Novel 3-aminomethyl- and 4-aminopiperidine analogues of 1-[2- (diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazines: Synthesis and evaluation as dopamine transporter ligands

Article abstract of DOI:10.1021/jm9902178

We have undertaken a program to develop cocaine antagonists based on the premise that such compounds should block cocaine binding but permit reuptake of dopamine at the dopamine transporter (DAT). To evaluate the structural features of potential cocaine a

Full text of DOI:10.1021/jm9902178

J . Med. Chem. 2000, 43, 205-213
205
Novel 3-Am in om eth yl- a n d 4-Am in op ip er id in e An a logu es of
1-[2-(Dip h en ylm eth oxy)eth yl]-4-(3-p h en ylp r op yl)p ip er a zin es: Syn th esis a n d
Eva lu a tion a s Dop a m in e Tr a n sp or ter Liga n d s
Sung-Woon Choi,† David R. Elmaleh,† Robert N. Hanson,‡ and Alan J . Fischman*,†
Division of Nuclear Medicine of the Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts 02114,
and Department of Pharmaceutical Sciences, Bouve College of Pharmacy and Health Sciences, Northeastern University,
Boston, Massachusetts 02115
Received April 29, 1999
We have undertaken a program to develop cocaine antagonists based on the premise that such
compounds should block cocaine binding but permit reuptake of dopamine at the dopamine
transporter (DAT). To evaluate the structural features of potential cocaine antagonists,
3-aminomethylpiperidine and 4-aminopiperidine moieties were incorporated at the central
bridge region (piperazine ring) of GBR 12935. The compounds were assayed as inhibitors of
[
125I]RTI-55 binding at the DAT and monoamine transport. The results indicated that most of
the new compounds preferentially inhibited norepinephrine reuptake by its transporter (NET)
but in some cases retained binding selectivity for the DAT. In general, the binding selectivity
and potency of [3H]NE reuptake inhibition were very sensitive to modifications of the central
bridge diamine moiety (position of two basic nitrogen atoms). Compound 6 exhibited the highest
ratio (14-fold) of DA reuptake inhibition to RTI-55 binding inhibition at the DAT; however, in
an in vitro assay of cocaine antagonism, this compound failed to reduce inhibition of [3H]DA
uptake by cocaine. These results demonstrated that separation of biological activities into the
binding and reuptake inhibition can be achieved by alterations in the internal diamine
component of GBR 12935, but additional modifications are necessary before these agents
constitute lead compounds for development as cocaine antagonists.
In tr od u ction
in vivo at DAT sites in the CNS.14-18 Unfortunately,
these compounds are highly lipophilic and bind to
nonspecific “piperazine acceptor” sites.19,20 Other stud-
ies, focused on analogues that incorporate modifications
of the central diamine and aralkyl moieties,21-23 estab-
lished that a two-carbon distance appeared to be optimal
for DAT activity. Although these studies made some
correlations between ligand structure and inhibition of
DAT binding, the key question remained unanswered,
i.e., Is it possible to dissociate binding and reuptake
inhibition by modifying the chemical structure of GBR
compounds? Separation of these two activities is an
important issue because the ability to inhibit cocaine
binding without significantly reducing dopamine re-
uptake may constitute the basis for a functional cocaine
antagonist.24
Because of its powerful reinforcing properties and
stimulant effect, cocaine is one of the most widely
abused drugs. The consequences of cocaine abuse have
had profound negative effects on public health and
safety.1-5 The reinforcing properties and other behav-
ioral effects of cocaine have been attributed to its action
on dopaminergic neurons (dopamine hypothesis).1,6
However, the actions of cocaine are believed to include
the blockade of reuptake of a variety of neurotransmit-
ters, including dopamine (DA), serotonin (5-HT), and
norepinephrine (NE).1-5 As a result, many research
efforts have been directed to the synthesis of analogues
and the development of structure-activity relationships
(SAR) focused on elucidating the nature of cocaine
binding at these sites.7-10 Although it has been shown
that the stimulant activities of cocaine and cocaine-like
compounds are directly related to their in vitro binding
affinity for the dopamine transporter (DAT),11 it has
been suggested that cocaine antagonists may be struc-
turally dissimilar to cocaine.11
In an effort to develop GBR analogues which have a
dissociation of binding and reuptake inhibition activi-
ties, we focused on probing the role of the central
diamine unit.25,26 Our primary objective was to identify
structural features which confer a strong binding activ-
ity at the DAT but have a weaker effect on DA reuptake.
As a first step in evaluating the positional/conforma-
tional requirements for each nitrogen atom in the ring
(positional flexibility for activity and selectivity), piper-
idine-based GBR analogues with flexible amine side
chains at the 3-position were selected (Figure 1). By
making substitutions with head ((bisphenylmethoxy)-
ethyl) and tail (phenylpropyl) groups at both sides of
the central ring, the resulting analogues were expected
to have limited and selective flexibility at the N1/N2
The development of cocaine antagonists12 has been
of considerable interest because of their potential use
in the treatment of cocaine addiction. Among these, the
GBR-type compounds13 have been extensively studied
because of their unique binding properties in vitro and
* Requests for reprints should be addressed to Alan J . Fischman,
M.D., Ph.D, Division of Nuclear Medicine, Massachusetts General
Hospital, Boston, MA 02114. Tel: (617) 726-8353. Fax: (617) 726-1650.
E-mail: fischman@petw6.mgh.harvard.edu.
† Massachusetts General Hospital.
‡ Northeastern University.
10.1021/jm9902178 CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/28/1999

206 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Choi et al.
F igu r e 1. Design strategy for the GBR analogues prepared in this study.
nitrogen atoms. The 4-aminopiperidine ring was chosen
as a diamine substitution of the piperazine ring in the
central portion of the GBR structure. Although the
amine at the exocyclic chain of the piperidine ring is
more conformationally restricted than that of 3-ami-
nomethylpiperidine, it is more flexible than the pipera-
zine ring. The spacial orientation of each nitrogen atom
of the piperidine analogues containing a fixed three-
carbon unit (distance from N1 to N2) is to be evaluated.
The extended form will provide a 3-aminomethylpiper-
idine ring core whereas the side-chain-rotated form will
result in a more conformationally restrained 4-aminopi-
peridine ring structure. Two series of semi-rigid piperi-
dine ring analogues with exocyclic amines, which are
expected to have low binding activity at the “piperazine
acceptor” site,27 were synthesized and evaluated for in
vitro binding and reuptake inhibition at dopamine,
serotonin, and norepinephrine transporters (DAT, SERT,
and NET).
lithium aluminum hydride to give primary amine
intermediates 3, 9, and 10 in good yields (84%, 80%,
and 79%, respectively). Monoalkylation of these primary
amines 3, 9, and 10 with appropriate head or tail
alkylating agents produced the target GBR analogues
4, 5, 11, and 12. Synthesis of N-methylated piperidine
analogue 6 was achieved using formaldehyde sodium
cyanoborohydride (80% yield).28
1
The H NMR spectrum of N-alkylated nipectotamide
2 in the synthesis of 3- aminomethylpiperidine GBR-
type analogues showed some unexpected chemical shift
and splitting patterns. Interestingly, these patterns
resulted from the internal hydrogen bonding between
the amide proton and the basic tertiary amine. This
hydrogen bonding appeared to stabilize structures that
are relatively unfavorable on steric and energetic
1
grounds. H NMR spectroscopy aided in assigning the
ring structure in these N-alkylated nipecotamides. The
key feature was a significant difference in the absorption
of two protons in the -CONH2 group. Intramolecular
hydrogen bonding led to a specific downfield shift and
broadening of the peak corresponding to one of the
amide protons. This effect was also reflected by differ-
ence in chemical shift, peak shapes, and coupling
constants for the Hc proton adjacent to Ha/Hb and Hd/
He proton pairs. In the absence of internal hydrogen
bonding, axial and equatorial configurations are pre-
ferred for the Hc proton and the amide functionality,
respectively. This is due to steric hindrance between the
amide and ring moieties. With the Hc proton in an axial
position, coupling to the Ha proton is expected to
produce a triplet with 2 large J values, which arise from
Ch em istr y
The synthetic methods for preparation of 3-amino-
methylpiperidine ring analogues of GBR 12935 are
illustrated in Scheme 1. Nipecotamide 1 was used as
starting material in the synthesis of compounds 4-6,
11, and 12. The N-alkylated amide intermediates 2, 7,
and 8 were prepared in good yields (88%, 79%, and 85%,
respectively) by alkylation of the nipecotamide with
head or tail alkylating agents such as (bisphenyl-
methoxy)ethyl bromide, 3-phenyl-1-bromopropane, or
4-fluorophenethyl bromide in the presence of K2CO3.
The resulting amides 2, 7, and 8 were reduced with

Synthesis of Dopamine Transporter Ligands
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 207
Sch em e 1a
a
(a) (Ph)2CHOCH2CH2Br, K2CO3, DMF; (b) LAH, THF; (c) HCHO, NaCNBH3; (d) 1, K2CO3, DMF.
ing materials in the synthesis of 15-17, 20, and 22.
N-Alkylation of 4-piperidone monohydrate HCl with
2-bromoethyl diphenylmethyl ether or 1-bromo-3-phen-
ylpropane proceeded in good yield (92%, 88%) to give
ketone intermediates 14 and 18, respectively. The
alkylated ketone intermediates 14 and 18 underwent
reductive amination with the appropriate primary
amine.29 Reductive aminations of ketone intermediate
14 were performed using a 4-fold excess of the amines,
3-phenyl-1-propylamine, or 4-fluorophenethylamine, and
gave products 15 and 16 in 38% and 25% yields,
respectively. Introduction of the methyl group into the
secondary amine 16 using a formaldehyde sodium
cyanoborohydride system gave tertiary amine 17 in
moderate yield (54%).25 As an alternate route to improve
yields, an indirect pathway was developed using reduc-
tive amination with methyl amine followed by alkylation
with tail-aralkyl halides. Reductive amination of ketone
F igu r e 2. Proposed structure for intermediate 5 illustrating
internal hydrogen bonding.
geminal and ax-ax couplings, and a doublet of doublets
with 1 large and 1 small J value from ax-ax and ax-eq
1
couplings. In contrast, the measured H NMR spectrum
of the Ha proton revealed a doublet of doublets with 1
large and 1 small J value from geminal and ax-eq
couplings. These findings are consistent with the inter-
nal hydrogen bonding depicted in Figure 2.
Preparation of the 4-aminopiperidine ring analogues
of GBR 12935, Scheme 2, used 4-piperidone monohy-
drate HCl and N-benzyl-4-aminopiperidine as the start-

208 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Choi et al.
Sch em e 2a
a
(a) (Ph)2CHOCH2CH2Br, K2CO3, DMF; (b) NaCNBH3, ethanolic HCl, molecular sieve; (c) HCHO, NaCNBH3; (d) PhCH2CH2CH2Br,
K2CO3, DMF; (e) CH3NH, NaCNBH3.
intermediate 18 proceeded smoothly in good yield (80%),
and the subsequent N-alkylation with (bisphenylmeth-
oxy)ethyl bromide yielded target compound 20 in a high
yield (93%). Monoalkylation of the N-benzyl-4-amino-
piperidine (5 equiv) with (bisphenylmethoxy)ethyl bro-
mide yielded GBR analogue 22 in moderate yield (70%).
porters (hSERT), or human norepinephrine transporters
(hNET). All compounds were tested for ability to dis-
place [125I]RTI and inhibit uptake of [3H]DA, [3H]5-HT,
and [3H]NE. The results of the in vitro biological assays
are shown in Tables 1 and 2. In the 3-aminomethylpi-
peridine series, compounds 4, 5, and 6, in which the
head group (bisphenylmethoxy ethyl moiety) is linked
directly to the piperidine ring, demonstrated high
affinity at the DAT (Ki ) 0.070-0.090 µM) and modest
to significant affinity for the other transporters as well
(SERT Ki ) 0.300-0.800 µM, NET Ki ) 0.040-0.190
Biologica l Resu lts a n d Discu ssion
Biological evaluation of these compounds was per-
formed with HEK 293 cells expressing cDNA for human
dopamine transporters (hDAT), human serotonin trans-

Synthesis of Dopamine Transporter Ligands
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 209
Ta ble 1. Binding Affinities and Selectivities of GBR Analogues at the DA (DAT), 5-HT (SERT), and NE (NET) Transporters
Measured by Inhibition of [125I]RTI-55 Binding (Ki ( SD, µM)a
binding (Ki ( SD, µM)
selectivity ratio
compound
DAT
SERT
NET
SERT/DAT
NET/DAT
4
5
6
11
12
15
16
17
0.087 ( 0.009
0.070 ( 0.027
0.090 ( 0.013
0.100 ( 0.030
0.018 ( 0.002
0.110 ( 0.014
0.160 ( 0.044
0.340 ( 0.140
0.030 ( 0.007
0.083 ( 0.028
0.073 ( 0.039
0.760 ( 0.093
0.960 ( 0.190
0.370 ( 0.033
0.89 ( 0.37
0.32 ( 0.10
0.53 ( 0.22
0.35 ( 0.16
0.21 ( 0.055
0.220 ( 0.110
0.055 ( 0.011
0.087 ( 0.023
1.40 ( 0.27
2.16 ( 0.33
2.09 ( 0.76
0.390 ( 0.088
0.490 ( 0.067
0.400 ( 0.097
0.190 ( 0.098
0.041 ( 0.015
0.110 ( 0.029
0.150 ( 0.043
0.023 ( 0.003
0.079 ( 0.038
0.120 ( 0.017
0.042 ( 0.011
0.029 ( 0.009
0.180 ( 0.081
0.630 ( 0.059
1.77 ( 0.37
10.2
4.5
5.9
3.3
11.9
2.0
0.4
0.3
47.1
26
28.6
2.1
0.6
1.3
1.5
1.3
0.7
0.8
0.1
1.0
2.1
8.6
20
22
GBR 12935
cocaine
cocaineb
cocainec
1.58 ( 0.26
1.60 ( 0.23
a
The Ki values for the test ligands were determined with assays described in the Methods section. Results are mean ( SD for three
independent experiments assayed in triplicate. Cocaine as reference for 15, 17, 20, and 22. c Cocaine as reference for GBR 12935.
b
Ta ble 2. DA, 5-HT, and NE Reuptake Inhibition and Ratios of Reuptake to Binding of GBR Analogues at DA and 5-HT Transporters
(IC50 ( SD, µM)a
discrimination ratio
[3H]DA
reuptake/
DAT binding
[3H]5-HT
reuptake/
SERT binding
reuptake inhibition (IC50 ( SD, µM)
compound
[3H]DA
[3H]5-HT
[3H]NE
4
5
6
11
12
15
16
17
0.364 ( 0.069
0.105 ( 0.018
1.26 ( 0.34
1.83 ( 0.58
0.190 ( 0.003
0.048 ( 0.012
0.350 ( 0.079
0.200 ( 0.060
0.097 ( 0.030
0.170 ( 0.055
0.230 ( 0.072
0.180 ( 0.069
0.200 ( 0.037
0.087 ( 0.010
0.165 ( 0.017
0.230 ( 0.041
0.220 ( 0.060
4.2
1.5
14.0
4.8
8.1
2.9
2.5
0.6
11.1
3.2
0.25
2.1
2.5
4.0
3.3
3.8
2.1
7.1
3.6
1.4
0.9
1.8
0.770 ( 0.190
2.13 ( 0.47
0.500 ( 0.059
0.150 ( 0.029
0.320 ( 0.130
0.390 ( 0.009
0.210 ( 0.021
0.330 ( 0.120
0.270 ( 0.070
0.018 ( 0.003
0.190 ( 0.021
0.220 ( 0.029
1.13 ( 0.20
0.820 ( 0.160
0.470 ( 0.190
0.390 ( 0.012
0.310 ( 0.120
1.91 ( 0.71
20
22
1.86 ( 0.59
3.71 ( 1.43
0.340 ( 0.078
0.220 ( 0.036
GBR 12935
cocaine
cocaineb
a
IC50 values for the test ligands were determined with assays described in Methods. Results are mean ( SD of three independent
b
experiments assayed in triplicate. Cocaine as reference for 9.
µM). This phenomenon may be related to control of
ligand recognition and neurotransmitter reuptake by
different structural regions of transporter molecules.
Compound 5, which possesses a three-carbon spacer
between the N2 nitrogen and tail benzene ring, had
approximately 2-fold stronger binding to the NET
compared to the DAT. In contrast, its regioisomer,
compound 12, retained selectivity for the DAT as well
as potent reuptake inhibition at the NET. The piperi-
dine ring GBR derivatives, 3-aminomethylpiperidines
5 and 12, represented an interesting pair of regioiso-
mers. These compounds demonstrated a 4-fold differ-
ence in binding affinity and a 2-fold difference in [3H]DA
reuptake inhibition at the DAT. The other regioisomer
pair 4 and 11 showed little selectivity among the three
transporters. Compound 12, in which the piperidine ring
was directly linked to the tail-aralkyl group, possessed
the strongest binding affinity at the DAT (Ki ) 0.018
µM) and better binding selectivity at the DAT compared
to its regioisomer 5. Replacement of the p-fluorophen-
ethyl moiety with a phenylpropyl moiety improved
slightly the binding activity at the DAT, but signifi-
cantly enhanced activity at the NET site (compound 5).
Methylation of N2 (compounds 4 to 6) did not improve
binding affinity to the DAT but decreased potency for
inhibition of [3H]DA reuptake (4, IC50 ) 0.36 µM and
6, IC50 ) 1.26 µM). Of particular note, one member of
this series, compound 6, exhibited the highest discrimi-
nation ratio (14-fold) of DA uptake inhibition to binding
inhibition, reduced selectivity for the NET, and moder-
ate affinity for the DAT.
The conformationally more rigid 4-aminopiperidine
GBR analogue 16 demonstrated 2- and 3-fold stronger
binding at the SERT compared to the NET and DAT,
respectively, and mild selectivity for inhibition of [3H]-
NE reuptake (2-fold vs [3H]DA and [3H]5-HT) was
maintained. This higher binding affinity at the SERT
was shifted to the NET and less so to the DAT when
the phenylpropyl group was replaced with a 4-fluo-
rophenyl moiety (16 to 15). When the N2 secondary
amine of analogue 16 was methylated, binding affinity
at the NET site was improved ∼3-fold; however, DAT
and SERT binding were reduced. Compound 20, the
regioisomer of 17, demonstrated strong binding affinity
at the DAT and NET but very weak binding at the
SERT (16-fold lower). Interestingly, this regioisomer
pair showed an improved discrimination ratio for re-
uptake to binding at the DAT (0.6 to 11.1). This was
also observed in the 3-aminomethylpiperidine series (5
vs 12). Replacement of phenylpropyl group by a benzyl
moiety in compound 22 resulted in retention of trans-
porter binding which supports findings by Dutta et al.30

210 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Choi et al.
However, selectivity for inhibition of [3H]NE reuptake
and selective binding at the DAT (2-fold vs NET site)
were maintained.
Atlantic Microlabs, Atlanta, GA, for carbon, hydrogen, and
nitrogen and were within (0.4% theoretical values unless
noted otherwise. 1H and 13C NMR spectra were determined
on a Varian XL-300 spectrometer. Chemical shifts are ex-
pressed in parts per million (ppm) on the δ scale relative to a
TMS internal reference standard. In general, CDCl3 was used
for the free bases and DMSO-d6 was used for salts. Coupling
constants (J values) were given in hertz. Thin layer chroma-
tography (TLC) was performed on 250 µm thickness silica gel
plates or alumina precoated plates (Whatman, AL SIL G/UV
or J . T. Baker, Baker-flex, SILICA GEL IB-F) containing
fluorescent indicator (2 × 8 cm) developed with appropriate
solvent mixtures. Column separations were performed on silica
gel (Baker, 40 µm flash chromatography). The collected frac-
tions were analyzed using TLC and visualized by ninhydrin
(0.5 g in 100 mL of methanol) for primary and secondary
amine(s), ultraviolet light, or iodine vapor. For salt formation,
free bases were dried and dissolved in ethyl acetate or/and
diethyl ether and filtered. Oxalic acid solution in same solvent
or HCl in ether was added to the filtrates until no further
turbidity or precipitation occurred. The resultant solids were
collected by filtration and recrystallized from appropriate
solvents. Unfilterable gels were evaporated to dryness and
recrystallized.
Syn th etic Ch em ica l Meth od s. N-[2-(Bisp h en ylm eth -
oxy)eth yl]-3-p ip er id in eca r boxa m id e (2). A suspension of
nipecotamide (1.24 g, 9.67 mmol), 1-(bisphenylmethoxy)-2-
bromoethane (2.56 g, 8.79 mmol), and K2CO3 powder (2.43 g,
17.6 mmol) in anhydrous DMF (20 mL) was stirred at ambient
temperature for 48 h. The reaction mixture was diluted with
200 mL of ethyl acetate and washed with 60 mL of saturated
NaCl solution (×3), dried over MgSO4, and evaporated. The
residue was purified by silica gel column chromatography
using CHCl3:MeOH:Et3N (96:4:0.4) as eluent, to give pure
product 2 as a colorless oil (88%) that gradually solidified on
cooling. Mp: (HCl salt): 223-224 °C; (free base): 94-97 °C.
1H NMR (CDCl3): δ 1.49-1.61 (2H, m), 1.68-1.81 (1H, m),
1.95 (1H, br d, J ) 9.6), 2.15 (1H, br t, J ) 10.7), 2.28 (1H, dd,
J ) 2.6, 11.7), 2.47 (1H, m), 2.54 (1H, ddd, J ) 4.4, 6.0, 13.3),
2.67 (1H, ddd, J ) 4.6, 6.7, 13.3), 2.80 (1H, br d, J ) 10.5),
3.02 (1 H, br d, J ) 11.0), 3.50-3.62 (2H, m), 5.31 (1H, br s),
5.34 (1H, s), 7.20-7.40 (10H, m), 8.26 (1H, br s). 13C NMR
(CDCl3): δ 22.6, 26.8, 41.4, 54.0, 54.5, 57.5, 65.7, 83.9, 84.0,
126.8, 127.5, 128.4, 142.0, 177.9.
N-(4-F lu or op h en yleth y)-3-p ip er id in eca r boxa m id e (7)
was prepared as described for 2 to give the title compound 7
(2.13 g, 8.5 mmol, 85%) as a sligthly yellow solid. Mp (free
base): 91-93 °C. 1H NMR (CDCl3): δ 1.55-1.79 (3H, m),
1.83-1.90 (1 H, m), 2.19 (1 H, br t, J ) 9.5), 2.39 (1H, br d,
J ) 11.1), 2.48 (1H, apparent br quintet, J ) 4.2), 2.52-2.83
(6H, m), 5.38 (1H, br s), 6.94-7.00 (2H, m), 7.11-7.27 (2H,
m), 7.53 (1 H, br s). 13C NMR (CDCl3): δ 22.6, 26.9, 32.3, 41.7,
53.4, 55.2, 59.9, 115.0, 115.3, 129.9, 130.0, 135.7,135.7, 177.6.
N-(3-P h en ylp r op yl)-3-p ip er id in eca r boxa m id e (8) was
prepared as described for 2 to give the title compound 8 (1.94
g, 7.9 mmol, 79%) as a slightly yellow solid. Mp (free base):
88-91 °C; (HCl salt): hygroscopic. 1H NMR (CDCl3): δ 1.54-
1.67 (2H, m), 1.69-1.89 (4H, m), 2.17 (1H, br t, J ) 10.4), 2.35
(3H, t overlapping with br d peak, J ) 7.5), 2.48 (1H, apparent
br quintet, J ) 4.2), 2.61 (3H, t, overlapping with br d peak,
J ) 7.6), 2.80 (1H, br d, J ) 8.3), 6.04 (1H, br s), 7.15-7.21
(3H, m), 7.26-7.30 (2H, m), 7.80 (1H, br s). 13C NMR (CDCl3):
δ 22.8, 26.9, 28.4, 33.7, 41.80, 53.7, 55.3, 58.2, 125.80, 128.2,
128.3, 141.8, 178.0.
Although the contribution of positional flexibility of
each amino group to binding could not be determined,
several interesting structural features responsible for
biological activities were identified. The piperidine ring
analogues with a branched basic amine side chain
demonstrated the importance of the central bridge ring
for modulating the relationship between binding affinity
and potency of reuptake inhibition at all three trans-
porter sites. A propylene moiety between two basic
nitrogen atoms appears to be the optimal linkage for
inhibition of [3H]NE reuptake. However, binding prop-
erties were altered by the position of each nitrogen atom
in the ring structure.
Based upon its apparent selectivity for inhibition of
DA reuptake vs DAT binding (IC50/Ki ) 14.0), compound
6 was selected for further evaluation using an in vitro
cocaine antagonism assay. The ability of this compound
to reduce the effectiveness of cocaine to inhibit the
reuptake of dopamine was determined at a concentra-
tion ranging from 0.010-0.100 µM. Unfortunately, none
of the concentrations of drug reduced the inhibition of
[3H]DA uptake by cocaine. These data suggest that
uptake-to-binding ratios derived from cells transfected
with recombinant transporters must be interpreted with
caution when used to guide the development of possible
cocaine antagonists.14,31 Interactions of ligands at bind-
ing site(s) in the recombinant transporters may be
different from those present in rat synaptosomes which
may also differ from human transporters in vivo.
Con clu sion s
A series of GBR 12935 analogues were synthesized
to investigate substitution effects with bioisosteric di-
amine units on cocaine binding and neurotransmitter
reuptake. The results demonstrated that substantial
modifications can be made without attenuating effects
at the DAT. Even though a comprehensive model for
explaining the contribution of flexibility at each nitrogen
atom was not established, a degree of separation
between cocaine binding and dopamine reuptake inhibi-
tion was achieved. Although compound 6 demonstrated
a high ratio (14-fold) of reuptake inhibition to binding,
this compound did not demonstrate cocaine antagonist
effects in vitro. Nevertheless, our results may provide
directions for the design of newer GBR analogues which
may have stronger binding at cocaine recognition sites
but much weaker inhibition of DA reuptake.
Exp er im en ta l Section
Gen er a l Ch em ica l Meth od s. All chemicals and solvents
were obtained from commercial suppliers and were used
without further purification. In general, all reactions were
performed under atmospheric conditions and at ambient
temperature unless otherwise noted. High boiling point sol-
vents (DMF, DMSO, etc.) that are water soluble were removed
from the reaction mixture by first pouring into ethyl acetate
and/or diethyl ether followed by washing with a saturated
NaCl solution. All organic extracts were dried over MgSO4 or
Na2SO4 except where otherwise noted. Volatile solvents were
removed by rotary evaporation under reduced pressure. Melt-
ing points were determined on a Thomas-Hoover melting point
apparatus or an electrothermal melting point apparatus and
were uncorrected. Elemental analyses were performed by
3-(Am in om eth yl)-1-[2-(bisp h en ylm eth oxy)eth yl]p ip er -
id in e (3). A solution of the amide 2, N-[2-(bisphenylmethoxy)-
ethyl]-3-piperidinecarboxamide (2.53 g, 7,48 mmol), in 20 mL
of THF was added dropwise to a 1.0 M solution of LiAlH4 (30
mL, 30 mmol) in THF, and the mixture stirred for 24 h at room
temperature. After careful sequential addition of water, NaOH
solution, and again water, the mixture was filtered and the
resultant filter cake was re-extracted with EtOAc (60 mL ×
2). The combined organic filtrate and extracts were dried,
filtered, and evaporated to give an oil. This crude product was

Synthesis of Dopamine Transporter Ligands
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 211
purified by column chromatography with silica gel using
CHCl3:MeOH:NH4OH (9:1:0.1) to give 2.05 g (6.3 mmol, 84%)
of pure clear oil. Mp (HCl salt): hygroscopic; (oxalate salt):
6.1), 2.94 (1H, br d, J ) 10.4), 3.57 (2H, t, J ) 5.2), 5.36 (1H,
s), 7.17-7.34 (15H, m). 13C NMR (CDCl3): δ 25.2, 28.6, 29.3,
33.9, 36.5, 49.5, 54.0, 54.4, 58.7, 58.8, 68.3, 83.8, 125.7, 126.9,
127.4, 128.2, 128.4, 142.2, 142.2. Anal. (C34H42N2O9‚1/2H2O) C,
H, N.
1
hygroscopic. H NMR (CDCl3): δ 0.89 (1 H, qd, J ) 4.1, 11.5),
1.53-1.81 (5H, m), 2.02 (1H, td, J ) 3.1, 11.1), 2.55 (2H, d,
J ) 6.4), 2.67 (4H, t overlapping with br s peak, J ) 6.2), 2.84
(1 H, br d, J ) 11.3), 2.93 (1 H, br d, J ) 9.3), 3.61 (2H, t, J )
6.1), 5.38 (1H, s), 7.20-7.35 (10H, m). 13C NMR (CDCl3): δ
24.9, 28.3, 38.8, 45.9, 54.6, 58.3, 58.4, 66.9, 83.8, 127.0, 127.4,
128.3, 142.1.
1-[2-(Bisp h en ylm et h oxy)et h yl]-3-[[N-m et h yl-N-[2-(4-
flu or op h en yl)eth yl]a m in o]m eth yl]p ip er id in e (6). To a
stirred solution of 1-[2-(bisphenylmethoxy)ethyl]-3-[[N-[2-(4-
fluorophenyl)ethyl]amino]methyl]piperidine (0.45 g, 1 mmol)
4 and 37% aqueous formaldehyde (0.4 mL, 5 mmol) in 6 mL
of isopropanol was added sodium cyanoborohydride (0.126 g,
2 mmol). The resultant turbid reaction mixture was stirred
for 24 h at room temperature. The volatiles were removed by
rotary evaporation, 100 mL of ethyl acetate was added, and
the organic layer was washed with water (30 mL × 3). The
organic layer was dried over MgSO4, filtered, and evaporated
to dryness to give the crude product which was purified by
column chromatography using silica gel and the appropriate
eluent to give pure product as an oil. Mp (oxalate salt): 149-
151 °C. 1H NMR (CDCl3): δ 0.85 (1H, qd, J ) 4.3, 11.8), 1.55-
1.83 (5H, m), 2.01 (1H, td, J ) 2.6, 11.0), 2.19 (2H, d, J ) 6.7),
2.25 (3H, s), 2.45-2.73 (6H, m), 2.92 (1 H, br d, J ) 11.3),
2.98 (1 H, br d, J ) 11.6), 3.62 (2H, t, J ) 6.1), 5.39 (1H, s),
6.92-6.98 (2H, m), 7.10-7.16 (2H, m), 7.21-7.37 (10H, m).
13C NMR (CDCl3): δ 25.0, 29.3, 32.7, 34.1, 42.8, 54.7, 58.3,
59.3, 59.9, 62.1, 66.7, 84.0, 114.8, 115.1, 127.0, 127.0, 127.4,
3-(Am in om et h yl)-1-[2-(4-flu or op h en yl)et h yl]p ip er i-
d in e (9) was prepared as described for 3 to give the title
compound 9 (1.20 g, 5.1 mmol, 79%) as a slightly yellow oil.
Mp (oxalate salt): 60-70 °C broad range. 1H NMR (CDCl3):
δ 0.92 (1H, qd, J ) 4.1, 11.7), 1.39 (2H, br s), 1.54-1.81 (5H,
m), 1.97 (1H, td, J ) 3.0, 11.3), 2.52-2.60 (4H, m), 2.76-2.81
(2H, m), 2.92 (1H, br d, J ) 11.1), 3.02 (1H, br d, J ) 8.8),
6.93-6.99 (2H, m), 7.13-7.17 (2H, m). 13C NMR (CDCl3): δ
25.1, 28.6, 32.7, 39.7, 46.4, 54.4, 58.1, 61.1, 114.9, 115.2, 129.9,
130.0, 136.1, 136.1.
3-(Am in om eth yl)-1-(3-ph en ylpr opyl)piper idin e (10) was
prepared as described for 3 to give the title compound 10 (1.39
g, 6.0 mmol, 80%) as a slightly yellow oil. Mp (bis-oxalate
salt): 43-48 °C. 1H NMR (CDCl3): δ 0.88 (1H, qd, J ) 4.3,
11.7), 1.50-1.90 (10H, m), 2.35 (2H, t, J ) 7.8), 2.55 (2H, d,
J ) 5.5), 2.61 (2H, t, J ) 7.7), 2.84 (1H, br d, J ) 11.1), 2.92
(1H, br d, J ) 7.2), 7.14-7.19 (3H, m), 7.24-7.29 (2H, m). 13
C
128.2, 128.3, 130.0, 130.1, 136.1, 136.2, 142.1. Anal. (C34H41
N2O9F) C, H, N.
-
NMR (CDCl3): δ 25.0, 28.5, 28.6, 33.7, 39.6, 46.3, 54.3, 58.1,
58.5, 125.6, 128.1, 128.2, 142.1.
1-[2-(Bisph en ylm eth oxy)eth yl]piper idin e-4-on e (14) was
prepared as described for 2 to give the title compound 14 (1.20
g, 5.1 mmol, 79%) as a pale yellow oil (2.85 g, 9.2 mmol, 92%).
Mp (oxalate salt): 135-137 °C. 1H NMR (CDCl3): δ 2.43 (4H,
t, J ) 6.2), 2.78-2.84 (6H, apparent q, J ) 6.7), 3.63 (2H, t,
J ) 5.8), 5.38 (1H, s), 7.21-7.37 (10H, m). 13C NMR (CDCl3):
δ 41.2, 53.4, 56.7, 67.3, 83.9, 84.0, 126.9, 127.4, 128.3, 142.0,
209.0.
1-(3-P h en ylp r op yl)p ip er id in e-4-on e (18) was prepared
as described for 2 to give the title compound 18 (3.82 g, 17.6
mmol, 88%) as a pale yellow oil. Mp (oxalate salt): 149-153
°C. 1H NMR (CDCl3): δ 1.85 (2H, quintet, J ) 7.5), 2.42-2.49
(6H, m), 2.67 (2H, t, J ) 7.6), 2.72 (4H, t, J ) 6.0), 7.16-7.20
(3H, m), 7.26-7.30 (2H, m). 13C NMR (CDCl3): δ 29.0, 33.4,
41.1, 53.9, 56.5, 125.7, 128.2, 128.3, 141.8, 209.0.
1-[2-(Bisp h en ylm eth oxy)eth yl]-3-[[(N-4-flu or op h en yl-
eth yl)a m in o]m eth yl]p ip er id in e (4) was prepared as de-
scribed for 2 to give the title compound 4 (0.33 g, 0.74 mmol,
60%) as a colorless oil. Mp (bis-oxalate salt): 200.5-202.5 °C.
1H NMR (CDCl3): δ 0.83-0.93 (1H, m), 1.48-1.79 (6H, m),
2.00 (1H, td, J ) 2.9, 11.1), 2.47 (2H, d, J ) 5.9), 2.66 (2H, t,
J ) 6.1), 2.70-2.82 (4H, m), 2.85 (1H, br d, J ) 12.2), 2.91
(1H, br d, J ) 7.3), 3.59 (2H, t, J ) 6.2), 5.37 (1H, s), 6.93-
6.99 (2H, m), 7.10-7.15 (2H, m), 7.20-7.36 (10H, m). 13C NMR
(CDCl3): δ 25.1, 29.0, 35.4, 36.4, 51.3, 53.9, 54.7, 58.3, 59.1,
67.0, 83.8, 83.9, 115.0, 115.3, 127.0, 127.1, 127.4, 128.3, 129.9,
130.0, 135.6, 135.7, 142.2. Anal. (C33H39N2O9F) C, H, N.
1-[2-(Bisp h en ylm eth oxy)eth yl]-3-[[(N-3-p h en ylp r op yl)-
a m in o]m eth yl]p ip er id in e (5) was prepared as described for
2 to give the title compound 5 (0.23 g, 0.52 mmol, 61%) as a
colorless oil with the exception that an amine to alkyl halogen
ratio of 3:1 was employed. Mp (bis-oxalate salt): 147-150 °C.
1H NMR (CDCl3): δ 0.90-1.01 (1H, m), 1.53-1.89 (8H, m),
2.08 (1H, td, J ) 2.5, 10.8), 2.50 (2H, d, J ) 6.2), 2.60 (2H, t,
J ) 7.3), 2.61 (2H, t, J ) 7.7), 2.67 (2H, t, J ) 6.1), 2.82 (1 H,
br d, J ) 11.3), 2.91 (1H, br d, J ) 9.3), 3.60 (2H, t, J ) 5.9),
5.37 (1H, s), 7.15-7.36 (15H, m). 13C NMR (CDCl3): δ 24.8,
28.8, 31.1, 33.5, 35.8, 49.4, 53.8, 54.7, 58.3, 58.9, 66.9, 83.9,
125.8, 127.0, 127.4, 128.3, 141.9, 142.2. Anal. (C34H42N2O9‚
1/2H2O) C, H, N.
1-[2-(4-F lu or op h en yl)et h yl]-3-[[N-[2-(b isp h en ylm et h -
oxy)eth yl]a m in o]m eth yl]p ip er id in e (11) was prepared as
described for 2 to give the title compound 11 (0.49 g, 1.1 mmol,
69%) as a colorless oil. Mp (oxalate salt): 209-211 °C, 1H NMR
(CDCl3): δ 0.93 (1H, qd, J ) 3.6, 11.5), 1.59-1.83 (6H, m),
1.97 (1H, td, J ) 3.1, 11.2), 2.48-2.51 (2H, m), 2.53-2.56 (2H,
m), 2.75-2.78 (2H, m), 2.82 (2H, td, J ) 1.7, 5.2), 2.91 (1H, br
d, J ) 11.3), 3.03 (1H, br d, J ) 8.9), 3.59 (2H, t, J ) 5.2), 5.37
(1H,s), 6.92-6.98 (2H, m), 7.11-7.16 (2H, m), 7.20-7.35 (10H,
m). 13C NMR (CDCl3): δ 25.2, 29.2, 32.7, 36.5, 49.5, 53.9, 54.4,
58.6, 61.1, 68.3, 83.8, 83.8, 114.9, 115.1, 126.9, 127.4, 128.3,
129.9, 130.0, 136.1, 136.1, 142.2. Anal. (C33H39N2O9F) C, H,
N.
1-(3-P h en ylp r op yl)-3-[[N-[2-(bisp h en ylm eth oxy)eth yl]-
a m in o]m eth yl]p ip er id in e (12) was prepared as described
for 2 to give the title compound 12 (0.55 g, 1.24 mmol, 70%)
as a colorless oil. Mp (oxalate salt): 187-189 °C. 1H NMR
(CDCl3): δ 0.89 (1H, qd, J ) 4.3, 11.8), 1.55-1.91 (9H, m),
2.35 (2H, t, J ) 7.8), 2.46 (2H, dd, J ) 2.8, 6.4), 2.61 (2H, t,
J ) 7.7), 2.81 (3H, dd, overlapping with br d peak, J ) 4.6,
1-[2-(Bisp h en ylm eth oxy)eth yl]-4-[N-(3-p h en ylp r op yl)-
a m in o]p ip er id in e (16). To a solution of 3-phenyl-1-propyl-
amine (5.40 g, 40 mmol) and 20 g of 3 Å molecular sieves in
100 mL of iPrOH was added 1.0 M HCl-ethanol dropwise to
adjust pH up to 8. This was followed by the addition of 1-[2-
(bisphenylmethoxy)ethyl]-4-piperidone (2.53 g, 8.2 mmol) and
NaBH3CN (0.63 g, 10 mmol). The resulting solution was stirred
for 72 h, diluted with 100 mL of MeOH, filtered, and evapo-
rated to give a crude oil. The crude oil was dissolved in 100
mL of ethyl acetate, washed sequentially with 30 mL of 0.1 N
NaOH (×3) and saturated NaCl, dried, and evaporated to yield
an oil that was purified by column chromatography. The
isolated pure compound 16 (1.34 g, 3.1 mmol, 38%) was treated
with oxalic acid in ethyl acetate or ether to give bis-oxalate
salt which was recrystallized from iPrOH-H2O. Mp (oxalate
1
salt): 227-230 °C, H NMR (CDCl3): δ 1.35 (3H, qd overlap-
ping with br s peak, J ) 3.7, 11.9), 1.76-1.86 (4H, quintet
overlapping with br d peak, J ) 7.4), 2.07 (2H, td, J ) 2.2,
11.6), 2.41 (1 H, tt, J ) 4.1, 10.6), 2.62-2.68 (6H, m), 2.88
(2H, br d, J ) 11.9), 3.58 (2H, t, J ) 6.2), 5.38, (1H, s), 7.17-
7.35 (15H, m). 13C NMR (CDCl3): δ 31.9, 32.8, 33.7, 46.2, 53.0,
54.8, 57.9, 67.1, 83.8, 125.7, 127.0, 127.3, 128.3, 142.3. Anal.
(C33H40N2O9) C, H, N.
1-[2-(Bisph en ylm eth oxy)eth yl]-4-[N-[2-(4-flu or oph en yl)-
eth yl]a m in olp ip er id in e (15) was prepared as described for
16 to give the title compound 15 (0.16 g, 0.37 mmol, 25%) as
a slightly yellow oil. Mp (oxalate): 235-237 °C, 1H NMR
(CDCl3): δ 1.44 (2H, qd, J ) 3.5, 10.4), 1.88 (2H, br d, J )
9.9), 2.21 (2H, br t, J ) 10.9), 2.56 (1H, tt, J ) 3.9, 10.1), 2.72
(2H, t, J ) 5.9), 2.76-2.81 (2H, m), 2.85-2.90 (2H, m), 2.96
(2H, br d, J ) 12.2), 3.60 (2H, t, J ) 5.9), 5.38 (1H, s), 6.94-

212 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Choi et al.
6.96 (2H, m), 7.13-7.18 (2H, m), 7.20-7.35 (10H, m). 13C NMR
(CDCl3): δ 31.6, 35.2, 47.9, 52.4, 57.6, 57.6, 66.5, 84.0, 115.1,
115.4, 126.9, 127.5, 128.4, 130.0, 130.0, 135.1, 135.1, 142.0.
Anal. (C32H37N2O9F‚1/2H2O) C, H, N.
[3H]Neu r otr a n sm itter Up ta k e for HEK 293 Cells Ex-
p r essin g Recom bin a n t Biogen ic Am in e Tr a n sp or ter s.
HEK 293 cells expressing hDAT, hSERT, or hNET were plated
on 150 mm dishes and grown until confluent. The medium was
removed, and the cells were washed twice at room temperature
with phosphate buffered saline (PBS). Following addition of
PBS (3 mL), the plates were placed in a 25 °C water bath for
5 min. The cells were gently scraped and then triturated with
a pipette. One plate provided sufficient cells for 48 measure-
ments, and cells from multiple plates were combined. Krebs
HEPES buffer (350 µL) and drugs (50 µL) were added to 1.0
mL assay vials and placed in a 25 °C water bath. Aliquots of
cell suspension (50 µL) were added, incubation was continued
for 10 min, and [3H]DA, [3H]5HT, or [3H]NE (50 µL, 0.020 µM
final concentration) was added. After an additional 10 min of
incubation, the reaction was terminated by filtration onto GF/C
filters using a Tom-tech harvester using filters presoaked in
0.05% polyethyleneimine. Scintillation fluid was added to each
square, and radioactivity remaining on the filter was measured
using a Wallac µ- or â-plate reader. All assays were performed
in triplicate with six drug concentrations. The results were
analyzed using GraphPAD Prism. Additional details about this
assay have been published elsewhere.32,33
1-[2-(Bisp h en ylm eth oxy)eth yl]-4-[N-m eth yl-N-(3-p h en -
ylp r op yl)a m in o]p ip er id in e (17) was prepared as described
for 6 to give the title compound 17 (0.25 g, 0.56 mmol, 54%)
1
as a clear colorless oil. Mp (bis-oxalate salt): 201-203 °C. H
NMR (CDCl3): δ 1.56 (2H, qd, J ) 3.5, 12.0), 1.70 (2H, br d,
J ) 1 1.3), 1.80 (2H, quintet, J ) 7.7), 2.03 (2H, td, J ) 2.1,
11.7), 2.26 (3H, s), 2.38 (1H, tt, J ) 3.8, 1.5), 2.48 (2H, t, J )
7.6), 2.61 (2H, t, J ) 8.0), 2.66 (2H, t, J ) 6.2), 2.99 (2H, br d,
J ) 11), 3.58 (2H, t, J ) 6.1), 5.36 (1H, s), 7.17-7.36 (15H,
m). Anal. (C34H42N2O9) C, H, N.
1-(3-P h en ylp r op yl)-4-m eth yla m in op ip er id in e (19) was
prepared as described for 6 to give the title compound 19
(2.05g, 8.82 mmol, 80%) as a slightly yellow oil without pH
adjustment step. Mp (bis-oxalate salt): 189-191 °C. 1H NMR
(CDCl3): δ 1.56 (2H, qd, J ) 3.8, 10.9), 1.77 (2H, quintet, J )
7.7), 1.95 (2H, br d, J ) 11.5), 2.10 (2H, br t, J ) 11.0), 2.40
(2H, t, J ) 7.7), 2.46 (3H, s), 2.56 (2H, t, J ) 7.6), 2.68 (1H, tt,
J ) 4.0, 10.6), 2.95 (2H, br d, J ) 12.5), 7.09-7.13 (3H, m),
7.18-7.23 (2H, m). 13C NMR (CDCl3): δ 28.0, 29.2, 31.5, 33.3,
51.3, 55.9, 57.3, 125.9, 128.4, 141.4.
Deter m in a tion of Coca in e An ta gon ism of [3H]Dop a -
m in e Up ta k e in HEK293 Cells. HEK 293 cells expressing
hDAT were grown on 150 mm plates to 100% confluence. The
media was removed, and the cells were washed twice with
phosphate buffered saline (PBS, pH 7.4) at room temperature.
Following addition of 3 mL of Krebs Hepes buffer (pH 7.4),
the plates were warmed in a 25 °C water bath for 5 min and
the cells were gently scraped and triturated with a pipette.
Cells from multiple plates were combined, and four plates
provided sufficient cells for the 144 assay wells which were
required to generate four cocaine dose-response curves. The
experimental conditions for the four curves included a control
curve to determine the effect of 0.01% DMSO and three cocaine
dose-response curves in the presence of one of three selected
concentrations of the test compound, which centered around
the Ki determined in the [125I]RTI-55 binding assay. The assay
was conducted in 96 1 mL vials. Krebs HEPES (300 µL) and
cocaine, mazindol, or buffer (50 µL) were added to each vial
in triplicate. Nine concentrations of cocaine ranging from 0.01
to 10 µM were used. Vehicle or one of three concentrations of
test compound were added to the vials, and the mixtures were
placed in a 25 °C water bath. Specific uptake was defined as
the difference in uptake observed in the presence and absence
of 5 µM mazindol. Cells (50 µL) were added and preincubated
with the test compound for 10 min. The assay was initiated
by the addition of [3H]dopamine (50 µL, 0.02 µM final
concentration) and terminated after 10 min by filtration
through Whatman GF/C filters that were presoaked in 0.05%
polyethylenimine. Cocaine IC50 values were calculated using
GraphPAD Prism software. Three to four independent deter-
minations were performed for each dose-response curve.
Statistical analysis of curves generated in the presence or
absence of test compound was performed using two-way
ANOVA. Cocaine IC50 values were compared using one-way
ANOVA or t-tests, as appropriate. Additional details about this
assay have been published elsewhere.32,33
1-(3-P h en ylp r op yl)-4-[N-m eth yl-N-[2-(bisp h en ylm eth -
oxy)eth yl]a m in o]p ip er id in e (20) was prepared as described
for 2 to give the title compound 20 (1.95 g, 4.41 mmol, 93%)
as a pale yellow oil. Mp (bis-oxalate salt): 222-225 °C. 1H
NMR (CDCl3): δ 1.55 (2H, qd, J ) 3.5, 12.1), 1.70-1.90 (6H,
m), 2.28 (3H, s), 2.32 (2H, t, J ) 7.7), 2.38 (1 H, tt, J ) 3.8,
11.5), 2.61 (2H, t, J ) 7.7), 2.75 (2H, t, J ) 6.3), 2.95 (2H, br
d, J ) 11.7), 3.54 (2H, t, J ) 6.2), 5.36 (1H, s), 7.16-7.36 (15H,
m). 13C NMR (CDCl3): δ 27.8, 28.8, 33.8, 38.6, 53.2, 53.3, 58.0,
61.3, 68.0, 83.8, 83.8, 125.6, 126.9, 127.3, 128.2, 128.2, 128.3,
142.1, 142.3. Anal. (C34H42N2O9) C, H, N.
1-Ben zyl-4-[2-(bisp h en ylm eth oxy)eth yl]a m in op ip er i-
d in e (22) was prepared as described for 2 to give the title
compound 22 (0.83 g, 2.07 mmol, 70%) as a sligthly yellow oil.
Mp (bis-oxalate salt): >240 °C, 1H NMR (CDCl3): δ 1.40 (2H,
qd, J ) 3.2, 12.2), 1.64 (1H, br s), 1.82 (2H, br d, J ) 12.5),
2.00 (2H, td, J ) 2.3, 11.6), 2.44 (1H, tt, J ) 4.1, 10.5), 2.85
(4H, t overlapping with br d peak, J ) 5.3), 3.49 (2H, s), 3.58
(2H, t, J ) 5.3), 5.36 (1H, s), 7.22-7.34 (15H, m). 13C NMR
(CDCl3): δ 32.7, 46.3, 52.4, 54.7, 63.0, 68.7, 83.9, 126.9,126.9,
127.4, 128.1, 128.3, 129.1, 142.2. Anal. (C31H36N2O9) C, H, N.
Biologica l Meth od s. [125I]RTI-55 Bin d in g Assa y. All
drugs were prepared as 10 mM stock solutions in DMSO. The
final DMSO concentration in the assay was 0.01%. Pipetting
was performed with a Biomek 2000 robotic workstation. HEK
293 cells expressing hDAT, hSERT, or hNET were grown on
150 mm diameter tissue culture dishes. The medium was
poured off, the cells were washed with 10 mL of phosphate
buffered saline, and 10 mL of lysis buffer (2 mM HEPES, 1
mM EDTA) was added. After 10 min, the cells were scraped
from the plates, poured into centrifuge tubes, and centrifuged
for 20 min at 30000g. The supernatant was removed, and the
pellet was resuspended in 0.32 M sucrose and dispersed with
a Polytron at setting 7 for 10 s. Each assay contained 50 µL of
membrane preparation (approximately 15 µg of protein), 25
µL of drug, and 25 µL of [125I]RTI-55 (40-80 pM final
concentration) in a final volume of 250 µL Krebs HEPES
buffer, which was used as the assay diluent. The membranes
were preincubated with drug for 10 min prior to addition of
Ack n ow led gm en t. This work was supported in part
by a Benedict Cassen Postdoctoral Fellowship (SWC)
sponsored by the Education and Research Counsel of
the Society of Nuclear Medicine. The in vitro assay was
performed by the Cocaine Treatment Discovery Program
(CTDP) of the National Institute on Drug Abuse (NIDA),
Medication Development Division (MDD), and Aaron
J anowsky, Ph.D., Oregon Health Sciences University,
Portland, OR.
[
125I]RTI-55. The assay tubes were incubated for 90 min at
room temperature in the dark, and incubation was terminated
by filtration onto GF/C filters using a Tom-tech harvester.
Scintillation fluid was added to each square, and radioactivity
remaining on the filter was measured using a Wallac µ- or
â-plate reader. Competition experiments were conducted in
duplicate. The results were analyzed using GraphPAD Prism,
and IC50 values were converted to Ki’s using the Cheng-
Prusoff equation. Additional details about this assay have been
published elsewhere.32,33
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis.
This material is available free of charge via the Internet at
http://pubs.acs.org.

Synthesis of Dopamine Transporter Ligands
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 213
(18) Zhang, Y. The Identification of GBR 12909 as Potential
a
Refer en ces
Therapeutic Agent for Cocaine Abuse. Med. Chem. Res. 1998,
8, 66-76.
(1) (a) J ohanson, C. E.; Fischman, M. W. The Pharmacology of
Cocaine Related to its Abuse. Pharmacol. Rev. 1989, 41, 3-52.
(b) Clouet, D., Asghar, K., Brown, R., Eds. Mechanisms of
Cocaine Abuse and Toxicity. NIDA Res. Monogr. 1988, 88.
(2) Ritz, M. C.; Lamb, R. J .; Goldberg, S. R.; Kuhar, M. J . Cocaine
receptors on dopamine transporters are related to self-admin-
istration of cocaine. Science 1987, 237, 1219-1222.
(3) Kennedy, L. T.; Hanbauer, I. Sodium-Sensitive Cocaine Binding
to Rat Striatal Membrane: Possible Relationship to Dopamine
Uptake Sites. J . Neurochem. 1983, 41, 172-178.
(19) Niznik, H. B.; Tyndale, R. F.; Sallee, F. R.; Gonzalez, F. J .;
Hardwick, J . P., Inaba, T.; Kalow, W. The Dopamine Transporter
and Cytochrome P45011D1 (Debrisoquine 4-Hydroxylase) in
Brain: Resolution and Identification of Two Distinct [3H]GBR-
12935 Binding Proteins. Arch. Biochem. Biophys. 1990, 276,
424-432.
(20) Andersen, P. H.; J ansen, J . A.; Nielsen, E. B. [3H]GBR 12935
binding in vivo in mouse brain: Labeling of a piperazine acceptor
site. Eur. J . Pharmacol. 1987, 144, 1-6.
(4) (a) Reith, M. E. A.; Sershen, H.; Lajtha, A. Saturable [3H] cocaine
binding in central nervous system of mouse. Life Sci. 1980, 27,
1055-1062. (b) Reith, M. E. A.; Meisler, B. E.; Sershen, H.;
Lajtha, A. Sodium-Independent Binding of [3H]Cocaine in Mouse
Striatum is Serotonin Related. Brain Res. 1985, 342, 145-148.
(5) Giros, B.; J aber, M.; J ones, S. R.; Wightman, R. M.; Caron, M.
G. Hyperlocomotion and indifference to cocaine and amphet-
amine in mice lacking the dopamine transporter. Nature 1996,
379, 606-612.
(6) Kuhar, M. J .; Ritz, M. C.; Boja, J . W. The dopamine hypothesis
of the reinforcing properties of cocaine. Trends Neurosci. 1991,
14, 299-302.
(7) Carroll, F. I.; Lewin, A. H.; Boja, J . W.; Kuhar, M. J . Cocaine
receptor: Biochemical characterization and structure-activity
relationships of cocaine analogs at the dopamine transporter.
J . Med. Chem. 1992, 35, 969-981.
(8) Clarke, R. L.; Daum, S. J .; Gambino, A. J .; Aceto, M. D.; Pearl,
J .; Levitt, M.; Cumiskey, W. R.; Bogado, E. F. Compounds
affecting the central nervous system. 4. 3â-phenyl-tropane-2-
carboxylic esters and analogs. J . Med. Chem. 1973, 16, 1260-
1267.
(9) Lever, J . R.; Scheffel, U.; Stathus, M.; Seltzman, H. H.; Wyrick,
C. D.; Abraham, P.; Parham, K.; Thomas, B. F.; Boja, J . W.;
Kuhar, M. J .; Carroll, F. I. Synthesis and in vivo studies of a
selective ligand for the dopamine transporter: 3â-(4-[125I]-
Iodophenyl)tropane-2â-carboxylic acid isopropyl ester ([125I]RTI-
121). Nucl. Med. Biol. 1996, 23, 277-284.
(10) Neumeyer, J . L.; Tamagnan, G.; Wang, S.; Gao, Y.; Milius, R.
A.; Kula, N. S.; Baldessarini, R. J . N-substituted analogs of 2â-
carbomethoxy-3â-(4′-iodophenyl)tropane (â-CIT) with selective
affinity to dopamine or serotonin transporters in rat forebrain.
J . Med. Chem. 1996, 39, 543-548.
(11) 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 analogoues. New high affinity
and selective compounds for the dopamine transporter. J . Med.
Chem. 1995, 38, 379-388.
(12) Rothman, R. B. High affinity dopamine reuptake inhibitors as
potential cocaine antagonists: A strategy for drug development.
Life Sci. 1990, 46, 17-21.
(13) Van der Zee, P.; Koger, H. S.; Gootjes, J .; Hespe, W. Aryl 1.4-
dialk(en)yl piperazines as selective and very potent inhibitors
of dopamine uptake. Eur. J . Med. Chem. 1980, 15, 363-370.
(14) Rothman, R. B.; Becketts, K. M.; Radesca, L. R.; de Costa, B.
R.; Rice, K. C.; Carroll, F. I.; Dersch, C. M. Studies of the biogenic
amine transporters. II. A brief study on the use of [3H] DA-
uptake-inhibition to transporter-binding-inhibition ratios for the
in vitro evaluation of putative cocaine antagonists. Life Sci. 1993,
53, 267-272.
(15) Baumann, M. H.; Char, G. U.; de Costa, B. R.; Rice, K. C.;
Rothman, R. B. GBR 12909 Attenuates Cocaine-Induced Activa-
tion of Mesolimbic Dopamine Neurons in the Rat. J . Pharmacol.
Exp. Ther. 1994, 271, 1216-1222.
(16) Glowa, J . R.; Wojnicki, F. H.; de Costa, B. R.; Matecka, D.; Rice,
K. C.; Rothman, R. B. The effect of GBR 12909 on responding of
rhesus monkeys maintained under schedules of cocaine- and
food-delivery. NIDA Res. Monogr. 1994, 141, 12.
(17) Rothman, R. B.; Mele, A.; Reid, A. A.; Akunne, H. C.; Greig, N.;
Thurkauf, A.; de Costa, B. R.; Rice, K. C.; Pert, A. GBR 12909
Antagonizes the Ability of Cocaine to Elevate Extracellular
Levels of Dopamine. Pharmacol. Biochem. Behav. 1991, 40, 387-
396.
(21) Matecka, D.; Rothman, R. B.; Radesca, L.; de Costa, B. R.;
Dersch, C. M.; Partilla, J . S.; Pert, A.; Glowa, J . R.; Wojnicki, F.
H. E.; Rice, K. C. Development of Novel, Potent, and Selective
Dopamine Reuptake Inhibitors through Alteration of the Piper-
azine Ring of 1-[2-(Diphenylmethoxy)ethyl]- and 1-[2-[Bis(4-
fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR
12935 and GBR 12909). J . Med. Chem. 1996, 39, 4704-4716.
(22) Matecka, D.; Lewis, D.; Rothman, R. B.; Dersch, C. M.; Radesca,
L.; de Costa, B. R.; Dersch, C. M.; Wojnicki, F. H. E.; Glowa, J .
R.; DeVries, A. C.; Pert, A.; Rice, K. C. Heteroaromatic Analogs
of 1-[2-(Diphenylmethoxy)ethyl]- and 1-[2-[Bis(4-fluorophenyl)-
methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and
GBR 12909) as High Affinity Dopamine Reuptake Inhibitors.
J . Med. Chem. 1997, 40, 705-716.
(23) Dutta, A. K.; Meltzer, P. C.; Madras, B. K. Positional importance
of the nitrogen atom in novel piperidine analogs of GBR 12909:
Affinity and selectivity for the dopamine transporter. Med.
Chem. Res. 1993, 3, 209-222.
(24) Deutsch, H.; Schweri, M. M. Can stimulant binding and dopa-
mine transport be differentiated? Studies with GBR 12783
derivatives. Life Sci. 1994, 55, 115-121.
(25) Hanson, R. N.; Choi, S. W.; Elmaleh, D. E.; Fischman, A. J .
Synthesis and evaluation of novel N-[2-bis-aryl methoxy) ethyl-
N′-aralkyl-R,ω-alkanediamines as potent an selective dopamine
reuptake inhibitors: seco analogs of GBR 12935 and GBR 12909.
Bioorg. Med. Chem. Lett. 1998, 7, 2559-2562.
(26) Choi, S. W.; Elmaleh, D. R.; Hanson, R. N.; Fischman, A. J .
Design, synthesis and biological evaluation of novel nonpipera-
zine analogs of 1-[2-(diphenylmethoxy)ethyl]- and 1-[2-[bis(4-
fluorophenyl)methoxylethyl]-4-(3-phenyl propyl)piperazines as
dopamine transporter inhibitors. J . Med. Chem. 1999, 42, 3647-
3656.
(27) Madras, B. K.; Reith, M. E. A.; Meltzer, P. C.; Dutta, A. K. O-526,
a piperidine analog of GBR 12909, retains high affinity for the
dopamine transporter in monkey caudate-putamen. Eur. J .
Pharmacol. 1994, 267, 167-173.
(28) Borch, R. F.; Hassid, A. I. A New Method for the Methylation of
amines. J . Org. Chem. 1972, 37, 1673-1675.
(29) Borch, R. F.; Bernstein, M. D.; Durst, H. D. The Cyanohydri-
doborate Anion as a Selective Reducing Agent. J . Amer. Chem.
Soc. 1971, 93, 2897-2904.
(30) Dutta, A. K.; Coffey, L. L.; Reith, M. E. A. High Selective, Novel
Analogs of 4-[2-(Diphenylmethoxy)ethyl]-l-benzylpiperidine for
the Dopamine Transporter: Effect of Different Aromatic Sub-
stitutions on Their Affinity and Selectivity. J . Med. Chem. 1997,
40, 35-43.
(31) 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.
(32) Eshleman, A. J .; Carmolli, M.; Cumbay, M.; Martens, C. R.;
Neve, K. A.; J anowsky, A. Characteristics of drug interactions
with recombinant biogenic amine transporters expressed in the
same cell type. J . Pharmacol. Exp. Ther. 1999, 289, 877-885.
(33) J ohnson, R. A.; Eshleman, A. J .; Meyers, T.; Neve, K. A.;
J anowsky, A. [3H]substrate- and cell-specific effects of uptake
inhibitors on human dopamine and serotonin transporter-
mediated efflux. Synapse 1998, 30, 97-106.
J M9902178