Synthesis and biological evaluation of 2-substituted 3β-tolyltropane derivatives at dopamine, serotonin, and norepinephrine transporters

Article abstract of DOI:10.1021/jm010453u

A series of eight 2-substituted 3-tolyltropane derivatives were synthesized, and the in vitro and in vivo biological activities as dopamine uptake inhibitors were determined. From the in vitro structure-activity data, it is apparent that a tolyl group in

Full text of DOI:10.1021/jm010453u

J . Med. Chem. 2002, 45, 1203-1210
1203
Syn th esis a n d Biologica l Eva lu a tion of 2-Su bstitu ted 3â-Tolyltr op a n e
Der iva tives a t Dop a m in e, Ser oton in , a n d Nor ep in ep h r in e Tr a n sp or ter s
Lifen Xu,† Sari Izenwasser,‡ J onathan L. Katz,§ Theresa Kopajtic,§ Cheryl Klein-Stevens,# Naiju Zhu,#
Stacey A. Lomenzo,† Leyte Winfield,† and Mark L. Trudell*,†
Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148, Department of Neurology, University of
Miami School of Medicine, Miami, Florida 33136, National Institute on Drug Abuse, Intramural Research Program,
P.O. Box 5180, Baltimore, Maryland 21224, and Department of Chemistry, Xavier University of Louisiana,
New Orleans, Louisiana 70125
Received October 1, 2001
A series of eight 2-substituted 3-tolyltropane derivatives were synthesized, and the in vitro
and in vivo biological activities as dopamine uptake inhibitors were determined. From the in
vitro structure-activity data, it is apparent that a tolyl group in the 2-position, independent
of the stereochemical attachment to the tropane ring system, provided compounds (9-12, 14)
that exhibit high-affinity binding at the dopamine transporter (DAT). Although a slight
stereochemical preference in binding affinity at the DAT was observed for the 2â-(R)-alcohol
10 over the 2â-(S)-isomer 11, no significant differences in behavioral effects were observed.
Furthermore, despite a relatively low potency of 10 for the inhibition of dopamine uptake
compared to its affinity for the DAT, its behavioral profile did not vary significantly from cocaine.
These data indicate that a behavioral characterization of compounds is a critical feature of
efforts to discover pharmacological treatments for cocaine abuse.
In tr od u ction
mine transporter ligands have been established for
these positions, as well as for positions 6-8 of the
tropane ring system.3-23 From these studies it has been
noted generally that a 4-substituted aryl group (e.g.,
tolyl) or a 3,4-disubstituted aryl group (e.g., 3,4-dichlo-
rophenyl) at the 3â-position of the tropane ring affords
compounds that exhibit high-affinity binding at dopa-
mine transporters. The effects of substituents at the
2-position have also been extensively studied in terms
of dopamine transporter affinity and selectivity. It is
apparent from investigations at the 2-position of 3â-
aryltropanes that most substituents (alkyl, vinyl, aryl,
alcohol, and ester groups) with â-stereochemistry rela-
tive to the tropane ring are tolerated by the dopamine
transporter and provide compounds with high affinity.
Over the past decade, illicit drug use in the United
States has been steadily increasing and a significant
increase in cocaine (1) abuse, as much as 45% in some
A survey of the literature revealed that relatively
little structure-activity data were available for sub-
stituents at the 2â-position that possess specific stereo-
chemistry inherent to the attached functional group.
Kozikowski and co-workers reported that alcohol 3
exhibited high binding affinity at the DAT.4 However,
the DAT affinity for the epimeric alcohol was not
determined. Therefore, it was of interest to study a class
of DAT ligands that would further elucidate the SAR
of substituents at the 2-position in terms of stereochem-
ical effects. It was envisaged that characterization of
the in vitro activity (DAT affinity, dopamine uptake
inhibition) and the behavioral effects (drug discrimina-
tion, locomotor stimulation) would serve to minimally
define the pharmacological activity of the compounds
at the DAT and more accurately reveal the SAR of the
compounds. In addition, this twofold approach would
identify any inconsistencies that might arise between
the in vitro SAR of a compound and its behavioral
effects. Herein, we report the synthesis and biological
activity of a series of 2-substituted 3â-tolytropanes.
areas of the country, has been recently reported.1
Despite a significant advancement in understanding
the biological mechanisms of action of cocaine, a satis-
factory cocaine therapeutic agent has yet to be identi-
fied.2 The search for potential anti-cocaine medications
has led to the extensive study of the structure-activity
relationships (SAR) of 1 at the dopamine transporter
(DAT).3 In addition, the 2-substituted 3-aryltropanes
have been studied extensively as cocaine congeners and
developed as tools [e.g., [3H]WIN 35 428 (2)] to explore
the DAT. This broad class of compounds has provided
tremendous insight into the nature of the dopamine
transporter pharmacophore. The structural criteria for
the 2-substituted 3-aryltropanes as high-affinity dopa-
* To whom correspondence should be addressed. Phone: (504)-280-
7337. Fax: (504) 280-6860. E-mail: mtrudell @uno.edu.
† University of New Orleans.
‡ University of Miami School of Medicine.
§ National Institute on Drug Abuse, Intramural Research Program.
#
Xavier University of Louisiana.
10.1021/jm010453u CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/06/2002

1204 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Xu et al.
Sch em e 1
F igu r e 1. ORTEP diagram of 2â-(R)-hydroxymethytolyl-3â-
tolyltropane (10, freebase).
Sch em e 2
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of the 2-substituted 3â-
tolyltropane derivatives was completed in a straight-
forward fashion from anhydroecgonine methyl ester
(Scheme 1).24 The reaction of tolylmagnesium bromide
with anhydroecgonine methyl ester in dichloromethane
furnished the 2â-carbomethoxy-3â-tolyltropane (4â) ste-
reoselectively (R/â, 1:5). Concomitant reduction with
lithium aluminum hydride furnished the alcohol 5.
Swern oxidation of 5 afforded the aldehyde 6, and
subsequent olefination gave the vinyl ester 7 in 60%
overall yield. Hydrogenation of 7 over palladium on
carbon followed by reduction with lithium aluminum
hydride afforded the alcohol 8 in 94% yield.
The reaction of 4â with excess tolylmagnesium bro-
mide afforded the tolyl ketone 9 in 88% yield. Reduction
of 9 with lithium aluminum hydride gave the alcohol
10 in 95% yield and in >99% de. The stereoselectivity
of the reduction is thought to be due to lithium chelation
with the carbonyl oxygen and the bridging nitrogen
atom [N(8)]. This locks the conformation of the carbonyl
side chain such that delivery of hydride then can only
be achieved from the less-hindered si face to furnish 10.
The absolute configuration of 10 was unequivocally
confirmed by X-ray crystallography (Figure 1). It is
evident from the X-ray structure that the hydroxyl
proton is oriented toward the proximity of the lone pair
of electrons on N(8), indicating the presence of an
intramolecular hydrogen bond (H-N(8) distance of 1.94
Å). In addition, the aryl groups of 10 are closely aligned
in parallel fashion indicative of a π-π interaction. The
(R)-stereochemistry at C(R) of 10 was readily inverted
under Mitsonobu reaction conditions to provide the (S)-
isomer 11 in 60% yield. Deoxygenation of ketone 9 was
achieved with lithium aluminum hydride/aluminum
chloride to provide 12 in 78% yield.
As illustrated in Scheme 2, two 2R-substituted de-
rivatives were prepared from 4r. Lithium aluminum
hydride reduction of 4r gave the R-hydroxymethyl
derivative 13 in 90% yield. The reaction of 4r with
tolylmagnesium bromide followed by reduction with
lithium aluminum hydride gave the alcohol 14, which
was isolated as a single diastereoisomer in 90% yield.
The alcohol 14 could not be crystallized to unequivocally
establish the stereochemistry at C(R). The stereochem-
istry at C(R) of 14 was assigned empirically on the basis
of the chemical reactivity of the 3â-tolyltropne ring
system with supporting computed conformational data.25
A conformational search was executed around the C(2)-
C(R) bond in 5° increments. This revealed a preferred
conformation with a dihedral angle defined by C(1)-
C(2)-C(R)-O of 10° in which the tolyl groups of 15 are
aligned in parallel fashion indicative of a π-π interac-
tion, thus leaving the re face of the carbonyl more

3â-Tolyltropane Derivatives at Dopamine Transporters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1205
Ta ble 1. cLogP Values, Dopamine, Serotonin, and Norepinephrine Transporter Binding Affinities, and Dopamine Uptake Inhibition
of 2-Substituted 3â-Tolyltropanes (7-14)
DAT binding
[3H]WIN 35 428
Ki (nM)c
[3H]DA uptake
inhibition
SERT binding
[3H]citalopram
Ki (nM)c
NET binding
[3H]nisoxetine
Ki (nM)c
cLogP
compda
valuesb
IC50 (nM)c
cocained
32 ( 5
388 ( 221
123 ( 9
136 ( 21
17 ( 1
405 ( 91
7
8
9
10
11
12
13
14
3.33
3.29
4.70
4.71
4.71
5.75
2.51
4.71
311 ( 107
266 (89
82 ( 26
14 ( 2
144 ( 24e
107 ( 19e
16 ( 7e
66 ( 5
70 ( 9
464 ( 44
214 ( 24
85 ( 9
11 ( 2
3490 ( 280
25 ( 2
271 ( 27
4040 ( 900
58 ( 18
16000 ( 900
16800 ( 2000
a
b
All compounds were tested as the HCl salt. See ref 29. c All values are the mean ( SEM of three experiments performed in triplicate.
The Ki and IC50 values are reproduced from ref 30 and were collected under identical conditions. e Nonlinear.
d
exposed than the si face for nucleophilic attack. This
conformation is also consistent with a Felkin-Ahn
model for nucleophilic attack at the carbonyl. Therefore,
(S)-stereochemistry was confidently assigned to C(R) of
alcohol 14.
Biology. The DAT binding affinity was determined
for the 2-substituted 3â-tolyltropanes 7-14 by their
ability to displace bound [3H]WIN 35 428 (2) from rat
caudate-putamen tissue.26 The Ki values that are re-
ported in Table 1 are inhibition constants derived for
the unlabeled ligands. The ability of these compounds
to inhibit [3H]dopamine uptake in chopped tissue assays
was also measured.27 In addition, the binding affinities
of the 2-substituted 3â-tolyltropanes were determined
at serotonin transporters (SERT) and norepinephrine
transporters (NET).
In vivo characterization of the 2-substituted 3â-
tolyltropanes consisted of evaluation of the compounds
in two separate paradigms. First, the cocaine-like in-
teroceptive (subjective) effects of the 2-substituted 3â-
tolyltropanes 7-14 were examined. To this end, rats
trained to discriminate between the effects of cocaine
(29 µmol/kg) and those of saline injection in a two-
response-key operant conditioning chamber. Each of the
2-substituted 3â-tolyltropanes was examined for its
potency and efficacy in substituting for the discrimina-
tive effects of cocaine as an indication of its cocaine-
like interoceptive effects. Second, selected compounds
were evaluated for locomotor stimulant effects in mice.
In this procedure the horizontal (locomotor/ambulatory)
activity of mice after injection of a range of doses of each
of the selected 2-substituted 3â-tolyltropanes was mea-
sured and compared to that obtained after injection of
cocaine.
All of the 2-substituted 3â-tolyltropanes displaced [3H]-
WIN 35 428 from caudate-putamen membranes with
relatively high affinity. The Ki values (Table 1) ranged
from 11 nM (12) to 136 nM (8) among the 2â-substituted
derivatives. Generally, the analogues 9-12 that pos-
sessed a tolyl group in the 2-position were more potent
than the alkenyl ester 7 or the hydroxypropyl derivative
8. This was consistent with previous results that suggest
that lipophilic interactions at the 2-position are more
important than H-bond interactions.7,9,11,17 It is note-
worthy that the (R)-isomer of the 2-hydroxymethyltolyl
analogue 10 (Ki ) 14 nM) was approximately 6-fold
more potent than the (S)-isomer 11 (Ki ) 85 nM). This
result demonstrates that stereochemistry incorporated
in the functional group attached to the 2-position can
affect ligand affinity. Surprisingly, the (S)-isomer of 2R-
hydroxymethyltolyl derivative 14 (Ki ) 25 nM) exhibited
only slightly diminished affinity relative to 10, while
an appreciably lower affinity (3490 nM) was obtained
for the 2R-hydroxymethyl analogue 13. None of the
tolyltropane derivatives produced a displacement profile
that better fit a two-site model than a one-site model.
This is in contrast to the displacement data for 1, which
is often reported to better fit a two-site model than a
single-site model.27,28 Among the 2-substituted 3â-
tolyltropanes that were tested, all appeared to have
relatively high affinity for serotonin transporters with
Ki values approximating those obtained at the dopamine
transporter. Alternatively, the Ki values for the nor-
epinephrine transporters among those compounds ex-
amined were generally lower than that for the other
transporters (Table 1).
Each of the compounds inhibited dopamine uptake
inhibition with IC50 values that ranged from 16 nM (12)
to 4040 nM (13). There was a significant correlation (R2
) 0.9981; p < 0.001) among the potencies for the
inhibition of dopamine uptake and the affinities deter-
mined by displacement of [3H]WIN 35 428 (Table 1). It
is interesting to note that 10 had an affinity for binding
to the dopamine transporter that was about 10-fold
higher than its potency for the inhibition of dopamine
uptake. In addition, it was unusual to find that the
dopamine uptake inhibition of the 2R-analogue 14 (IC50
) 58 nM) was more potent than that observed for the
2â-isomers 10 (IC50 ) 144 nM) and 11 (IC50 ) 107 nM).
It must be noted, however, that the fact that the uptake
inhibition curves for the 2â-isomers 10 and 11 were not
linear. Thus, although there was not a statistically
significant two-component model for the inhibition of
dopamine uptake, it is likely that there is more than
one site involved in this function. Because of the
nonlinearity of the curves, these values should be
considered only as estimates of their IC50 values for
inhibiting uptake.
As has been shown previously, there was a dose-
related increase in the percentage of cocaine-appropriate
responses in rats trained to discriminate cocaine (10.0
mg/kg; 29 mmol/kg) from saline (Figure 2, filled circles).
Similarly, each of the 2-substituted 3â-tolyltropanes
analogues also produced a level of drug-appropriate
response significantly greater than that produced by
saline (exceeding 20%; Figure 2, Table 2). Among the

1206 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Xu et al.
F igu r e 3. Dose-dependent effects of tolyltropanes on locomo-
tor activity in mice. The ordinate represents horizontal loco-
motor counts after drug administration. The abscissa repre-
sents dose of drug in mmol/kg on a log scale. Each point
represents the average effect determined in eight mice. The
data are from the 30 min period during the first 60 min after
drug administration, in which the greatest stimulant effects
were observed.
F igu r e 2. Effects of tolyltropane analogues in subjects trained
to discriminate injections of cocaine from saline. Ordinates for
top panels are the percentage of responses on the cocaine-
appropriate key. Ordinates for the bottom panels are rates at
which responses were emitted (as a percentage of response rate
after saline administration). Abscissas represent drug dose in
mmol/kg (log scale). Each point represents the effect in four
to six subjects. The percentage of responses emitted on the
cocaine-appropriate key was considered unreliable and not
plotted if fewer than half of the subjects responded at that
dose.
activity to a level respectively exceeding and approach-
ing that produced by cocaine. Alternatively, the 2R-
derivative 13 produced only a marginal stimulation of
locomotor activity, achieving significance only at the 8.3
µmol/kg dose.
For all of the 2â-substituted 3â-tolyltropanes tested
in this study the behavioral effects were found to be
typically cocaine-like. However, the relative potency of
the behavioral effects did not correspond entirely to the
relative affinities of the compounds at the DAT. Most
notable were 10 and 12, which had a lower in vivo
potency than that which would be predicted on the basis
of their affinity for the DAT. These findings suggest that
these compounds may have characteristics (e.g., high
lipophilicity) that reduced their central availability. On
the basis of calculated partition coefficients (cLogP,
Table 1), compound 12 is an order of magnitude more
lipophilic than the oxygenated derivatives 9-11.29
However, the actual lipophilicity of 10 may be closer to
that of 12 because of the intramolecular H bond between
the nitrogen atom and the hydroxyl residue.
Ta ble 2. Substitution of 2-Substituted 3â-Tolyltropanes in
Rats Trained To Discriminate Cocaine
drug response
response rate
compda
ED50 (mmol/kg)
ED50 (mmol/kg)
% maximum effect
7
244
54.0
69.9 @ 89.3 mmol/kg
((70.3-2.77) × 104) (37.4-91.9)
8
9
21.5
23.6
(17.3-33.8)
5.06
(3.31-7.72)
18.3
93.9 @ 54.9 mmol/kg
100 @ 27.0 mmol/kg
97.9 @45.7 mmol/kg
66.7 @ 37.7 mmol/kg
82.8 @ 39.0 mmol/kg
2.96 @ 2.69 mmol/kg
(17.5-25.8)
10.9b
(4.96-56.1)
61.6b
10
11
12
13
((17.1-1.22) ×105) (14.6-22.3)
31.4
28.3b
(22.0-51.4)
12.0
(9.43-15.4)
20.6
((14.9-7.37) ×104)
23.5
(11.0-127)
NSc
(9.07-256)
a
b
All compounds were tested as the HCl salt. Nonsignificant
effect of dose. c Nonsignificant linear regression.
The ratio of potency for dopamine uptake inhibition
to the DAT binding affinity (DUI/DAT) for 10 (10)
suggested the potential for anomalous behavioral activ-
ity. Despite what was predicted on the basis of DAT
affinity, alcohol 10 was among the most potent of the
2â-analogues in the drug discrimination experiments
with an ED50 value of 18.3 mmol/kg for its full cocaine-
like substitution. Moreover, 10 was the most effective
of the compounds tested for stimulation of locomotor
activity, and it exceeded the magnitude of the effect of
cocaine. Therefore, despite a high DUI/DAT ratio, 10
exhibited typical stereoselective cocaine-like behavioral
activity. The high DUI/DAT ratio may have been
contributed by characteristics that limit distribution in
tissue, thereby decreasing potency for inhibition of
dopamine uptake without altering binding affinity,
which is assayed at equilibrium. This hypothesis is
consistent with the finding that 10 had a lower in vivo
2-substituted 3â-tolyltropanes, only 7 and 11 produced
a level of substitution less than 80%, and for those two,
despite a failure to achieve an average value indicating
full substitution, there was a full substitution in the
majority of the animals tested at the highest doses. In
contrast, the 2R-hydroxymethyl 3â-tolyltropane (13) did
not produce a level of drug-appropriate response that
was greater than that obtained with saline across a
range of doses from those having no effect to those that
virtually eliminated response.
Selected compounds 10, 11, and 13 were further
evaluated in locomotor stimulant assays. As has been
demonstrated previously, cocaine increased ambulatory
activity with a maximum of approximately 500 counts
per minute during the first 30 min of the session at 59
µmol/kg (Figure 3). Both 10 and 11 increased locomotor

3â-Tolyltropane Derivatives at Dopamine Transporters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1207
the aldehyde 6 (480 mg, 2.0 mmol). The reaction mixture was
allowed to stir for 24 h. The acetonitrile was removed under
reduced pressure, and the residue was diluted with water. The
aqueous solution was extracted with ether (3 × 40 mL). The
combined organic layers were washed with water and brine
and dried (Na2SO4), and the solvent was removed under
reduced pressure. The product was purified by flash column
chromatography (ether/triethylamine, 9:1) to afford 7 as a
potency than that which would be predicted on the basis
of its affinity for the DAT.
Con clu sion s
From the in vitro SAR data, it is apparent that a tolyl
group in the 2-position, independent of stereochemical
attachment to the tropane ring system, provides com-
pounds that exhibit high-affinity binding at the DAT.
Although a slight stereochemical preference was ob-
served for the (R)-alcohol 10 over the (S)-isomer 11 in
binding affinities at DAT, no significant differences in
behavioral effects were observed. Furthermore, despite
a high DUI/DAT affinity ratio observed for 10, the
behavioral profile did not vary significantly from co-
caine. Thus, in this case, a comparison of the DUI/DAT
binding affinity ratio was not useful for identification
of a cocaine antagonist. This demonstrates that char-
acterization of the behavioral effects in addition to in
vitro activity of potent DAT ligands is important for the
accurate assessment of the structure-activity relation-
ships of a compound and its potential for development
as cocaine medication.
colorless oil (370 mg, 65%). [R]21 +68° (c 1, MeOH, HCl salt);
D
1H NMR (CDCl3) δ 7.60 (m, 4H), 6.68-6.57 (m, 1H), 5.42 (d, J
) 16.0 Hz, 1H), 3.56 (s, 3H), 3.28 (m, 1H), 3.08 (s, 2H), 2.42
(m, 1H), 2.24 (s, 3H), 2.20 (s, 3H), 2.18-2.05 (m, 3H), 1.78-
1.67 (m, 2H), 1.59 (m, 1H); 13C NMR (CDCl3) δ 166.6, 150.7,
139.1, 135.4, 128.8, 127.6, 121.3, 67.5, 62.2, 50.9, 50.5, 42.0,
36.4, 26.5, 25.1, 20.9. Anal. (C19H25NO‚HCl) C, H, N.
2â-(3-Hyd r oxyp r op yl)-3â-tolyltr op a n e (8). A solution of
unsaturated ester 7 (560 mg, 2.0 mmol) in dry methanol (20
mL) was hydrogenated (1 atm) over 10% palladium on carbon
(50 mg). The reaction mixture was stirred at room temperature
overnight and then was filtered through a pad of Celite. The
solvent was removed under reduced pressure to afford the
saturated ester as a colorless oil. The oil was dissolved in dry
ether and was added via syringe to a stirred suspension of
LiAlH4 (76 mg, 2.0 mmol) in dry ether (10 mL) at 0 °C under
a nitrogen atmosphere. Stirring was continued overnight at
room temperature. The reaction mixture was cooled to 0 °C,
and an aqueous solution of NaOH (5%, 10 mL) was added
dropwise. The reaction mixture was filtered, and the residue
was washed with ether (5 × 50 mL). The combined filtrate
and washings were concentrated under reduced pressure to
furnish an oil. The oil was purified by column chromatography
(SiO2, CHCl3/CH3OH, 95:5) to furnish 8 (510 mg, 94%). Mp
Exp er im en ta l Section
All chemicals and reagents not otherwise noted were
purchased from Aldrich Chemical Co. The solvents THF, ether,
and 1,4-dioxane were dried by distillation from Na and
benzophenone. Dichloromethane and acetonitrile were dried
by distillation over P2O5. The spectral data for all compounds
205-206 °C (HCl salt); [R]21 +33.7° (c 1, CH3OH, HCl salt);
D
1
are reported for the free base. H and 13C NMR spectra were
1H NMR (CDCl3) δ 7.29 (d, J ) 8.0 Hz, 2H), 7.09 (d, J ) 8.0
Hz, 2H), 4.08-3.90 (m, 2H), 3.39 (m, 1H), 3.17(m,1H), 2.83 (s,
3H), 2.29 (s, 3H), 2.26-2.05 (s, 2H), 2.01 (m, 2H), 1.90 (m, 2H),
1.26-1.12 (m, 4H), 1.14 (m, 2H); 13C NMR (CDCl3) δ 138.5,
136.4, 129.4, 128.1, 66.6, 64.2, 61.5, 46.1, 43.3, 40.3, 39.5, 29.4,
26.3, 24.9, 26.3, 24.9, 21.1, 20.4. Anal. (C18H27NO‚HCl‚2.5H2O)
C, H, N.
recorded on a Varian multiprobe 400 MHz spectrometer. The
free base was then converted into the hydrochloride salt to
give a hygroscopic solid used for microanalysis and biological
testing. Microanalysis for C, H, and N were performed by
Atlantic Microlabs, Inc., Norcross, GA. Melting points were
recorded on a Hoover Mel-Temp apparatus and are uncor-
rected.
2â-Tolylca r bon yl-3â-tolyltr op a n e (9). A solution of 4â
(7.0 g, 26 mmol) in freshly distilled ether (50 mL) was added
dropwise via syringe to a stirred solution of tolylmagnesium
bromide (1 M, 50 mL) in dry ether (600 mL) at -40 °C under
a nitrogen atmosphere. The mixture was stirred at -40 °C
for 2.5 h. The reaction mixture was then cooled to -78 °C and
quenched with a solution of TFA (11.4 g, 100 mmol) in dry
ether (40 mL) over 5 min. The mixture was allowed to warm
to 0 °C and was diluted with water (150 mL). The aqueous
phase was acidified with HCl (concentrated) to pH 1, and the
ether layer was separated. The aqueous layer was made basic
with NH4OH (concentrated) at 0 °C and was extracted with
ether (4 × 100 mL). The organic layer was dried (Na2SO4) and
the solvent was removed under reduced pressure to provide
the crude product as a yellow oil. The crude product was
purified by flash column chromatography (SiO2, ether/Et3N,
9:1) to give the ketone 9 as colorless crystals (4.8 g, 56%), mp
186-187 °C. Ketone 9 was homogeneous by TLC (Rf 0.85, SiO2,
ether/Et3N, 9.5:0.5). [R]21D +27.94° (c 1, CH3OH, HCl salt); 1H
NMR (CDCl3) δ 7.73 (d, J ) 8.1 Hz, 2H), 7.19 (d, J ) 7.7 Hz,
4H), 7.00 (d, J ) 8.1 Hz, 2H), 3.88 (m, 1H), 3.46 (m, 1H), 3.39
(m, 1H), 3.12-3.06 (m, 1H), 2.82 (m, 1H), 2.35 (s, 3H), 2.22 (s,
3H), 2.08 (s, 3H), 1.86 (m, 2H), 1.80-1.60 (m, 3H); 13C NMR
(CDCl3) δ 198.4, 142.8, 140.3, 134.9, 129.1, 128.6, 128.1. 127.2,
65.0, 62.4, 53.8, 42.1, 34.3, 33.9, 26.5, 25.3, 21.5, 20.9. Anal.
(C23H27NO) C, H, N.
2â-Hyd r oxym eth yl-3â-tolyltr op a n e (5). To a stirred sus-
pension of LiAlH4 (76 mg, 2.0 mmol) in dry ether (10 mL) at
0 °C under a nitrogen atmosphere a solution of the ester 4â
(540 mg, 2.0 mmol) in dry ether (5 mL) was added dropwise
via syringe. Stirring was continued overnight at room tem-
perature. The reaction mixture was cooled to 0 °C, and an
aqueous solution of NaOH (5%, 10 mL) was added dropwise.
The reaction mixture was filtered, and the residue was washed
with ether. The combined filtrate and washings were concen-
trated under reduced pressure to furnish the alcohol 5 as a
colorless oil (0.45 g, 92%). [R]21D -66.03° (c 1, CHCl3); 1H NMR
(CDCl3) δ 7.24 (d, J ) 8.0 Hz, 2H), 7.12 (d, J ) 8.0 Hz, 2H),
3.74 (d, J ) 13.0 Hz, 1H), 3.45-3.31 (m, 3H), 3.10-3.00 (m,
1H), 2.52 (m, 1H), 2.32 (s, 3H), 2.26 (s, 3H), 2.25-2.05 (m, 3H),
1.73 (d, J ) 8.1 Hz, 2H), 1.62 (m, 1H), 1.48 (m, 1H); 13C NMR
(CDCl3) δ 139.6, 135.6, 129.0, 128.2, 68.5, 65.2, 62.0, 45.5, 41.2,
37.1, 36.4, 26.3, 25.2, 21.0. Anal. (C16H23NO) C, H, N.
2â-F or m yl-3â-tolyltr op a n e (6). To a solution of oxalyl
chloride (1.2 mL, 2.0 M in CH2Cl2,) under a nitrogen atmo-
sphere at -60 °C was added a solution of DMSO (0.31 mL,
340 mg, 4.4 mmol) in dry CH2Cl2 (1 mL). After 0.5 h, a solution
of the alcohol 5 (490 mg, 2.0 mmol) in dry CH2Cl2 (3 mL) was
added. Stirring was continued for 1 h at 60 °C followed by the
addition of triethylamine (1.3 mL, 9.2 mmol). The reaction
mixture was allowed to warm to room temperature and was
diluted with water. The organic layer was separated and dried
(Na2SO4), and the solvent was removed under reduced pres-
sure to yield the aldehyde 6 (461 mg, 95%). The material was
carried on to the olefination step without further purification.
2â-(2′Ca r bom eth oxyeth en yl)-3â-tolyltr op a n e (7). To a
stirred suspension of lithium chloride (0.10 g, 2.4 mmol) in
dry acetonitrile (10 mL) at room temperature under a nitrogen
atmosphere were added trimethyl phosphonoacetate (440 mg,
2.4 mmol), N,N-diisopropylethylamine (0.26 g, 2.0 mmol), and
2â-(R)-(Hyd r oxym eth yltolyl)-3â-tolyltr op a n e (10). To a
stirred suspension of LiAlH4 (76 mg, 2.0 mmol) in dry ether
at 0 °C under a nitrogen atmosphere was added a solution of
ketone 9 (670 mg, 2.0 mmol) in dry ether dropwise via syringe.
Stirring was continued overnight at room temperature. The
reaction mixture was then cooled to 0 °C, and an aqueous
solution of NaOH was added dropwise. The reaction mixture
was filtered, and the residue was washed with ether. The

1208 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Xu et al.
solvent was removed under reduced pressure, which afforded
the alcohol 10 as a white solid (640 mg, 95%). The alcohol was
homogeneous by TLC (Rf 0.3, SiO2, ether/Et3N, 9:1). Mp 135-
136 °C; [R]21D +55.56° (c 1, MeOH, HCl salt); 1H NMR (CDCl3)
δ 7.00-7.27 (m, 8H), 4.43 (d, J ) 7.0 Hz, 1H), 3.12 (m, 1H),
2.92 (m, 1H), 2.70-2.50 (m, 2H), 2.30 (s, 6H), 2.12 (s, 3H),
140.9, 135.9, 129.3, 127.7, 63.2, 62.8, 61.9, 48.6, 40.9, 40.8, 37.9,
25.9, 21.7, 21.0; [R]21 +22.63° (c 1, CHCl3). Anal. (C26H23NO)
D
C, H, N.
2r-(S)-(Hyd r oxym eth yltolyl)-3â-tolyltr op a n e (14). A
solution of 4r (540 mg, 2.0 mmol) in freshly distilled ether (5
mL) was added dropwise via syringe to a stirred solution of
tolylmagnesium bromide (1 M, 4.0 mL) in dry ether (6 mL) at
0 °C under a nitrogen atmosphere. The mixture was stirred
at 0 °C for 2.5 h. The mixture was allowed to warm to room
temperature and was diluted with water (15 mL). The aqueous
phase was acidified with HCl (concentrated) to pH 1, and the
ether layer was separated. The aqueous layer was made basic
with NH4OH (concentrated) at 0 °C and was extracted with
ether (4 ×100 mL). The organic layer was dried (Na2SO4), and
the solvent was removed under reduced pressure to provide
the crude ketone 15 as a yellow oil. The crude product was
purified by flash column chromatography (SiO2, ether/triethyl-
amine, 9:1) to give the ketone 15 as a colorless oil (560 mg,
85%). Ketone 15 was homogeneous by TLC (Rf 0.75, SiO2,
1.80-1.98 (m, 2H), 1.72-1.43 (m, 3H), 1.45-1.20 (m, 2H); 13
C
NMR (CDCl3) δ 141.7, 139.3, 136.7, 136.0, 130.0, 128.6, 127.8,
126.5, 62.6, 61.8, 52.5, 46.0, 42.6, 40.4, 39.8, 25.8, 22.8, 21.0,
20.9, 11.2. Anal. (C23H29NO) C, H, N.
2â-(S)-Hyd r oxym eth yltolyl-3â-tolyltr op a n e (11). (a) To
a solution of 10 (500 mg, 1.50 mmol), triphenylphosphine (920
mg, 3.5 mmol), and benzoic acid (460 mg, 3.7 mmol) in dry
THF (20 mL) was added diethyl azodicarboxylate (592 mg, 3.4
mmol) dropwise. The reaction mixture was stirred overnight
at room temperature. The solution was then concentrated, and
the residue was purified by column chromatography (petro-
leum ether/triethylamine, 9:1) to afford the benzoate ester (410
mg, 62%).
1
ether/triethylamine, 9.5:0.5). H NMR (CDCl3) δ 7.63 (d, J )
(b) To a solution of the benzoate ester (130 mg, 0.3 mmol)
in dry MeOH (20 mL) was added NaOCH3 (400 mg, 7.4 mmol).
The reaction mixture was refluxed for 24 h. The mixture was
allowed to cool to room temperature and diluted with water
(20 mL), and the pH was adjusted to pH 10 with concentrated
NH4OH. The mixture was extracted with ether (4 × 30 mL)
and dried (Na2SO4). The solvent was removed under reduced
pressure to afford a yellow oil. The crude product was purified
by flash column chromatography (SiO2, petroleum ether/
EtOAc/triethylamine, 8.5:0.5:1) to furnish the alcohol 11 as a
colorless oil (95 mg, 94%). [R]21D +28.74° (c 1, CHCl3); 1H NMR
(CDCl3) δ 7.00-7.30 (m, 8H), 4.44 (br, 1H), 3.10-3.20 (m, 1H),
2.88 (m, 1H), 2.45-2.38 (m, 2H), 2.36 (s. 6H), 2.29 (s, 3H),
8.0 Hz, 2H), 7.19 (m, 4H), 7.01 (d, J ) 8.1 Hz, 2H), 3.88 (m,
1H), 3.46 (m, 1H), 3.39 (m,1H), 3.06-3.12 (m, 1H), 2.82 (m,
1H), 2.31 (s, 3H), 2.20 (s, 3H), 2.08 (s, 3H), 1.86 (m, 2H), 1.60-
1.80 (m, 3H).
To a stirred suspension of LiAlH4 (76 mg, 2.0 mmol) in dry
Et2O at 0 °C under a nitrogen atmosphere was added a
solution of 15 (670 mg, 2.0 mmol) in dry ether dropwise via
syringe. Stirring was continued overnight at room tempera-
ture. The reaction mixture was cooled to 0 °C, and an aqueous
solution of NaOH (5%, 10 mL) was added dropwise. The
mixture was then filtered, and the residue was washed with
ether. The combined filtrate and washings were concentrated
to provide the alcohol 14 as a white solid (640 mg, 95%). The
alcohol was homogeneous by TLC (Rf 0.3, SiO2, petroleum
ether/triethylamine, 9:1). Mp 135-136 °C; [R]21D +26.34° (c 1,
MeOH, HCl salt); 1H NMR (CDCl3) δ 6.65-6.85 (m, 8H), 5.06
(br, 1H), 3.12 (m, 1H), 2.92 (m, 1H), 2.50-2.70 (m, 2H), 2.30
(s, 6H), 2.12 (s, 3H), 1.80-1.98 (m, 2H), 1.43-1.72 (m, 3H),
1.25-1.40 (m, 2H); 13C NMR (CDCl3) δ 141.7, 139.3, 136.7,
136.0, 130.0, 128.6, 127.8, 126.5, 62.6, 61.8, 52.5, 46.0 42.6,
40.4, 39.8, 25.8, 22.8, 21.0, 20.9, 11.2. Anal. (C23H27NO) C, H,
N.
[3H]WIN 35 428 Bin d in g Assa y. Male Sprague-Dawley
rats (200-250 g, Taconic, Germantown, NY) were decapitated,
and their brains were removed and placed in an ice-cooled dish
for dissection of the caudate-putamen. The tissue was homog-
enized in 30 volumes of ice-cold modified Krebs HEPES buffer
(15 mM HEPES, 127 mM NaCl, 5 mM KCl, 1.2 mM MgSO4,
2.5 mM CaCl2, 1.3 mM NaH2PO4, 10 mM glucose, pH adjusted
to 7.4) using a Teflon/glass homogenizer and centrifuged at
20 000g for 10 min at 4 °C. The resulting pellet was then
washed two more times by resuspension in ice-cold buffer and
centrifugation at 20 000g for 10 min at 4 °C. Fresh homoge-
nates were used in all experiments. Binding assays were
conducted in modified Krebs HEPES buffer on ice, essentially
as previously described.27 The total volume in each tube was
0.5 mL, and the final concentration of membrane after all
additions was approximately 0.3% (w/v) corresponding to 150-
300 µg of protein per sample. Increasing concentrations of the
drug being tested were added to triplicate samples of mem-
brane suspension. Five minutes later, [3H]WIN 35 428 (final
concentration of 1.5 nM) was added and the incubation was
continued for 1 h on ice. The incubation was terminated by
the addition of 3 mL of ice-cold buffer and rapid filtration
through Whatman GF/B glass fiber filter paper (presoaked in
0.1% BSA in water to reduce nonspecific binding) using a
Brandel cell harvester (Gaithersburg, MD). After filtration, the
filters were washed with three additional 3 mL washes and
transferred to scintillation vials. Absolute ethanol (0.5 mL) and
Beckman Ready Value scintillation cocktail (2.75 mL) were
added to the vials, which were counted the next day at an
efficiency of about 36%. Under these assay conditions, an
average experiment yielded approximately 6000 dpm total
binding per sample and approximately 250 dpm nonspecific
1.97-2.10 (m, 1H), 1.97-1.80 (m, 2H), 1.80-1.60 (m, 4H); 13
C
NMR (CDCl3) δ 141.8, 140.0, 135.5, 134.0, 129.3, 129.0, 127.5,
125.4, 76.6, 64.0, 60.3, 52.6, 39.8, 38.9, 35.2, 29.6, 26.3, 20.9.
Anal. (C23H29NO‚0.5H2O) C, H, N.
2â-(p-Meth ylp h en ylm eth yl)-3â-tolyltr op a n e (12). In a
dry 50 mL round-bottomed flask was placed ketone 9 (340 mg,
1.0 mmol), AlCl3 (470 mg, 3.5 mmol), and 1,4-dioxane (15 mL).
The solution was stirred for 10 min under a nitrogen atmo-
sphere followed by the addition of LiAlH4 (2.5 mL, 2.5 mmol).
The reaction mixture was refluxed overnight. The 1,4-dioxane
was removed under reduced pressure, and a solution of NaOH
(5%) was added dropwise to the residue. The aqueous solution
was extracted with ether (3 × 20 mL). The combined extracts
were dried (Na2SO4), and the solvent was removed under
reduced pressure. The crude product was purified by coluumn
chromatography (SiO2, petroleum ether/triethylamine, 9.5:0.5)
to provide 12 as white crystals (190 mg, 60%). Mp 175-177
1
°C; H NMR (CDCl3) δ 7.13 (s, 4H), 6.97 (d, J ) 7.9 Hz, 4H),
3.28 (br, 1H), 3.12 (m, 1H), 2.83 (m, 1H), 2.75 (t, J ) 12.5 Hz,
2H), 2.33 (s, 3H), 2.28 (s, 3H), 2.23 (m, 1H), 2.20 (s, 3H), 2.02
(m, 2H), 1.82 (m, 1H), 1.67-1.59 (m, 2H), 1.32 (m, 1H); 13C
NMR (CDCl3) δ 140.6, 133.2, 135.2, 134.5, 129.2, 128.9, 128.6,
127.6, 63.2, 62.2, 49.1, 41.7, 36.0, 33.6, 32.6, 26.4, 25.0, 20.9.
Anal. (C23H29N‚0.33H2O) C, H, N.
2r-Hyd r oxym eth yl-3â-tolyltr op a n e (13). To a stirred
suspension of LiAlH4 (76 mg, 2.0 mmol) in dry ether at 0 °C
under a nitrogen atmosphere was added a solution of 2R-
carbomethoxy-3â-phenyltropane (4r) (540 mg, 2.0 mmol) in
dry ether dropwise via syringe. Stirring was continued over-
night at room temperature. The reaction mixture was cooled
to 0 °C, and an aqueous solution of NaOH (5%, 10 mL) was
added dropwise. The reaction mixture was filtered, and the
residue was washed with ether. The combined filtrate and
washings were concentrated under reduced pressure, which
afforded alcohol 13 as a white solid (470 mg, 95%). The alcohol
was homogeneous by TLC (Rf 0.3, SiO2, petroleum ether/
triethylamine, 9:1). Mp 200-202 °C; 1H NMR (CDCl3) δ 7.08-
7.17 (d, J ) 7.9, 4H), 3.44 (m, 1H), 3.41-3.33 (m, 1H), 3.30-
3.20 (m, 2H), 2.37 (s, 3H), 2.32 (s, 3H), 2.35-2.20 (m, 2H),
2.05-2.16 (m, 1H), 1.88-2.04 (m, 2H), 1.74-1.84 (m, 1H),
1.56-1.64 (m, 2H), 1.38-1.45 (m, 1H); 13C NMR (CDCl3) δ

3â-Tolyltropane Derivatives at Dopamine Transporters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1209
binding. Nonspecific binding was defined as binding in the
presence of 100 µM cocaine. Ki values were derived from 14
point competition assays using increasing concentrations of
unlabeled compounds (0.05 nM to 100 µM) against 1.5 nM [3H]-
WIN 35 428. Displacement data were analyzed by the use of
the nonlinear least-squares curve-fitting computer program
LIGAND.31
presoaked in 0.05% polyethylenimine (PEI). Nonspecific bind-
ing was defined using 1 µM desipramine. For these assays,
an initial screen was conducted to assess displacement of
nisoxetine at a concentration of 1 µM of the unknown com-
pound. If there was greater than 50% displacement of nisox-
etine, a Ki value was determined in subsequent studies.
Coca in e Discr im in a tion Stu d ies. Sessions were con-
ducted daily, 5 days per week. Five minutes before sessions,
ip injections were given and the subjects were placed in
experimental chambers. The chambers contained two response
levers: a device for delivering food pellets and stimulus lights.
Session start was indicated by illumination of the lights.
Subjects were trained to press the response levers, with
responses on one lever reinforced with food presentation after
a presession injection of cocaine and responses on the other
lever reinforced after an injection of saline. Over the course
of several sessions the number of responses required for each
food pellet was gradually increased to 20. “Cocaine” and
“saline” sessions alternated in a sequence of double alterna-
tions. Sessions ended after 20 food presentations or 15 min,
whichever occurred first. Test sessions began once perfor-
mances met a criterion for stability (greater than 85% correct
responses prior to the first food presentation and throughout
the entire session). Test sessions were identical to training
sessions with the exception that 20 or 30 consecutive responses
on either lever were reinforced.
[3H]Dop a m in e Up t a k e In h ib it ion St u d ies. Rats were
sacrificed by decapitation, and their brains were removed and
placed in an ice-cooled dish for dissection of the caudate-
putamen. [3H]Dopamine uptake was measured in a chopped
tissue preparation as described previously.26 Briefly, the tissue
was chopped into 225 µm slices on a McIllwain tissue slicer
with two successive cuts at an angle of 90°. The strips of tissue
were suspended in oxygenated modified Krebs HEPES buffer
(see above), which was pregassed with 95% O2/5% CO2 and
warmed to 37 °C. After being rinsed, aliquots of tissue slice
suspensions were incubated in buffer in glass test tubes at 37
°C to which either the drug being tested or no drug was added,
as appropriate. After a 5 min incubation period in the presence
of drug, [3H]dopamine (final concentration of 15 nM) was added
to each tube. After 5 min the incubation was terminated by
the addition of 2 mL of ice-cold buffer to each tube and
filtration under reduced pressure over glass fiber filters
(presoaked in 0.1% polyethylenimine in water). The filters
were rinsed and placed in scintillation vials to which 1 mL of
methanol and 2 mL of 0.2 M HCl were added to extract the
accumulated [3H]dopamine. Radioactivity was determined by
liquid scintillation spectrometry at an efficiency of approxi-
mately 30%. The reported values represent specific uptake
from which nonspecific binding to filters was subtracted. Data
were analyzed using standard analysis of variance and linear
regression techniques.32 If there was a significant deviation
(p < 0.05) from linearity, the concentration-effect curve was
resolved, if possible, into two linear components, which were
analyzed independently. IC50 values were calculated only on
curve components that exhibited a significant linear regression
(p < 0.05).
[3H]Cita lop r a m Bin d in g Assa y. Brains from male Spra-
gue-Dawley rats weighing 200-225 g (Taconic Labs) were
removed, and the midbrain was dissected and rapidly frozen.
Membranes were prepared by homogenizing tissues in 20
volumes (w/v) of 50 mM Tris containing 120 mM NaCl and 5
mM KCl (pH 7.4 at 25 °C), using a Brinkman Polytron (setting
6 for 20 s) and centrifuged at 50 000g for 10 min at 4 °C. The
resulting pellet was resuspended in buffer, recentrifuged, and
resuspended in buffer to a concentration of 15 mg/mL. Ligand
binding experiments were conducted in assay tubes containing
0.5 mL of buffer for 60 min at room temperature. Each tube
contained 1.4 nM [3H]citalopram (NEN) and 1.5 mg of mid-
brain tissue (original wet weight). Nonspecific binding was
determined using 10 mM fluoxetine. Incubations were termi-
nated by rapid filtration through Whatman GF/B filters,
presoaked in 0.3% polyethylenimine, using a Brandel R48
filtering manifold (Brandel Instruments, Gaithersburg, Mary-
land). The filters were washed twice with 5 mL of cold buffer
and transferred to scintillation vials. Beckman Ready Safe (3.0
mL) was added, and the vials were counted the next day using
a Beckman 6000 liquid scintillation counter (Beckman Coulter
Instruments, Fullerton, California). Data were analyzed with
GraphPad Prism software (San Diego, California).
Locom otor Activity Stu d ies. Subjects were experimen-
tally naive group-housed male Swiss Webster mice approxi-
mately 14 weeks old. They were studied in 40 cm3 clear acrylic
chambers, which counted ambulatory activity with photoelec-
tric detectors placed 2.56 cm apart along the walls. One
activity count was registered each instance in which the
subject crossed a beam. Multiple interruptions of the same
beam (e.g., grooming, head bobbing) were not counted. Mice
were injected and placed in the apparatus for 60 min. Each
dose was studied in six mice.
Ack n ow led gm en t. We are grateful to the National
Institute on Drug Abuse (Grant DA11528) for financial
support of this research. The authors thank Bettye
Campbell and Dawn French for technical support, Patty
Ballerstadt for administrative and clerical support, and
Dawn French for expert data analysis.
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data for 10. This material is available free of charge
via the Internet at http://pubs.acs.org.
Refer en ces
(1) Mathias, R. Cocaine, Marijuana and Heroin Abuse Up, Meth-
amphetamine Abuse Down. NIDA Notes 2000, 15 (3), 4-5.
(2) Carroll, F. I.; Howell, L. L.; Kuhar, M. J . Pharmacotherapies
for the Treatment of Cocaine Abuse: Preclinical Aspects. J . Med.
Chem. 1999, 42, 2721-2736.
(3) Carroll, F. I.; Lewin, A. H.; Boja, J . W.; Kuhar, M. J . Cocaine
Receptors: Biochemical Characterization and Structure-Activ-
ity Relationships of Cocaine Analogues at the Dopamine Trans-
porter. J . Med. Chem. 1992, 35, 969-981.
(4) Kozikowski, A. P.; Roberti, M.; J ohnson, K. M.; Bergmann, J .
S.; Ball, R. C. SAR of Cocaine: Further Exploration of the
Structural Variations at the C-2 Center Provides Compounds
of Subnanomolar Binding Potency. Bioorg. Med. Chem. Lett.
1993, 1327-1332.
(5) Carroll, F. I.; Mascarella, S. W.; Kuzemko, M. A.; Gao, Y.;
Abraham, P.; Lewin, A. H.; Boja, J . W.; Kuhar, M. J . Synthesis,
Ligand Binding, and QSAR (CoMFA and Classical) Study of 3â-
(3′-Substituted phenyl)-, 3â-(4′-Substituted phenyl)-, and 3â-
(3′,4′-Disubstituted phenyl)tropane 2â-Carboxylic Acid Methyl
Esters. J . Med. Chem. 1994, 37, 2865-2873.
(6) Carroll, F. I.; Kotian, P.; Dehghani, A.; Gray, J . L.; Kuzemko,
M. A.; Parham, K. A.; Abraham, P.; Lewin, A. H.; Boja, J . W.;
Kuhar, M. J . Cocaine and 3â-(4′-Substituted phenyl)tropane 2â-
Carboxylic Acid Ester and Amide Analogues. New High Affinity
and Selective Compounds for the Dopamine Transporter. J . Med.
Chem. 1995, 38, 379-388.
(7) Kozikowski, A. P.; Saiah, M. K. E.; J ohnson, K. M.; Bergmann,
J . S. Chemistry and Biology of the 2â-Alkyl-3â-phenyl Analogues
of Cocaine: Subnanomolar Affinity Ligands That Suggest a New
Pharmacophore Model at the C-2 Position. J . Med. Chem. 1995,
38, 3086-3093.
[3H]Nisoxetin e Bin d in g Assa y. The frontal cortex of male
Sprague-Dawley rats was removed and frozen. Membranes
were prepared by homogenizing tissues in 50 mM Tris (120
mM NaCl, 5 mM KCl; pH 7.4 at 25 °C) and centrifuging
(50 000g) for 10 min at 4 °C. The resulting pellet was then
washed and centrifuged two more times. The final pellet was
resuspended to a concentration of 80 mg/mL (original wet
weight). Assays were conducted in the above Tris buffer.
Volume totaled 0.5 mL with tissue concentration of 8 mg/tube.
[3H]Nisoxetine (specific activity of 80 Ci/mmol; final concentra-
tion of 0.5 nM; New England Nuclear, Boston, MA) was added,
and the incubation continued for 1 h on ice. Incubations were
terminated by rapid filtration through Whatman GF/B filters,

1210 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Xu et al.
(8) Davies, H. M. L.; Kuhn, L. A.; Thornley, C.; Matasi, J . J .; Sexton,
T.; Childers, S. R. Synthesis of 3â-Aryl-8-azabicyclo[3.2.1]octanes
with High Binding Affinities and Selectivities for the Serotonin
Transporter Site. J . Med. Chem. 1996, 39, 2554-2558.
(9) Kotian, P.; Mascarella, S. W.; Abraham, P.; Lewin, A. H.; Boja,
J . W.; Kuhar, M. J .; Carroll, F. I. Synthesis, Ligand Binding and
Quantitative Structure-Activity Relationship Study of 3â-(3′-
Substituted phenyl)-2â-heterocyclic Tropanes: Evidence for an
Electrostatic Interaction at the 2â-Position. J . Med. Chem. 1996,
39, 2753-2763.
(10) Blough, B. E.; Abraham, P.; Lewin, A. H.; Kuhar, M. J .; Boja, J .
W.; Carroll, F. I. Synthesis and Transporter Binding Properties
of 3â-(4′-Alkyl-, 4′-alkenyl, and 4′-alkynylphenyl)nortropane 2â-
Carboxylic Acid Methyl Esters: Serotonin Transporter Selective
Analogues. J . Med. Chem. 1996, 39, 4027-4035.
(11) Xu, L.; Kelkar, S. V.; Lomenzo, S. A.; Izenwasser, S.; Katz, J .
L.; Kline, R. H.; Trudell, M. L. Synthesis, Dopamine Transporter
Affinity, Dopamine Uptake Inhibition, and Locomotor Stimulant
Activity of 2-Substituted 3â-Phenyltropane Derivatives. J . Med.
Chem. 1997, 40, 858-863.
(12) Emond, P.; Garreau, L.; Chalon, S.; Boazi, M.; Caillet, M.;
Bricard, J .; Frangin, Y.; Mauclaire, L.; Besnard, J . C.; Guilloteau,
D. Synthesis and Ligand Binding of Nortropane Derivatives.
N-Substituted 2â-Carboxymethyl-3â-(4′-iodophenyl)nortropane
and N-(3-Iodoprop-(2E)-enyl)-2â-carboxymethyl-3â-(3′,4′-disub-
stituted phenyl)nortropane. New High-Affinity and Selective
Compounds for the Dopamine Transporter. J . Med. Chem. 1997,
40, 1366-1372.
ation of 7-Fluoro-3-(p-flourophenyl)-2-propyltropanes. Bioorg.
Med. Chem. Lett. 2000, 10, 1443-1446.
(20) Tamagnan, G.; Baldwin, R. M.; Kula, N. S.; Baldessarini, R. J .;
Innis, R. B. Synthesis and Monoamine Transporter Affinity of
2â-Carbomethoxy-3â-(2′, 3′, or 4′-Substituted) Biphenyl-tro-
panes. Bioorg. Med. Chem. Lett. 2000, 10, 1783-1785.
(21) Zhao, L.; J ohnson, K. M.; Zhang, M.; Flippen-Anderson, J .;
Kozikowski, A. P. Chemical Synthesis and Pharmacology of 6-
and 7-Hydroxylated 2-Carbomethoxy-3-(p-tolyl)tropanes. An-
tagonism of Cocaine’s Locomotor Stimulant Effects. J . Med.
Chem. 2000, 43, 3283-3294.
(22) Davies, H. M. L.; Gilliat, V.; Kuhn, L. A.; Saikali, E.; Ren, P.;
Hammand, P. S.; Sexton., T.; Childers, S. R. Synthesis of 2â-
Acyl-3â-(substituted naphthyl)-8-azabicyclo[3.2.1]octanes and
Their Binding Affinities at Dopamine and Serotonin Transport
Sites. J . Med. Chem. 2001, 44, 1509-1515.
(23) Meltzer, P. C.; Wang, B.; Chen, Z.; Blundell, P.; J ayaraman, M.;
Gonzalez, M. D.; George, C.; Madras, B. K. Synthesis of 6- and
7-Hydroxy-8-bicyclo[3.2.1]octanes and Their Binding Affinity for
the Dopamine and Serotonin Transporters. J . Med. Chem. 2001,
44, 2619-2635.
(24) Xu, L.; Trudell, M. L. Stereoselective Synthesis of 2â-Car-
bomethoxy-3â-phenyltropane Derivatives. Enhanced Stereose-
lectivity Observed for the Conjugate Addition Reaction of
Phenylmagnesium Bromide Derivatives with Anhydroecgonine
Methyl Ester in Dichloromethane. J . Heterocycl. Chem. 1996,
33, 2037-2039.
(25) SYBYL, version 6.0; Tripos Associates, Inc.: St. Louis, MO, 1992.
(26) Izenwasser, S.; Rosenberger, J . G.; Cox, B. M. Inhibition of [3H]-
Dopamine and [3H]Serotonin Uptake by Cocaine: Comparison
between Chopped Tissue Slices and Synaptosomes. Life Sci.
1992, 50, 541.
(13) Lomenzo, S. A.; Izenwasser, S.; Katz, J . L.; Terry, P. D.; Zhu,
N.; Klein, C. L.; Trudell, M. L. Synthesis, Structure, Dopamine
Transporter Affinity and Dopamine Uptake Inhibition of 6-Alkyl-
3-benzyl-2-[(methoxycarbonyl)methyl]tropane Derivatives. J .
Med. Chem. 1997, 40, 4406-4414.
(14) Keverline-Frantz, K. I.; Boja, J . W.; Kuhar, M. J .; Abraham, P.;
Burgess, J . P.; Lewin, A. H.; Carroll, F. I. Synthesis and Ligand
Binding of Tropanes Ring Analogues of Paroxetine. J . Med.
Chem. 1998, 41, 247-257.
(27) Izenwasser, S.; Rosenberger, J . G.; Cox, B. M. The Cocaine
Analog WIN 35,428 Binds to Two Sites in Fresh Rat Caudate-
Putamen: Significance of Assay Procedures. Life Sci. 1993, 52,
PL 141.
(28) Madras, B. K.; Fahey, M. A.; Bergman, J .; Canfield, D. R.;
Spealman, R. D. Effects of Cocaine and Related Drugs in
Nonhuman Primates. I. [3H]Cocaine Binding Sites in Caudate-
Putamen. J . Pharmacol. Exp. Ther. 1989, 251, 131-141.
(29) ChemOffice, version 4.5; Cambridgesoft Corporation, Cambridge,
MA 1997. Ghose, A. K.; Crippe, G. M. Atomic Physicochemical
Parameters for Three-Dimensional-Structure-Directed Quanti-
tative Structure-Activity Relationships. 2. Modeling Dispersive
and Hydrophobic Interactions. J . Chem. Inf. Comput. Sci. 1987,
27, 21-35.
(30) Izenwasser, S.; Terry, P.; Heller, B.; Witkin, J . M.; Katz, J . L.
Differential Relationship among Dopamine Transporter Affini-
ties and Stimulant Potencies of Various Uptake Inhibitors. Eur.
J . Pharmacol. 1994, 263, 277-283.
(15) Prakash, K. R. C.; Araldi, G. L.; Smith, M. P.; Zhang, M.;
J ohnson, K. M.; Kozikowski, A. P. Synthesis and Biological
Activity of New 6- and 7-Substituted 2â-Butyl-3-phenyltropanes
as Ligands for the Dopamine Receptor. Med. Chem. Res. 1998,
8, 43-58.
(16) J iang, C.; Chang, A.-C.; Abraham, P.; Kuhar, M. J .; Carroll, F.
I. Synthesis and Transporter Binding of (R)-2â,3â- and (R)-
2R,3R-Diaryltropanes. Bioorg. Med. Chem. Lett. 1998, 8, 3689-
3692.
(17) Kozikowski, A. P.; Araldi, G. L.; Prakash, K. R. C.; Zhang, M.;
J ohnson, K. M. Synthesis and Biological Properties of New 2â-
Alkyl and 2â-Aryl-3-(substituted phenyl)tropane derivative.
Stereochemical Effect of C-3 on Affinity and Selectivity for
Neuronal Dopamine and Serotonin Transporters. J . Med. Chem.
1998, 41, 4973-4982.
(31) Munson, P. J .; Rodbard, D. LIGAND: A Versatile Approach for
Characterization of Ligand-Binding Systems. Anal. Biochem.
1980, 107, 220.
(32) Snedecor, G. W., Cochran, W. G. Statistical Methods, 6th ed.;
Iowa State University Press: Ames, IA, 1967; pp 135-171.
(18) Meltzer, P. C.; Blundell, P.; Yong, Y. F.; Chen, Z.; George, C.;
Gonzalez, M. D.; Madras, B. K. 2-Carbomethoxy-3-aryl-8-bicyclo-
[3.2.1]octanes: Potent Non-nitrogen Inhibitors of Monoamine
Transporters. J . Med. Chem. 2000, 43, 2982-2991.
(19) Prakash, K. R. C.; Trzcinska, M.; J ohnson, K. M.; Kozikowski,
A. P. An Enantioselective Synthesis and Biobehavioral Evalu-
J M010453U