1
1
Et3N in EtOAc). H NMR (CDCl3, 300 MHz): d 1.63
(m, 1H), 1.9±2.3 (m, 3H), 2.03 (d, J=18 Hz, 1H, H-4a),
2.47 (s, 3H, NCH3), 2.76 (dd, J=4.7, 18 Hz, 1H, H-4b),
3.34 (t, 1H, H-5), 3.46 (s, 3H, OCH3), 3.84 (d, J=5.5 Hz
1H, H-1), 7.1±7.5 (m, 2H) 7.22±7.34 (m, 3H). Anal.
.
(C16H19 NO2 1/10 H2O) C, H, N.
(1R,1S)-2-Carbomethoxy-3-(3-chlorophenyl)-8-oxabicyclo-
[3.2.1]-2-octene (12j). Compound 12j was prepared from
9 with 3-chlorophenylboronic acid as described above.
A light yellow oil was obtained (78%): Rf 0.43 (30%
EtOAc in hexanes); 1H NMR (CDCl3, 300 MHz): d 1.76
(m, 1H), 2.04±2.26 (m, 4H), 2.92 (dd, 1H, J=18 Hz);
3.52 (s, 3H), 4.63 (m, 1H), 5.00 (m, 1H), 6.90 (m, 1H),
7.08 (s, 1H), 7.25 (m, 2H). Anal. (C15H15ClO3) C, H, Cl.
(1R)-N-Methyl-2-carbomethoxy-3-(4-¯uorophenyl)-8-
azabicyclo[3.2.1]-2-octene (13b). Compound 13b was
prepared from 10(1R) with 4-¯uorophenylboronic acid
as described above. A yellow oil was obtained (72%). Rf
0.60 (10% Et3N in ether). 1H NMR (CDCl3, 100 MHz):
d 1.63±2.3 (m, 5H), 2.45 (s, 3H, NCH3), 2.6±2.9 (m,
1H), 3.35 (m, 1H), 3.50 (s, 3H, OCH3), 3.85 (m, 1H),
.
6.9±7.2 (m, 4H). Anal. (C16H18 NO2F 1/6 H2O) C, H, N.
(1R,1S)-2-Carbomethoxy-3-(2-anthracenyl)-8-oxabicyclo-
[3.2.1]-2-octene (12m). Compound 12m was prepared
from 9 with 2-anthracenylboronic acid as described
above. A yellow solid was obtained (33%): Rf 0.39
(1S)-N-Methyl-2-carbomethoxy-3-(4-¯uorophenyl)-8-aza-
bicyclo[3.2.1]-2-octene (13c). Compound 13c was pre-
pared from 10(1S) with 4-¯uorophenylboronic acid as
described above. A yellow oil was obtained (43%). Rf
0.49 (10% Et3N in ethyl acetate). H NMR (CDCl3,
100 MHz): d 1.63±2.3 (m, 5H), 2.45 (s, 3H, NCH3), 2.6±
2.9 (m, 1H), 3.35 (m, 1H), 3.50 (s, 3H, OCH3), 3.85 (m,
(30% EtOAc in hexanes); mp 185.5±186.5 ꢀC; H NMR
1
1
(CDCl3, 300 MHz): d 1.84 (m, 1H), 2.14±2.32 (m, 4H),
2.08 (dd, 1H, J=18 Hz); 3.46 (s, 3H), 4.71 (m, 1H), 5.07
(m, 1H), 7.23 (m, 1H), 7.46 (m, 2H), 7.73 (s, 1H), 7.97
(m, 3H), 8.38 (m, 2H). Anal. (C23H20O3) C, H.
.
1H), 6.9±7.2 (m, 4H). Anal. (C16H18 NO2F 1/3 H2O) C,
H, N.
(1R,1S)-2-Carbomethoxy-3-(3,4-methylenedioxyphenyl)-
8-oxabicyclo[3.2.1]-2-octene (12o). Compound 12o was
prepared from 9 with 3,4-methylenedioxyphenylboronic
acid as described above. An o€-white solid was
obtained (78%): Rf 0.37 (30% EtOAc in hexanes); mp
106.2±107.2 ꢀC; 1H NMR (CDCl3, 300 MHz): d 1.75 (m,
1H), 2.04±2.26 (m, 4H), 2.91 (dd, 1H, J=18 Hz); 3.56 (s,
3H), 4.62 (m, 1H), 4.97 (m, 1H), 5.95 (s, 2H), 6.59 (m,
2H), 6.75 (m, 1H). Anal. (C16H16O5) C, H.
(1R)-N-Methyl-2-carbomethoxy-3-(3,4-dichlorophenyl)-
8-azabicyclo[3.2.1]-2-octene (13d). Compound 13d was
prepared from 10(1R) with 3,4-dichlorophenylboronic
acid as described above. A yellow oil was obtained
1
(83%). Rf 0.56 (10% Et3N in ethyl acetate). H NMR
(CDCl3, 300 MHz): d 1.61 (m, 1H), 1.9±2.05 (m, 2H),
2.1±2.3 (m, 2H), 2.43 (s, 3H, NCH3), 2.76 (dd, J=19,
4.7 Hz, 1H), 3.36 (t, J=4.9 Hz, 1H), 3.52 (s, 3H, OCH3),
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.
(C16H17NO2Cl2) C, H, N.
2-Anthracenylboronic acid was prepared from 2-iodo-
anthracene and triisopropylborate according to a lit-
erature procedure.41 A yellow solid was obtained (34%):
1H NMR (DMSO-d6, 300 MHz): d 7.36 (s, 2H), 7.50 (m,
2H), 7.91 (m, 1H), 8.04 (m, 2H), 8.51 (s, 1H), 8.56 (s,
1H), 8.64 (s, 1H).
(1S)-N-Methyl-2-carbomethoxy-3-(3,4-dichlorophenyl)-
8-azabicyclo[3.2.1]-2-octene (13e). Compound 13e was
prepared from 10(1S) with 3,4-dichlorophenylboronic
acid as described above. A yellow oil was obtained
1
2-Iodoanthracene was prepared from 2-aminoanthra-
cene according to a literature procedure.48 A white solid
was obtained (7%): 1H NMR (CDCl3, 300 MHz): d 7.49
(69%). Rf 0.56 (10% Et3N in ethyl acetate). H NMR
(CDCl3, 300 MHz): d 1.61 (m, 1H), 1.9±2.05 (m, 2H),
2.1±2.3 (m, 2H), 2.43 (s, 3H, NCH3), 2.76 (dd, J=19,

588
P. C. Meltzer et al. / Bioorg. Med. Chem. 8 (2000) 581±590
4.7 Hz, 1H), 3.36 (t, J=4.9 Hz, 1H), 3.52 (s, 3H, OCH3),
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.
(C16H17NO2Cl2) C, H, N.
from 11 with 3,4-dichlorophenylboronic acid as descri-
bed above. An oil was obtained (69%): Rf 0.5 (10%
EtOAc in hexanes); 1H NMR (CDCl3, 300 MHz): d
1.48±1.70 (m, 3H), 1.76±2.03 (m, 3H), 2.14 (d, J=19 Hz,
1H), 2.40±2.65 (m, 1H), 2.60±2.67 (m, 1H), 2.99±3.02
(m, 1H), 3.49 (s, 3H), 6.90 (dd, J=8, 2 Hz, 1H), 7.17 (d,
J=2 Hz, 1H), 7.34 (d, J=8 Hz, 1H). Anal. (C16H16
O2Cl2) C, H, Cl.
(1R)-N-Methyl-2-carbomethoxy-3-(2-naphthyl)-8-azabi-
cyclo[3.2.1]-2-octene (13f). Compound 13f was prepared
from 10(1R) with 2-naphthylboronic acid as described
above. A yellow oil was obtained (51%). Rf 0.48 (10%
1
Et3N in ethyl acetate). H NMR (CDCl3, 100 MHz): d
1.3±3.6 (m, 7H), 2.5 (s, 3H, NCH3), 3.45 (s, 3H, OCH3),
(1R,1S)-2-Carbomethoxy-3-(2-naphthyl)-bicyclo[3.2.1]-2-
octene (14d). Compound 14d was prepared from 11 with
2-naphthylboronic acid as described above. An oil was
.
3.8±4.0 (m, 1H), 7.2±8.0 (m, 7H). Anal. (C20H21NO2 l/2
H2O) C, H, N.
1
obtained (54%): Rf 0.41 (10% EtOAc in hexanes); H
NMR (CDCl3, 300 MHz): d 1.63±1.74 (m, 2H), 1.82±
2.17 (m, 4H), 2.38 (d, J=18.7 Hz, 1H), 2.52±2.55 (m, 1H),
2.79±2.87 (m, 1H), 3.14 (t, J=4.5 Hz, 1H), 3.44 (s, 3H),
7.25±7.29 (m, 1H), 7.47±7.50 (m, 2H), 7.62 (d, J=1.4Hz,
1H), 7.80±7.86 (m, 3H). Anal. (C20H20O2) C, H.
(1R)-2-Carbomethoxy-3-(3,4-dichlorophenyl)-8-norazabi-
cyclo[3.2.1]-2-octene (13g). 2-Methoxycarbonyl-3-(3,4-
dichlorophenyl)tropene 13d (200 mg, 0.61 mmol) was
combined with 1-chloroethyl chloroformate (4 mL) and
the resulting solution was brought to re¯ux for 2 h. The
excess chloroformate was removed in vacuo and the
residue was brought to re¯ux in methanol (30 mL) for
45 min. The methanol was then removed in vacuo
and the residue was partitioned in CHCl3/NaHCO3/
Na2CO3(aqueous) (pH=9). The aqueous layer was
extracted with CHCl3 (3Â10 mL) and the combined
organic extracts were dried (Na2SO4), ®ltered and con-
centrated. The residue was chromatographed (eluent: 5±
15% Et3N in EtOAc). Like fractions were combined to
yield a yellow oil (50%). Rf 0.3 (10% Et3N in EtOAc);
1H NMR (CDCl3, 100 MHz) d 1.50±2.3 (m, 5H), 2.5±
2.9 (m, 1H), 3.53 (s, 3H), 3.8 (m, 1H), 4.2 (m, 1H), 6.9
(dd, 1H), 7.2 (dd, 1H), 7.4 (d, 1H). Anal. (C15H15
General procedure for synthesis of the 2-amide-2-octenes,
15a, b and c. To a solution of 2-carbomethoxy-3-(3,4-
dichlorophenyl)-8-oxabicyclo[3.2.1]oct-2-ene 12f (285mg,
0.91 mmol) in THF/MeOH (2 mL/0.67 mL) was added a
solution of lithium hydroxide (0.67 mL of a 4.8 M solu-
tion) and the resulting solution was stirred at room
temperature overnight. TLC (20% ethyl acetate in hex-
anes with 0.5% acetic acid) showed completion of
hydrolysis. Water and ether were then added and the
layers separated. The aqueous phase was then acidi®ed
with 1 M HCl and extracted with ether. The ether layer
was washed with brine and dried over MgSO4. Con-
centration gave the crude product (258 mg) which was
puri®ed by column chromatography (SiO2, 1 g, eluent:
50% ethyl acetate in hexanes with 1% acetic acid) and
gave 128 mg (47%) of the acid which was used immedi-
ately for the next step.
.
NO2Cl2 1/5 H2O) C, H, N.
(1R)-2-Carbomethoxy-3-(4-¯uorophenyl)-8-norazabicyclo-
[3.2.1]-2-octene (13h). Compound 13h was prepared
from 13b as described above. A yellow oil was obtained
(68%). Rf 0.7 (10% MeOH in hexanes+0.5%
The pure acid was taken up in CH2Cl2 (2 mL) and trea-
ted with oxalyl chloride (0.058 mL) and a drop of DMF.
After stirring at room temperature for 1 h the volatile
compounds were evaporated and the residue pumped
for 4 h. The acid chloride was then dissolved in CH2Cl2
(2 mL) and then the required amine added (4 equiva-
lents) neat and the solution stirred overnight. The crude
amide was puri®ed by chromatography (30±50% ethyl
acetate in hexanes) and gave:
ꢀ
1
NH4OH); mp 67±68 C; H NMR (CDCl3) d 1.50±3.0
(m, 6H), 3.5 (s, 3H), 3.8 (m, 1H), 4.2 (m, 1H), 6.8±7.2
(m, 4H). Anal. (C15H16NO2F) C, H, N.
(1R,1S)-2-Carbomethoxy-3-phenylbicyclo[3.2.1]-2-octene
(14a). Compound 14a was prepared from 11 with phe-
nylboronic acid as described above. An oil was obtained
(75%): Rf 0.5 (10% EtOAc in hexanes); 1H NMR
(CDCl3, 300 MHz): d 1.52±1.66 (m, 2H), 1.73 (d,
J=11 Hz, 1H), 1.79±2.06 (m, 3H), 2.23 (d, J=18.7 Hz,
1H), 2.44±2.47 (m, 1H), 2.66±2.74 (m, 1H), 2.99±3.02
(m, 1H), 3.43 (s, 3H), 7.07±7.10 (m, 2H), 7.20±7.32 (m,
3H). Anal. (C16H18O2) C, H.
(1R,S)-N-Cyclobutyl-2-carboxamido-3-(3,4-dichlorophe-
nyl)-8-oxabicyclo[3.2.1]-2-octene (15a). As a white solid.
Yield=72%. Rf 0.50 (ethyl acetate), mp 174±176 ꢀC; 1H
NMR (CDCl3, 100 MHz): d 1.2±2.4 (m, 11H), 3.0 (dd,
J=5, 17 Hz, 1H, H-4b), 4.1±4.5 (m, 1H, C(H)-N-), 4.6±
4.8 (m, 1H, H-5), 4.85 (d, J=5 Hz, 1H, H-1), 5.08 (br d,
J=7 Hz, 1H, NH), 7.1 (dd, J=10, 2 Hz, 1H), 7.35 (d,
J=2.4 Hz, 1H), 7.46 (d, J=10 Hz, 1H). Anal. (C18H19
NO2Cl2) C, H, N, Cl.
(1R,1S)-2-Carbomethoxy-3-(4-¯uorophenyl)-bicyclo[3.2.1]-
2-octene (14b). Compound 14b was prepared from 11
with 4-¯uorophenylboronic acid as described above. An
oil was obtained (73%): Rf 0.5 (10% EtOAc in hex-
anes); 1H NMR (CDCl3, 300 MHz): d 1.50±2.04 (m,
6H), 2.20 (d, J=18.7 Hz, 1H), 2.44±2.48 (m, 1H), 2.63±
2.71 (m, 1H), 3.00 (t, J=4.5 Hz, 1H), 3.46 (s, 3H), 6.94±
7.08 (m, 4H). Anal. (C16H17O2 F) C, H.
(1R,S)-3-(3,4-Dichlorophenyl)-8-oxabicyclo[3.2.1]oct-2-
ene-2-carboxylic acid pyrrolidine amide (15b). As a
white solid (90%). Rf 0.30 (ethyl acetate), mp 140±
142 ꢀC; 1H NMR (CDCl3, 100 MHz): d 1.4±2.5 (m, 9H),
2.7±3.5 (m, 5H), 4.55±4.80 (m, 2H), 7.15 (dd, 1H), 7.36
(d, 1H), 7.45 (d, 1H). Anal. (C18H19NO2Cl2) C, H, N, Cl.
(1R,1S)-2-Carbomethoxy-3-(3,4-dichlorophenyl)-bicyclo-
[3.2.1]-2-octene (14c). Compound 14c was prepared

P. C. Meltzer et al. / Bioorg. Med. Chem. 8 (2000) 581±590
589
(1R,S)-3-(3,4-Dichlorophenyl)-8-oxabicyclo[3.2.1]oct-2-
ene-2-carboxylic acid morpholine amide (15c). As a
white solid (60%). Rf 0.32 (ethyl acetate), mp 141±
143 ꢀC; 1H NMR (CDCl3, 100 MHz): d 1.6±3.7 (m,
14H), 4.3±4.8 (m, 2H), 7.12 (dd, 1H), 7.36 (d, 1H), 7.4
(d, 1H). Anal. (C18H19NO3Cl2) C, H, N, Cl.
water-soluble drugs were dissolved in water or bu€er
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 bu€er and added
(0.2 mL) to the assay medium as described above. IC50
values were computed by the EBDA computer pro-
gram and are the means of experiments conducted in
triplicate.
Tissue sources and preparation
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 tis-
sue. Membranes were prepared as described previously.
Brie¯y, the caudate-putamen was homogenized in 10
volumes (w/v) of ice-cold Tris±HCl bu€er (50 mM, pH
7.4 at 4 ꢀC) and centrifuged at 38,000Âg for 20 min in
the cold. The resulting pellet was suspended in 40
volumes of bu€er, and the entire procedure was repe-
ated 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 bu€er just
before assay and was dispersed with a Brinkmann
Polytron homogenizer (setting #5) for 15 s. All experi-
ments were conducted in triplicate and each experiment
was repeated in each of 2±3 preparations from indivi-
dual brains.
Serotonin transporter assay
The serotonin transporter was assayed in caudate-puta-
men membranes using conditions similar to those for
the dopamine transporter. The anity of [3H]citalo-
pram (spec. act.: 82 Ci/mmol, DuPont-NEN) for the
serotonin transporter was determined in experiments by
incubating tissue with a ®xed concentration of [3H]cita-
lopram and a range of concentrations of unlabeled
citalopram. The assay tubes received, in Tris±HCl bu€er
(50 mM, pH 7.4 at 0±4 ꢀC; NaCl 100 mM), the following
constituents at a ®nal assay concentration: citalopram,
0.2 mL (1 pM, 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 terminated by rapid ®l-
tration over Whatman GF/B glass ®ber ®lters pre-
soaked in 0.1% polyethyleneimine. The ®lters were
washed twice with 5 mL Tris±HCl bu€er (50 mM),
incubated overnight at 0±4 ꢀC in scintillation ¯uor
(Beckman Ready-Value, 5 mL) and radioactivity was
measured by liquid scintillation spectrometry (Beckman
1801). Cpm were converted to dpm following determi-
nation of counting eciency (>45%) of each vial by
external standardization. Total binding was de®ned as
[3H]citalopram bound in the presence of ine€ective
concentrations of unlabeled citalopram (1 or 10 pM).
Non-speci®c binding was de®ned as [3H]citalopram
bound in the presence of an excess (10 mM) of ¯uox-
etine. Speci®c binding was the di€erence between the
two values. Competition experiments to determine the
anities of other drugs at [3H]citalopram binding sites
were conducted using procedures similar to those out-
lined above. IC50 values were computed by the EBDA
computer program and are the means of experiments
conducted in triplicate.
Dopamine transporter assay
The dopamine transporter was labeled with [3H]WIN
35,428 ([3H]CFT, 2b-carbomethoxy-3b-(4-¯uorophenyl)-
N-[3H]methyltropane, 81±84 Ci/mmol, DuPont-NEN).
The anity of [3H]WIN 35,428 for the dopamine trans-
porter was determined in experiments by incubating
tissue with a ®xed concentration of [3H]WIN 35,428 and
a range of concentration of unlabeled WIN 35,428. The
assay tubes received, in Tris±HCl bu€er (50 mM, pH 7.4
at 0±4 ꢀC; NaCl 100 mM), the following constituents at
a ®nal assay concentration: WIN35,428, 0.2 mL (1 pM,
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 addi-
tion of membranes and terminated by rapid ®ltration
over Whatman GF/B glass ®ber ®lters pre-soaked in
0.1% bovine serum albumin (Sigma Chem. Co.). The
®lters were washed twice with 5 mL Tris.HCl bu€er
(50 mM), incubated overnight at 0±4 ꢀC in scintillation
¯uor (Beckman Ready-Value, 5 mL) and radioactivity
was measured by liquid scintillation spectrometry
(Beckman 1801). Cpm were converted to dpm following
determination of counting eciency (>45%) of each
vial by external standardization. Total binding was
de®ned as [3H]WIN 35,428 bound in the presence of
ine€ective concentrations of unlabeled WIN 35,428
(1 or 10 pM). Non-speci®c binding was de®ned as
[3H]WIN 35,428 bound in the presence of an excess
(30 mM) of ( )-cocaine. Speci®c binding was the di€er-
ence between the two values. Competition experiments
to determine the anities of other drugs at [3H]WIN
35,428 binding sites were conducted using procedures
similar to those outlined above. Stock solutions of
Acknowledgement
This work was supported by the National Institute of
Drug Abuse (PM: DA7-8081; DA11542; BKM: DA
09462, RR00168).
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