Design, synthesis, and biological evaluation of novel non-piperazine analogues of 1-[2-(diphenylmethoxy)ethyl]- and 1-[2-[bis(4- fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines as dopamine transporter inhibitors

Article abstract of DOI:10.1021/jm990161h

A series of novel diamine, amine-amide, and piperazinone analogues of N- [2-(bisarylmethoxy)-ethyl]-N'-(phenylpropyl)piperazines, GBR 12909 and 12935, were synthesized and evaluated as inhibitors of presynaptic monoamine neurotransmitter transporters. The primary objective of the study was to determine the structural requirements for selectivity of ligand binding and potency for neurotransmitter reuptake inhibition. In general, the target compounds retained transporter affinity; however, structural variations produced significant effects on reuptake inhibition and transporter selectivity. For example, analogues prepared by replacing the piperazine ring in the GBR structure with an N,N'-dimethylpropyldiamine moiety displayed enhanced selectivity for binding and reuptake inhibition at the norepinephrine (NE) transporter site (e.g. 4 and 5). Congeners in which the amide nitrogen atom was attached to the aralkyl moiety of the GBR molecule showed moderate affinity (K(i) = 51-61 nM) and selectivity for the dopamine transporter (DAT) site. In contrast, introduction of a carbonyl group adjacent to either nitrogen atom of the piperazine ring (e.g. 25 and 27) was not well tolerated. From the compounds prepared, analogue 16 was selected for further evaluation. With this congener, locomotor activity induced by cocaine at a dose of 20 mg/kg was attenuated with an-AD₅₀ (dose attenuating cocaine-induced stimulation by 50%) of 60.0 ± 3.6 mg/kg.

Full text of DOI:10.1021/jm990161h

J . Med. Chem. 1999, 42, 3647-3656
3647
Design , Syn th esis, a n d Biologica l Eva lu a tion of Novel Non -P ip er a zin e
An a logu es of 1-[2-(Dip h en ylm eth oxy)eth yl]- a n d
1-[2-[Bis(4-flu or op h en yl)m eth oxy]eth yl]-4-(3-p h en ylp r op yl)p ip er a zin es a s
Dop a m in e Tr a n sp or ter In h ibitor s
Sung-Woon Choi,† David R. Elmaleh,† Robert N. Hanson,‡ and Alan J . Fischman*,†
Division of Nuclear Medicine, 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 2, 1999
A series of novel diamine, amine-amide, and piperazinone analogues of N-[2-(bisarylmethoxy)-
ethyl]-N′-(phenylpropyl)piperazines, GBR 12909 and 12935, were synthesized and evaluated
as inhibitors of presynaptic monoamine neurotransmitter transporters. The primary objective
of the study was to determine the structural requirements for selectivity of ligand binding and
potency for neurotransmitter reuptake inhibition. In general, the target compounds retained
transporter affinity; however, structural variations produced significant effects on reuptake
inhibition and transporter selectivity. For example, analogues prepared by replacing the
piperazine ring in the GBR structure with an N,N′-dimethylpropyldiamine moiety displayed
enhanced selectivity for binding and reuptake inhibition at the norepinephrine (NE) transporter
site (e.g. 4 and 5). Congeners in which the amide nitrogen atom was attached to the aralkyl
moiety of the GBR molecule showed moderate affinity (Ki ) 51-61 nM) and selectivity for the
dopamine transporter (DAT) site. In contrast, introduction of a carbonyl group adjacent to
either nitrogen atom of the piperazine ring (e.g. 25 and 27) was not well tolerated. From the
compounds prepared, analogue 16 was selected for further evaluation. With this congener,
locomotor activity induced by cocaine at a dose of 20 mg/kg was attenuated with an AD50 (dose
attenuating cocaine-induced stimulation by 50%) of 60.0 ( 3.6 mg/kg.
In tr od u ction
pharmacological,12,13 neurochemical,14-16 biological,17,18
and molecular pharmacological19 investigations have
been conducted using these agents both to probe the
DAT and to develop promising lead compounds for drug
development.20 Several structure-activity relationship
(SAR) studies have been conducted to evaluate the three
key structural features of the GBR compounds (Figure
1).21 These studies suggested that the internal pipera-
zine ring moiety plays a key role for modulating selec-
tive binding and in producing slow dissociation from the
site.22-24 Later studies evaluated the role of the pipera-
zine ring and the tail-phenylpropyl moiety.23,25-27 For
example, the piperazine moiety was replaced by five-,
seven-, or eight-membered rings, trans-2,5-dimethylpip-
erazine, homopiperazine, and ring-opened alkyldiamines.
Although these studies failed to produce pure cocaine
antagonists, a number of potent, selective, and high-
affinity DAT inhibitors were identified.24,28
Cocaine is one of the most powerful stimulant/
reinforcer drugs, and its abuse is a major public health
and social problem.1 The primary pharmacological
properties of cocaine have been associated with its
propensity for binding to neurotransmitter transporters
in the central nervous system (CNS), especially the
dopamine transporter (DAT). According to the dopamine
hypothesis, “cocaine receptors” located on the DAT in
the mesolimbic system of the brain are believed to be
responsible for the drug’s reinforcing and self-adminis-
tration properties and perhaps some of its euphorigenic
effects. As a result, this protein has become a target for
the development of the medications for treating cocaine
addiction.2-9 Over the past decade, several different
classes of DAT-specific ligands have been identified. Of
these compounds, 3â-aryltropanes4,10 and N,N′-disub-
stituted piperazine derivatives11 have been the most
extensively evaluated.
Two types of cocaine antagonists have been postu-
lated, pure pharmacological antagonists and partial
agonists (noncompetitive inhibitors).29 Comparison of
IC50 values for [3H]DA uptake inhibition with IC50
values for inhibition of transporter ligand (e.g. [125I]RTI-
55) binding has been an important criterion for screen-
ing test ligands as cocaine antagonists.22 In this context,
good candidate cocaine antagonists are expected to
display high “uptake-to-binding ratios” when both as-
says are performed under identical experimental condi-
tions. On the basis of these ratios, GBR 12935 has been
In 1980, Van der Zee et al. developed the GBR series
of compounds in which the tropine of benztropine was
replaced by substituted piperazine.11 Since the initial
study which identified the GBR compounds as potent
and highly selective DA reuptake inhibitors, numerous
* Requests for reprints should be addressed to Alan J . Fischman,
M.D., Ph.D., Massachusetts General Hospital. Phone: (617) 726-8353.
Fax: (617) 726-1650. E-mail: fischman@petw6.mgh.harvard.edu.
† Massachusetts General Hospital.
‡ Northeastern University.
10.1021/jm990161h CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/14/1999

3648 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Choi et al.
F igu r e 1. General structure of test ligands with modification sites indicated.
from the tail side was used to evaluate the effects of
basicity at the N2 nitrogen atom and its flexibility
(Figure 1). Also, the amide present in these tail-amide
analogues can serve as synthetic intermediates for the
amine in seco GBR analogue 20. Introduction of a
carbonyl group into the piperazine ring (piperazinones)
effectively eliminates the basicity of the adjacent nitro-
gen atom, and this modification was used to evaluate
the effect of basicity of each nitrogen atom without
significantly changing flexibility of the ring. This modi-
fication was also used to evaluate the possibility of
different interactions at the binding sites with the DAT
protein.
In this paper, we report the synthesis and the
pharmacological evaluation of three series of GBR
derivatives (Figure 1). In order to gain insight into some
of the structural parameters which govern (1) binding
selectivity at dopamine, norepinephrine, and serotonin
(the three neurotransmitter transporters) and (2) po-
tency of neurotransmitter reuptake inhibition, chemical
modifications were systematically performed on these
structures. Also, new functional groups were introduced
to test for the possibility of additional modes of binding
(additional primary recognition contacts) between the
ligand and transporter which might have different
effects on binding and reuptake inhibition at the
DAT.
F igu r e 2. Average horizontal activity counts/10 min over 30
min as a function of dose, starting 20 min after pretreatment
with 16. The slope of the descending portion of the dose-effect
curve (30-100 mg/kg dose range) indicated an ID50 of 47.4 (
5.2 mg/kg.
considered to be a possible antagonist or partial ago-
nist.22 Previously published SAR studies with chiral
piperazines related to GBR 12909 supported the notion
of cocaine antagonism and established the feasibility of
developing SAR studies of cocaine antagonists based on
comparisons of binding and uptake inhibition.23
Ch em istr y
The requisite starting materials 2a ,2b in the first
series of GBR analogues 4, 5, 8, and 9 were prepared
according to literature procedures with slight modifica-
tions.11 For example, 2-bromoethanol was used instead
of chloroethanol to synthesize the more reactive 2-bro-
moethyl diphenylmethyl ether 2a or its bis(4-fluoro)
derivative 2b, and purification was achieved by using
column chromatography instead of vacuum distillation.
Commercially available N,N′-dimethylethylenediamine
and N,N′-dimethyl-1,3-propanediamine were used as
starting materials (10 equiv, excess) for the first
monoalkylation step, and the final yields could be
increased to 80% from the starting material (Scheme
1).
Synthesis of the second series of GBR analogues is
shown in Scheme 2. Compounds 15-19 were prepared
by monoacylation of ethylene- or propylenediamine with
acid chlorides (22-35% yield) followed by mono-N-
alkylation with 2b (73-85% yield). Compound 14 was
converted to GBR analogue 20 by alkylation with 2b
followed by LAH reduction. Because the monoacylated
The goal of the present study was to develop and
identify novel GBR analogues with sufficient structural
variations to more precisely define the structural re-
quirements for potent and selective binding to the DAT
and to investigate the structural features for ligand
interaction that block cocaine binding site(s), without
interfering with DA uptake (cocaine antagonists). Previ-
ously, it was shown that the (bisphenylmethoxy)ethyl
group (head, Figure 1) and diamine moiety are essential
pharmacophores for retention of DAT binding.24,32 Also,
at least one nitrogen atom (N2, Figure 1) connected to
the tail-phenylpropyl moiety was shown to be re-
quired.25,33 We began our investigation by opening the
piperazine ring between the ethylenediamine and pro-
pylenediamine moieties with secondary and tertiary
amines.32 On the basis of previous findings, we decided
to modify the ethylenediamine moiety of seco GBR
analogues (Figure 2). To evaluate the effect of distance
between the bridge and tail tether, length of the
methylene tether (n, n′) was varied. Introduction of a
carbonyl group external to the ring-opened diamine unit

Non-Piperazine Analogues as DAT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3649
Sch em e 1a
a
(a) HOCH2CH2Br, H2SO4, PhCH3; (b) MeNH(CH2)3NHMe, K2CO3, DMF; (c) Ph(CH2)2Br; K2CO3, DMF; (d) Ph(CH2)3Br, K2CO3, DMF;
(e) MeNH(CH2)2NHMe, K2CO3, DMF; (f) 2a , K2CO3, DMF.
diamines were highly soluble in water, an extensive
extraction process was used to improve yields.
Biologica l Resu lts
The final products were converted to oxalate or
hydrochloride salts and submitted to the NIDA/MDD
for the determination of binding affinity (Ki) and
potency of reuptake inhibition (IC50) at dopamine,
serotonin, and norepinephrine transporter sites (Tables
1 and 2). The results shown in Table 2 indicate that
most of the new seco compounds inhibited dopamine
uptake at concentrations (120-830 nM) comparable to
or lower than those reported for cocaine (190-300 nM).
Interestingly, N,N′-dimethyl ring-opened GBR ana-
logues such as 4 and 5, with a propylene moiety between
two nitrogen atoms, exhibited high affinity at the NET
(Ki ) 194, 30 nM) and inhibited [3H]NE reuptake
selectively (Tables 1 and 2). Because analogues contain-
ing acyclic diamine spacers exhibited NET activity,
“linear” distance factors alone appear to produce NET
selectivity. However, compound 20, with a three-carbon
spacer between the two basic nitrogens (secondary
amines), showed selectivity for DAT binding and inhibi-
The synthetic route for preparing piperazinone ring
GBR analogues is shown in Scheme 3. The requisite
ketopiperazine was synthesized in good yield (84%,
crude) using the method described by Aspinal31 with
some modifications. Instead of using distillation for the
cyclization of 22 and purification of 23, the crude
reaction mixture was stirred at ambient temperature
for 24 h in DMF, the solvent was removed, and the
resultant solid was purified by chromatography to give
crude product 23. The intermediate 23 was used in the
following step without further purification. The N-
alkylation step afforded amide ring intermediates 24
and 26 in moderate yields, which may be partially
attributed to the impurity of ketopiperazine 23. Subse-
quent alkylation of 24 and 26 with appropriate alkyl
halides proceeded in moderate yields to give target GBR
analogues 25 and 27, respectively. The structures of all
final products were confirmed by spectroscopic and
analytic methods.

3650 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Choi et al.
Sch em e 2a
a
(a) SOCl2, CHCl3, cat. DMF; (b) 2b, K2CO3, DMF; (c) LAH, THF.
Sch em e 3a
a
(a) H2NCH2CH2NH2, abs. EtOH; (b) DMF, 60-70 °C; (c) 2a , K2CO3, DMF; (d) Ph(CH2)3Br, NaH, DMF; (e) Ph(CH2)2Br, K2CO3, DMF;
(f) 2b, NaH, DMF.
tion of [3H]DA reuptake. The other ring-opened conge-
ners, 8 and 9, with an ethylene moiety between two
nitrogen atoms, retained high affinity (Ki ) 26, 30 nM)
at DAT sites and high selectivity for DAT vs SERT sites
(18-, 30-fold). Interestingly, analogue 9 possessed a
reuptake-to-binding ratio at the DAT site of about 32:
1, similar to results reported by Matecka et al.24
Insertion of one more carbon into the alkyl chain of the
tail-phenylethyl group in 4 resulted in the tail-phenyl-
propyl moiety of 5 (three-carbon spacer between tail
benzene ring and adjacent basic nitrogen atom). This
modification improved binding affinity for NET (6-fold),
DAT (3-fold), and SERT (4-fold) sites. However, activity
as an inhibitor of reuptake was slightly improved only

Non-Piperazine Analogues as DAT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3651
Ta ble 1. Affinities and Selectivities of GBR Analogues at the DA, 5-HT, and NE Transporters Measured by Inhibition of [125I]RTI-55
Bindinga
binding (Ki ( SD, nM)
selectivity ratio
compd
DAT
SERT
NET
SERT/DAT
NET/DAT
4
5
8
9
15
16
17
18
19
20
25
27
cocaine
cocaineb
cocainec
322 ( 57
122 ( 12
30 ( 10
1530 ( 421
438 ( 122
550 ( 70
770 ( 180
351 ( 81
590 ( 150
556 ( 174
523 ( 28
1150 ( 212
125 ( 12
5900 ( 790
114 ( 49
387 ( 88
204 ( 40
488 ( 67
194 ( 79
30 ( 5
4.7
3.6
18.3
30.1
6.5
0.6
0.2
12.3
10.2
15.6
3.5
11.2
10.7
7.8
370 ( 126
261 ( 81
26 ( 7.8
54 ( 21
844 ( 224
1760 ( 940
604 ( 234
545 ( 176
477 ( 80
510 ( 160
54 ( 23
1.2
10.3
10.3
18.8
4.5
0.7
0.1
51 ( 6
61 ( 11
28 ( 7
82 ( 12
2.9
1.0
4.9
8200 ( 1300
1430 ( 590
759 ( 93
619 ( 93
960 ( 190
5850 ( 450
7050 ( 2620
1770 ( 369
2040 ( 300
2580 ( 260
a
The Ki values for the test ligands were determined with assays described in the Experimental Section. Results are mean ( SEM for
three independent experiments assayed in triplicate. Cocaine as reference for 9. c Cocaine as reference for 16 and 25.
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
Transportersa
ratio
reuptake inhibition (IC50 ( SD, nM)
[3H]DA
reuptake/DAT binding
[3H]5-HT
reuptake/SERT binding
compd
[3H]DA
[3H]5-HT
[3H]NE
4
5
8
9
15
16
17
18
19
20
25
27
cocaine
cocaineb
cocainec
349 ( 18
237 ( 41
119 ( 11
830 ( 340
47 ( 16
1700 ( 370
1700 ( 560
1050 ( 130
>10 µM
210 ( 33
143 ( 27
279 ( 67
2600 ( 710
299 ( 82
>10 µM
1.1
1.9
4.0
32.4
0.9
2.7
1.5
1.8
1.2
2.4
1.1
3.9
1.9
>13.0
1990 ( 317
4500 ( 1400
1300 ( 66
1140 ( 392
1230 ( 135
249 ( 31
5.7
7.6
2.3
2.2
1.1
2.0
1380 ( 390
80 ( 22
399 ( 117
205 ( 44
192 ( 52
116 ( 52
>10 µM
90 ( 24
73 ( 17
68 ( 8
>10 µM
>10 µM
190 ( 21
299 ( 61
218 ( 29
>10 µM
>1.2
>1.7
>1.7
>34.5
3940 ( 856
336 ( 78
>10 µM
230 ( 41
125 ( 8.6
217 ( 60
301 ( 42
217 ( 36
a
IC50 values for the test ligands were determined with assays described in the Experimental Section. Results are mean ( SEM of
three independent experiments assayed in triplicate. Cocaine as reference for 9. c Cocaine as reference for 16 and 25.
b
at the NET sites (Table 2, 4 and 5). Also, this structural
change at the tail moiety exhibited a 2-fold improvement
in the ratio of [3H]DA reuptake inhibition to binding
inhibition at the DAT. This finding suggests that the
3-phenylpropyl group may bind to domain(s) of the DAT
which are more important for binding than for uptake
inhibition.23
and tested (Tables 1 and 2). This series of amine-amide
GBR analogues showed moderate affinity (Ki ) 51-61
nM, Table 1) and selectivity for the DAT, independent
of the distance (n, Figure 1) between the basic nitrogen
and the amide function of the tail moiety. Fluoro
substitution at the benzene ring in the tail moiety
improved potency for [3H]DA reuptake inhibition about
2-fold without affecting binding affinity (IC50 ) 47 nM,
15-17). Introduction of a two-carbon (ethylene) linkage
(n′, Figure 1) between the amide and tail benzene ring,
congener 17, had little effect at the DAT but produced
a 2-fold improvement in reuptake inhibition at the NET.
This modification produced a slight improvement in
binding at all three transporters, similar to observations
with seco GBR analogues (4 and 5, Tables 1 and 2). A
three-carbon (propylene) linkage between the basic
nitrogen atom and the amide function nitrogen atom,
such as in congener 19, produced 2-fold improvement
in selectivity for DAT vs SERT binding affinity com-
pared to compound 17. However, DAT vs NET selectiv-
ity was decreased from 11.2 to 7.8-fold. These observa-
tions were in agreement with previous findings from
seco GBR analogues, but the effects were minimal. This
tolerance of an amide function may be attributable to a
strong hydrogen-bonding interaction between the trans-
In previous studies it was shown that only one
nitrogen atom attached to the phenylpropyl (tail) moiety
of the GBR molecule was required for the retention of
high-affinity DAT binding.25 These new second-genera-
tion piperidine-based GBR-type compounds were more
selective but equipotent compared to the original GBR
12909.33 This was in contrast to the original notion,
proposed by Van der Zee et al.,11 that the 3-phenylpropyl
substitution maximized the potency. Interestingly, ini-
tial evaluation of binding assay data (NOVA Screen) for
ring-opened seco GBR analogues with a tail-amide
function (compound 16, Table 1), in which the N2
nitrogen atom was replaced by a nonbasic amide func-
tion with one basic nitrogen atom (N1) attached to the
head moiety, demonstrated retention of DAT binding
(IC50 ) 450 nM, NOVA Screen not shown). This
prompted a further evaluation of this series of com-
pounds and four additional congeners were prepared

3652 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Choi et al.
porter and the ligand’s amide function as donor/acceptor
in the ligand binding site(s) of the DAT.
Because we were trying to detect general trends in
biological activity, multiple simultaneous changes were
incorporated in the target compounds, e.g., fluorine for
hydrogen on the aryl groups or the introduction of a
different alkyl chain in the aralkyl tail. Previous stud-
ies11,24,32 have described the individual effects of these
changes on transporter inhibition. Introduction of a
carbonyl group adjacent to each nitrogen atom on the
piperazine ring produced the piperazinone ring GBR
analogues 25 and 27 and showed a profound effect on
biological properties. This modification which was used
to evaluate the effect of basicity of each nitrogen atom
without significantly changing flexibility of the ring was
not successful in producing compounds with the desired
properties. Introduction of a carbonyl group into the
piperazine ring (piperazinone) significantly attenuated
potency of reuptake inhibition at the DAT. Replacement
of the phenylpropyl moiety by a phenylethyl moiety
significantly shifted selectivity toward the SERT in
these piperazinone ring GBR analogues. Interestingly,
27 possessed a reuptake-to-binding ratio at the SERT
site of about 35:1. This analogue was practically inactive
(>10 µM) for the inhibition of reuptake to the DAT and
NET sites and had weak binding affinity (DAT 1.4 µM,
NET 7.0 µM).
Based on the results in Tables 1 and 2, it appears that
compound 16 has a reasonable ratio of [3H]DA reuptake
to DAT binding (2.7) with DAT binding comparable to
that of cocaine. Although compounds 8 and 9 have
superior selectivity ratios (4.0 and 32.4) both agents
have much higher potency DAT binding compared with
cocaine, similar to GBR 1290934 (IC50(DAT) ) 43 ( 10
nM and IC50(SERT) ) 741 ( 56 nM). Thus, since our
goal was to develop pure cocaine antagonists which have
DAT binding similar to that of cocaine but reduced
potency for [3H]DA reuptake inhibition, compound 16
was selected for preliminary behavioral screening which
involved testing of the compound alone and in combina-
tion with cocaine for its effect on locomotion activity in
mice. Figure 2 shows average horizontal activity counts/
10 min over 30 min as a function of dose, starting 20
min after ip administration of compound 16 without
cocaine. Significant suppression of spontaneous loco-
motor activity was obtained at doses of 30 and 100 mg/
kg. The mean average horizontal activity counts/10 min
on the descending portion of the dose-effect curve (30-
100 mg/kg dose range) for this 30-min period were fit
to a linear function of log10 dose. Analogue 16 sup-
pressed spontaneous locomotor activity with an ID50
(dose producing half-maximal depressant activity, where
maximal depression ) 0 counts/30 min) of 47.4 ( 5.2
mg/kg (mean ( SEM). Since this compound is not
expected to significantly alter synaptic dopamine levels,
the observed reduction in locomotor activity suggests
that compound 16 may have other biological properties
in addition to binding to the DAT.
F igu r e 3. Average horizontal activity counts/10 min over 30
min as a function of dose condition, 20 min after pretreatment
with 16. The bar above “coc” represents the effect of vehicle
20 min prior to saline injection. The bars above “3, 10, 30, and
100” represent the effects of 16 at the designated doses 20 min
prior to cocaine injection (20 mg/kg). The mean average
horizontal activity counts/10 min on the descending portion
of the dose-effect curve (10-100 mg/kg dose range) were fit
to a linear function of log dose to yield an AD50 of 60.0 ( 3.6
mg/kg.
kg. In contrast, at low doses of 3, 10, and 30 mg/kg, very
weak stimulations of cocaine-induced locomotor activity
were observed. Analogue 16 attenuated locomotor activ-
ity induced by 20 mg/kg cocaine with an AD50 (dose
attenuating cocaine-induced stimulation by 50%) of 60.0
( 3.6 mg/kg (mean ( SEM).
Con clu sion s
The rigid piperazine ring (bridge, Figure 1) of GBRs
was substituted with conformationally flexible polym-
ethylenediamines and amine-amide moieties without
compromising biological activity. New analogues with
a three-carbon linkage at the bridge portion of the
structure displayed a tendency for NET site activities,
and this bridge was proved to be a “selectivity controller”
moiety. Tolerance of amide function at the N2 (Figure
1) nitrogen atom may be due to strong hydrogen-
bonding capabilities at the binding site(s) of the DAT
protein. The current studies have provided additional
information on pharmacophoric groups which are re-
sponsible for selective binding and potency of reuptake
inhibition at the DAT. These acyclic analogues along
with the results of in vivo studies with 16 may provide
a basis for the design of novel probes and lead com-
pounds which are structurally different DAT inhibitors
with selective and strong binding to the DAT and weak
potency for DA uptake.
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. The volatile solvents
Figure 3 shows the results of a cocaine/compound 16
interaction study. The period of 0-30 min was selected
for analysis of dose-response data, because this is the
time interval in which coicaine produces its maximal
effects. Significant suppression of cocaine-induced lo-
comotor activity was obtained only at a dose of 100 mg/

Non-Piperazine Analogues as DAT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3653
1
were removed by rotary evaporation under reduced pressure.
Melting points were determined on a Thomas-Hoover melting
point apparatus or an Electrothermal melting point apparatus
and are uncorrected. Elemental analyses were performed by
Atlantic Microlabs, Atlanta, GA, for carbon, hydrogen, and
nitrogen and were within (0.4% of theoretical values unless
noted otherwise. 1H,13C NMR spectra were determined on a
Varian XL-300 spectrometer. Chemical shifts are expressed
in parts per million (ppm) on the δ scale relative to a TMS
internal reference standard. In general, CDCl3 was used for
free bases and DMSO-d6 was used for salts. Coupling constants
(J values) are given in Hz. Thin layer chromatography (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
developed with appropriate solvent mixtures. Column separa-
tions were performed on silica gel (Baker, 40 µm flash
chromatography). The collected fractions were analyzed by
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 the 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 recrystal-
lized from appropriate solvents. Unfilterable gels were evapo-
rated to dryness and recrystallized.
oil. H NMR (CDCl3): δ 1.65 (2H, quintet, J ) 7.5), 2.27 (3H,
s), 2.29 (3H, s), 2.40 (4H, t, J ) 7.5), 2.56-2.61 (2H, m), 2.67
(2H, t, J ) 6.1), 2.73-2.78 (2H, m), 3.56 (2H, t, J ) 6.1), 5.36
(1H, s), 7.17-7.36 (15H, m). 13C NMR (CDCl3): δ 25.17, 33.76,
42.20, 42.91, 55.57, 56.13, 56.92, 59.60, 67.39, 83.92, 125.89,
126.93, 127.33, 128.30, 128.67, 140.52, 142.30. Mp (bisoxalate
salt): 198-200 °C. Anal. Calcd for C32H40N2O9: C, 64.42; H,
6.76; N, 4.69. Found: C, 64.53; H, 6.77; N, 4.63.
N,N′-Dim et h yl-N-[2-(b isp h en ylm et h oxy)et h yl]-N′-(3-
p h en ylp r op yl)-1,3-p r op a n ed ia m in e (5) was prepared as
described for 3 to give the title compound 5 (70%) as a colorless
1
oil. H NMR (CDCl3): δ 1.63 (2H, quintet, J ) 7.5), 1.78 (2H,
quintet, J ) 7.7), 2.19 (3H, s), 2.26 (3H, s), 2.30-2.43 (6H,
m), 2.61 (2H, t, J ) 7.8), 2.67 (2H, t, J ) 6.1), 3.56 (2H, t, J )
6.1), 5.36 (1H, s), 7.17-7.36 (15H, m). 13C NMR (CDCl3): δ
25.18, 28.98, 33.68, 42.17, 42.91, 55.71, 56.17, 56.90, 57.23,
67.38, 83.90, 125.66, 126.91, 127.31, 128.24, 128.28, 128.34,
142.28. Mp (bisoxalate salt): 191-193 °C. Anal. Calcd for
C
33H42N2O9: C, 64.90; H, 6.93; N, 4.59. Found: C, 64.76; H,
6.98; N, 4.55.
N,N′-Dim eth yl-N-[2-(bisp h en ylm eth oxy)eth yl]-N′-[2-(4-
flu or op h en yl)eth yl]-1,2-eth ylen ed ia m in e (8) was prepared
as described for 3 to give the title compound 8 (85%) as a
colorless oil. 1H NMR (CDCl3): δ 2.28 (3H, s), 2.29 (3H, s),
2.53-2.59 (6H, m), 2.69-2.75 (4H, m), 3.56 (2H, t, J ) 6.1),
5.35 (1H, s), 6.91-6.97 (2H, m), 7.09-7.13 (2H, m), 7.19-7.36
(10H, m). 13C NMR (CDCl3): δ 32.92, 42.49, 43.24, 55.31,
55.74, 57.36, 60.09, 67.33, 83.96, 114.87, 115.15, 126.89,
127.33, 128.28, 129.90, 130.00, 136.01, 136.06, 142.25, 159.65,
162.88. Mp (bisoxalate salt): 225-230 °C. Anal. Calcd for
1-[Bis(4-flu or op h en yl)m eth oxy]-2-br om oeth a n e (1b). A
mixture of 2-bromoethanol (15.0 g, 120 mmol), 1 mL of
concentrated sulfuric acid, and 4,4′-difluorobenzhydrol (4.4 g,
20 mmol) in 50 mL of toluene was heated at reflux for 6 h.
The reaction mixture was cooled and washed successively with
saturated sodium bicarbonate solution (50 mL) and water (50
mL). The organic layer was dried over MgSO4 and filtered,
and the solvent was removed by rotary evaporation. The
resultant colorless oil was applied on a silica gel column for
separation. Elution with 3% ethyl acetate/hexane afforded 5.3
C
31H37N2O9F: C, 61.99; H, 6.21; N, 4.66. Found: C, 62.15; H,
6.26; N, 4.72.
N,N′-Dim eth yl-1,2-bis[2-[bis(4-flu or op h en yl)m eth oxy]-
eth yl]eth a n ed ia m in e (9) was prepared as described for 3
to give the title compound 9 (85%) as a colorless oil. 1H
NMR (CDCl3): δ 2.25 (3H, s), 2.52 (2H, s), 2.66 (2H, t, J )
6.0), 3.50 (2H, t, J ) 6.1), 5.30 (1H, s), 6.96-7.02 (4H, m),
7.24-7.29 (4H, m). 13C NMR (CDCl3): δ 43.17, 55.81, 57.32,
67.32, 82.55, 115.08, 115.36, 128.44, 128.55, 137.83, 137.87,
160.46, 163.72. Mp (bisoxalate salt): 207-210 °C. Anal. Calcd
for C38H40N2O10F4: C, 60.00; H, 5.30; N, 3.68. Found: C, 60.11;
H, 5.26; N, 3.67.
1
g (17.0 mmol, 85%) of the product as a colorless oil. H NMR
(CDCl3): δ 3.45 (2H, t, J ) 6.0), 3.70 (2H, t, J ) 6.0), 5.36
(1H, s), 6.94-7.00 (4H, m), 7.26-7.31 (4H, m). 13C NMR
(CDCl3): δ 30.55, 68.64, 82.27, 115.19, 115.30, 128.45, 128.55,
137.23, 137.27, 160.42, 163.68.
1-(Bisp h en ylm eth oxy)-2-br om oeth a n e (1a ) was pre-
pared as described for 1b to give the title compound 1a as a
clear colorless oil. 1H NMR (CDCl3): δ 3.42 (2H, t, J ) 6.1),
3.69 (2H, t, J ) 6.1), 5.37 (1H, s), 7.19-7.34 (10H, m). 13C NMR
(DMSO-d6): δ 32.67, 68.71, 82.25, 126.59, 127.32, 128.28,
142.06.
N-(4-F lu or op h en yla cetyl)-1,2-eth a n ed ia m in e (10). A
mixture of thionyl chloride (11.9 g, 100 mmol) and 4-fluorophe-
nylacetic acid (4.62 g, 30 mmol) in 40 mL of CHCl3 was stirred
at room temperature for 6 h. The reaction mixture was
concentrated in vacuo to give the acid chloride as a slightly
yellow solid. This material was dissolved in 20 mL of anhy-
drous CH2Cl2 and was added over 2 h to the mixture of
ethylenediamine (18.03 g, 300 mmol) and triethylamine (6.07
g, 60 mmol) in 50 mL of anhydrous CH2Cl2. The reaction
mixture was allowed to stir for 24 h at ambient temperature.
The solvent was removed using rotary evaporation. Ethyl
acetate (200 mL) and saturated NaCl solution (30 mL) were
added to the resultant crude product and stirred 2 h. The
organic layer was separated, washed with saturated NaCl (30
mL), dried over MgSO4, filtered, and evaporated to dryness.
The crude product was purified by chromatography on silica
gel. Elution with CHCl3:MeOH:NH4OH (9:1:0.1) afforded 1.3
g (6.63 mmol, 22.1%) of desired product 10 as a slightly yellow
sticky oil that solidified upon cooling. 1H NMR (CDCl3): δ 1.59
(2H, br s), 2.75 (2H, t, J ) 5.9), 3.25 (2H, q, J ) 5.9), 3.50 (2H,
s), 6.57 (1H, br s), 6.98-7.04 (2H, m), 7.20-7.25 (2H, m). 13C
NMR (CDCl3): δ 41.05, 42.07, 42.48, 115.27, 115.56, 130.56,
130.66, 160.12, 163.37, 171.10. Mp (oxalate salt): 147-150 °C.
N,N′-Dim eth yl-N-[2-(bisp h en ylm eth oxy)eth yl]-1,3-p r o-
p a n ed ia m in e (3). To a mixture of amine (4.49 g, 44 mmol),
N,N′-dimethyl-1,3-propanediamine, and K2CO3 (1.82 g, 13.2
mmol) in 15 mL of DMF was added dropwise 1-(bisphenyl-
methoxy)-2-bromoethane (1.28 g, 4.4 mmol) in 10 mL of DMF.
The mixture was stirred for 48 h and poured into 200 mL of
EtOAc. The organic phase was washed with saturated NaCl
solution (60 mL × 3) dried over MgSO4, filtered, and evapo-
rated to dryness. The residue was purified by silica gel column
chromatography, eluted with CHCl3:MeOH:NH4OH (9:1:0.1)
to give the desired product 3 (1.23 g, 3.94 mmol, 89%) as a
1
clear oil. H NMR (CDCl3): δ 1.8 (1H, br s), 1.94 (3H, s), 2.01
(2H, quintet, J ) 5.6), 2.35 (3H, s), 2.66 (2H, t, J ) 5.1), 2.68
(2H, t, J ) 4.8), 2.97 (2H, t, J ) 5.5), 3.58 (2H, t, J ) 5.1),
5.40 (1H, s), 7.25-7.42 (10H, m). Mp (HCl salt): 185-188 °C.
N,N′-Dim et h yl-N-[2-(4-flu or op h en yl)et h yl]-1,2-et h yl-
en ed ia m in e (7) was prepared as described for 3 to give the
title compound 7 (88%) as a slightly yellow oil. 1H NMR
(CDCl3): δ 1.73 (1H, br s), 2.28 (3H, s), 2.36 (3H, s), 2.49-
2.62 (6H, m), 2.70-2.75 (2H, m), 6.93-7.00 (2H, m), 7.11-
7.16 (2H, m). 13C NMR (CDCl3): δ 33.00, 36.41, 42.13, 49.32,
56.88, 59.52, 114.79, 115.07, 129.90, 130.00, 136.16, 136.21,
159.65, 162.87. Mp (bisoxalate salt): 207-209 °C.
N -[3-(4-B r o m o p h e n y l)-2-p r o p e n y l]-1,2-e t h a n e d i-
a m in e (11) was prepared as described for 10 to give the
product 11 (25%) as a white solid. 1H NMR (CDCl3): δ 1.34
(2H, br s), 2.90 (2H, t, J ) 5.9), 3.43 (2H, q, J ) 5.8), 6.48 (1H,
d, J trans ) 15.7), 6.85 (1H, br s), 7.29-7.32 (2H, m), 7.42-7.46
(2H, m), 7.54 (1H, d, J trans ) 15.6). 13C NMR (CDCl3): δ 41.26,
42.22, 121.49, 123.59, 129.02, 131.87, 133.65, 139.32, 165.91.
Mp (free base): 129-132 °C, (oxalate salt) 183-186 °C.
N,N′-Dim et h yl-N-[2-(b isp h en ylm et h oxy)et h yl]-N′-(2-
p h en yleth yl)-1,3-p r op a n ed ia m in e (4) was prepared as
described for 3 to give the title compound 4 (83%) as a colorless

3654 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Choi et al.
N-(P h en yla cetyl)-1,2-eth a n ed ia m in e (12) was prepared
as described for 10 to give the product 12 (23%) as a slightly
yellow oil, with the exception that the commercially available
phenylacetyl chloride was used. 1H NMR (CDCl3): δ 1.32 (2H,
br s), 2.75 (2H, t, J ) 6.0), 3.25 (2H, q, J ) 5.9), 3.57 (2H, s),
6.03 (1H, br s), 7.26-7.38 (5H, m). 13C NMR (CDCl3): δ 41.18,
42.18, 43.73, 127.18, 128.86, 129.26, 134.95, 171.26. Mp
(oxalate salt): 135-138 °C.
N-(3-P h en ylp r op ion yl)-1,2-eth a n ed ia m in e (13) was pre-
pared as described for 10 to give the product 13 (28%) as a
slightly yellow oil with the exception that the commercially
available 3-phenylpropionyl chloride was used. 1H NMR
(CDCl3): δ 1.29 (2H, br s), 2.48 (2H, t, J ) 7.7), 2.72 (2H, t, J
) 5.9), 2.97 (2H, t, J ) 7.7), 3.24 (2H, q, J ) 5.8), 6.06 (1H, br
s), 7.17-7.31 (5H, m). 13C NMR (CDCl3): δ 31.73, 38.44, 41.21,
41.98, 126.15, 128.28, 128.42, 140.81, 172.29. Mp (oxalate
salt): 127-132 °C.
N-(P h en yla cet yl)-1,3-p r op a n ed ia m in e (14) was pre-
pared as described for 10 to give the product 14 (35%) as a
slightly yellow oil with the exception that the commercially
available phenylacetyl chloride was used. 1H NMR (CDCl3):
δ 1.30 (2H, br s), 1.54 (2H, quintet, J ) 6.5), 2.68 (2H, t, J )
6.4), 3.31 (2H, q, J ) 6.2), 3.55 (2H, s), 6.39 (1H, br s), 7.24-
7.37 (5H, m). 13C NMR (CDCl3): δ 32.11, 37.95, 39.89, 43.78,
127.08, 128.79, 129.35, 135.05, 171.05. Mp (oxalate salt): 105-
108 °C.
N-[2-[Bis(4-flu or op h en yl)m et h oxy]et h yl]-N′-(4-flu o-
r op h en yla cetyl)-1,2-eth a n ed ia m in e (15). (4-Fluorophenyl-
acetyl)-1,2-ethanediamine (1.1 g, 5.6 mmol) (10) was dissolved
in 20 mL of anhydrous DMF and stirred with K2CO3 powder
(2.30 g, 16.8 mmol) for 0.5 h. To this turbid solution was added
1-[bis(4-fluorophenyl)methoxy]-2-bromoethane (1.2 g, 3.7 mmol)
in 10 mL of DMF dropwise. The reaction mixture was stirred
for 24 h at ambient temperature. The turbid reaction mixture
was poured into 200 mL of ethyl acetate. The organic layer
was separated, washed with saturated NaCl solution (60 mL
× 3), dried over MgSO4, and evaporated to dryness. The crude
oil was applied to a silica gel column for purification. Elution
with CHCl3:MeOH:Et3N (97:3:0.3) afforded the desired
monoalkylated product 15 (1.20 g, 2.71 mmol) as a colorless
oil. 1H NMR (CDCl3): 1.61 (1H, br s), 2.69 (2H, t, J ) 5.9),
2.75 (2H, t, J ) 5.2), 3.29 (2H, q, J ) 5.8), 3.46 (2H, t, J )
5.2), 3.49 (2H, s), 5.30 (1H, s), 6.04 (1H, br s), 6.95-7.05 (6H,
m), 7.17-7.29 (6H, m). 13C NMR (CDCl3): δ 39.07, 42.76, 48.01,
48.67, 68.34, 82.52, 115.18, 115.46, 115.75, 128.41, 128.52,
130.77, 130.88, 137.62, 137.66, 160.51, 163.77, 170.75. Mp
(oxalate salt): 157-159 °C. Anal. Calcd for C27H27N2O6F3: C,
60.90; H, 5.11; N, 5.26. Found: C, 60.74; H, 5.20; N, 5.16.
scribed for 15 to give the title compound 18 (73%) as a slightly
yellow oil.
1H NMR (CDCl3): δ 1.96 (1H, br s), 2.44 (2H, t, J
) 7.8), 2.69 (2H, t, J ) 5.8), 2.78 (2H, t, J ) 5.2), 2.94 (2H, t,
J ) 7.8), 3.30 (2H, q, J ) 5.7), 3.50 (2H, t, J ) 5.2), 5.32 (1H,
s), 5.98 (1H, br s), 6.97-7.05 (4H, m), 7.15-7.29 (9H, m). Mp
(oxalate salt): 144-146 °C. Anal. Calcd for C28H30N2O6F2: C,
63.63; H, 5.72; N, 5.30. Found: C, 63.42; H, 5.84; N, 5.25.
N-[2-[Bis(4-flu or op h en yl)m et h oxy]et h yl]-N′-(p h en yl-
a cetyl)-1,3-p r op a n ed ia m in e (19) was prepared as described
1
for 15 to give the product 19 (85%) as a slightly yellow oil. H
NMR (CDCl3): δ 1.57 (2H, quintet, J ) 6.4), 1.60 (1H, br s),
2.60 (2H, t, J ) 6.4), 2.70 (2H, t, J ) 5.3), 3.30 (2H, q, J )
6.1), 3.44 (2H, t, J ) 5.3), 3.50 (2H, s), 5.30 (1H, s), 6.50 (1H,
br s), 6.96-7.05 (4H, m), 7.20-7.33 (9H, m). 13C NMR
(CDCl3): δ 28.83, 38.84, 43.87, 47.88, 49.09, 68.21, 82.46,
115.15, 115.43, 127.08, 128.41, 128.52, 128.79, 129.34, 135.16,
137.67, 137.71, 160.49, 163.76, 170.90. Mp (oxalate salt): 148-
149 °C. Anal. Calcd for C28H30N2O6F2: C, 63.63; H, 5.72; N,
5.30. Found: C, 63.51; H, 5.68; N, 5.22.
N-[2-[Bis(4-flu or op h en yl)m eth oxy]eth yl]-N′-(2-p h en yl-
eth yl)-1,3-p r op a n ed ia m in e (20). N-[2-[Bis(4-fluorophenyl)-
methoxy]ethyl]-N′-(phenylacetyl)-1,3-propanediamine (0.76 g,
1.7 mmol) (19) in 10 mL of anhydrous ether was added
dropwise to 6 mL (6 mmol) of 1 M solution of LiAH4 in ether,
and the mixture was stirred at room temperature for 24 h.
After careful workup (water, 20% NaOH addition to destroy
excess LiAH4), the mixture was filtered and reextracted with
ether from the filter cake. The combined organic filtrate and
extracts were dried over MgSO4, filtered, and evaporated to
give an oil that was purified by silica gel column chromatog-
raphy (CHCl3:MeOH:Et3N, 96:4:0.4). The reduced product 20
was purified as a colorless oil (0.36 g, 0.85 mmol, 50%) and
converted to the bismalonate salt which was recrystallized
from iPrOH-H2O. 1H NMR (CDCl3): δ 1.49 (2H, br s), 1.66
(2H, quintet, J ) 7.0), 2.64 (2H, t, J ) 7.0), 2.68 (2H, t, J )
7.0), 2.76-2.89 (6H, m), 3.53 (2H, t, J ) 5.3), 5.32 (1H, s),
6.97-7.04 (4H, m), 7.17-7.32 (9H, m). Mp (bismalonate salt):
153-155 °C. Anal. Calcd for C32H38N2O9F2: C, 60.75; H, 6.05;
N, 4.43. Found: C, 60.86; H, 6.12; N, 4.38.
2-Ketop ip er a zin e (23). A solution of ethyl chloroacetate
(21) (4.9 g, 40 mmol) in 40 mL of absolute ethanol was slowly
added dropwise over a period of 3.5 h at ambient temperature
to ethylenediamine (24 g, 400 mmol) in 100 mL of absolute
ethanol. The reaction mixture was allowed to stand for 2 h
after addition was completed. Sodium ethoxide (15 mL, 40
mmol, 21 wt % solution in denatured ethyl alcohol) was added.
The precipitated sodium chloride was filtered off, the solvent
was removed by evaporation, and 40 mL of DMF was added
to the resultant red oil. The reaction mixture was allowed to
stir for 24 h at ambient temperature and then heated at about
60-70 °C while removing the volatile materials with N2 gas.
The resultant yellow solid was applied to silica gel column for
separation. The crude product (3.3 g, 33 mmol, 82%) was
obtained by elution with a solvent mixture (CHCl3:MeOH:NH4-
OH, 9:1:0.1). This crude yellow solid was used for next
synthesis without further purification. Recrystalization three
times from acetone gave well-defined, pure-white crystals. 1H
NMR (CDCl3): δ 1.70 (1H, br s), 3.03 (2H, t, J ) 5.4), 3.37
(2H, td, J ) 2.3, 5.4), 3.52 (2H, s), 6.54 (1H, br s). 13C NMR
(CDCl3): δ 42.31, 43.05, 49.83, 170.00. Mp: 132-134 °C
(uncorrected) (lit.13 mp 136 °C, corrected).
N-[2-[Bis(4-flu or op h en yl)m eth oxy]eth yl]-N′-[3-(4-br o-
m op h en yl)-2-p r op en yl]-1,2-eth a n ed ia m in e (16) was pre-
pared as described for 15 to give the product 16 (82%) as a
colorless oil. 1H NMR (CDCl3): δ 1.64 (1H, br s), 2.80-2.88
(4H, m), 3.46 (2H, q, J ) 5.7), 3.54 (2H, t, J ) 5.1), 5.33 (1H,
s), 6.32 (1H, br s), 6.34 (1H, d, J trans ) 15.6), 6.97-7.03 (4H,
m), 7.25-7.33 (6H, m), 7.46-7.50 (2H, m), 7.54 (1H, d, J trans
) 15.6). 13C NMR (CDCl3): δ 39.13, 48.16, 48.75, 68.32, 82.55,
115.20, 115.49, 121.40, 123.68, 128.44, 128.55, 129.12, 131.97,
133.78, 137.63, 137.68, 139.50, 160.53, 163.80, 165.55. Mp
(oxalate): 170-172 °C. Anal. Calcd for C28H27N2O6F2Br: C,
55.55; H, 4.49; N, 4.63. Found: C, 55.56; H, 4.52; N, 4.66.
N-[2-[Bis(4-flu or op h en yl)m et h oxy]et h yl]-N′-(p h en yl-
a cetyl)-1,2-eth a n ed ia m in e (17) was prepared as described
for 15 to give the product 17 (74%) as a slightly yellow oil.
1H NMR (CDCl3): δ 1.50 (1H, br s), 2.67 (2H, t, J ) 6.0),
2.74 (2H, t, J ) 5.2), 3.28 (2H, q, J ) 5.8), 3.45 (2H, t,
J ) 5.4), 3.52 (2H, s), 5.30 (1H, s), 6.06 (1H, br s), 6.97-7.04
(4H, m), 7.21-7.32 (9H, m). 13C NMR (CDCl3): δ 39.07, 43.63,
48.01, 48.63, 68.29, 82.41, 115.08, 115.36, 127.03, 128.35,
128.45, 128.73, 129.20, 134.96, 137.59, 137.63, 160.41, 163.67,
170.92. Mp (oxalate salt): 143-145 °C. Anal. Calcd for
4-[2-(Bisp h en ylm eth oxy)eth yl]-2-k etop ip er a zin e (24).
A solution of 1-(bisphenylmethoxy)-2-bromoethane (1.97 g, 6.8
mmol) in 10 mL of DMF was added slowly to crude 2-ketopip-
erazine (1.0 g, 10 mmol) and potassium carbonate (2.8 g, 20
mmol) in 15 mL of DMF. The reaction mixture was stirred
overnight at ambient temperature. About 30 mL of water was
added slowly to the reaction mixture which was then extracted
with ether (60 mL × 3). The ether layer was washed with 50%
NaCl solution (60 mL × 1), dried over magnesium sulfate, and
filtered. The slightly yellow solid which was obtained from
evaporation of ether was applied for column chromatography
on silica gel. Elution with a solvent mixture (CHCl3:MeOH:
C
27H28N2O6F2: C, 63.03; H, 5.48; N, 5.44. Found: C, 62.91; H,
5.48; N, 5.40.
N-[2-[Bis(4-flu or op h en yl)m et h oxy]et h yl]-N′-(h yd r o-
cin n a m oyl)-1,2-et h a n ed ia m in e (18) was prepared as de-

Non-Piperazine Analogues as DAT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3655
Et3N, 97:3:0.3) afforded 1.85 g (5.96 mmol, 87.6%) of product
24 as fine white crystals. 1H NMR (CDCl3): δ 2.71 (2H, t, J )
5.4), 2.73 (2H, t, J ) 5.6), 3.21 (2H, s), 3.28-3.33 (2H, m), 3.59
(2H, t, J ) 5.6), 5.35 (1H, s), 7.14 (1H, br s), 7.20-7.36 (10H,
m). 13C NMR (CDCl3): δ 41.11, 49.24, 56.69, 57.12, 66.74,
84.00, 127.00, 127.40, 128.29, 141.90, 169.81. Mp (free base):
93-94 °C.
were incubated for 90 min at room temperature in the dark,
and the 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 converted
to Ki values using the Cheng-Prusoff equation.
4-(2-P h en yleth yl)-2-k etop ip er a zin e (26) was prepared
as described for 24 to give the title compound 26 (67%) as a
[3H]Neu r otr a n sm itter u p ta k e by HEK 293 cells ex-
p r essin g r ecom bin a n t biogen ic a m in e tr a n sp or ter s:
HEK 293 cell lines, transfected with NET, DAT, and SERT
transporters,35,36 were plated on 150-mm dishes and grown
until confluent. The medium was removed, and the cells were
washed twice at room temperature with 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 pipet. One plate provided sufficient cells for
48 measurements, and cells from multiple plates were com-
bined. Krebs HEPES buffer (350 µL) and drugs (50 µL) were
added to a 1.0-mL assat vial 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]5-HT, or [3H]NE (50
µL, 20 nM 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% poly(ethylenimine). 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 6 drug concentrations. The
results were analyzed using GraphPAD Prism.
Locom otor a ctivity: These measurements were performed
with 16 Digiscan locomotor activity testing chambers (40.5 ×
40.5 × 30.5 cm). Panels of infrared beams (16 beams) and
corresponding photodetectors were located in the horizontal
direction along the sides of each activity chamber. Twenty
minutes prior to locomotor activity testing, groups of nonha-
bituated male Swiss Webster mice (n ) 8 each) received an
intraperitoneal (ip) injection of either vehicle (methylcellulose)
or compound 16 (3, 10, 30, or 100 mg/kg). Immediately before
placement in the apparatus, all animals were injected with
saline (0.5 mL, ip). In all studies, horizontal activity (inter-
ruption of 1 photocell beam) was measured for 1 h within 10-
min periods. Testing was conducted with one mouse per
activity chamber. In the cocaine/compound 16 interaction
study; 20 min following ip vehicle or 16 injections (3, 10, 30,
or 100 mg/kg), groups of 8 nonhabituated male Swiss Webster
mice were injected with either 0.9% saline or cocaine (20 mg/
kg, ip) and placed in the Digiscan apparatus for a 1-h session.
1
slightly yellow solid. H NMR (CDCl3): δ 2.67-2.71 (4H, m),
2.78-2.83 (2H, m), 3.20 (2H, s), 3.33-3.37 (2H, m), 7.19-7.22
(3H, m), 7.27-7.31 (2H, m), 7.52 (1H, br s). 13C NMR (CDCl3):
δ 33.34, 41.04, 48.97, 56.69, 59.12, 126.11, 128.33, 128.52,
139.51, 169.88. Mp (free base): 113-115 °C.
4-[2-(Bisp h en ylm et h oxy)et h yl]-1-(3-p h en ylp r op yl)-2-
k et op ip er a zin e (25). 4-[2-(Bisphenylmethoxy)ethyl]-2-ke-
topiperazine (24) (0.68 g, 2.2 mmol) was dissolved in 10 mL of
anhydrous DMF and stirred with sodium hydride powder (0.06
g, 2.5 mmol) under a N2 atmosphere. After 0.5 h, 1-bromo-3-
phenylpropane (0.44 g, 2.2 mmol) in 5 mL of DMF was added
dropwise to the turbid reaction mixture at ambient tempera-
ture. The reaction mixture was allowed to stir for 16 h under
N2 gas atmosphere. The remaining sodium hydride was
destroyed with methanol. About 20 mL of water was added
slowly to the reaction mixture which was then extracted with
ether (50 mL × 3). The separated ether layer was dried over
magnesium sulfate, filtered, and evaporated to yellow viscous
oil. Silica gel column purification using a solvent mixture
(hexane:EtOIAc:Et3N, 50:50:2) afforded 600 mg (1.4 mmol,
64%) of pure product as a slightly yellow, viscous oil. 1H NMR
(CDCl3): δ 1.88 (2H, quintet, J ) 7.7), 2.63 (2H, t, J ) 7.8),
2.69 (2H, t, J ) 5.7), 2.72 (2H, t, J ) 5.4), 3.20 (2H, s), 3.26
(2H, t, J ) 5.4), 3.41 (2H, t, J ) 7.5), 3.58 (2H, t, J ) 5.7),
5.35 (1H, s), 7.15-7.34 (15H, m). 13C NMR (CDCl3): δ 28.37,
33.11, 46.03, 46.45, 50.12, 56.73, 57.72, 66.79, 84.06, 84.09,
125.85, 126.88, 127.46, 128.23, 128.35, 141.47, 141.94, 166.77.
Mp (oxalate salt): 126-128 °C. Anal. Calcd for C30H34N2O6:
C, 69.48; H, 6.61; N, 5.40. Found: C, 69.55; H, 6.66; N, 5.43.
1-[2-[Bis(4-flu or op h en yl)m et h oxy]et h yl]-4-(2-p h en yl-
eth yl)-2-k etop ip er a zin e (27) was prepared as described for
25 to give the title compound 27 (65%) as a slightly yellow oil.
1H NMR (CDCl3): δ 2.62-2.68 (2H, m), 2.72 (2H, t, J ) 5.4),
2.78-2.83 (2H, m), 3.21 (2H, s), 3.51 (2H, t, J ) 5.4), 3.61 (4H,
s), 5.31 (1H, s), 6.96-7.03 (4H, m), 7.19-7.31 (9H, m). 13C
NMR (CDCl3): δ 33.49, 46.80, 48.50, 50.00, 57.36, 59.23, 67.10,
82.48, 82.52, 115.19, 115.48, 128.38, 128.46, 128.62, 137.57,
137.61, 139.57, 160.51, 163.77, 167.02. Mp (oxalate salt): 138-
141 °C, (HCl salt) 150-152 °C. Anal. Calcd for C29H30N2O6F2
(oxalate salt): C, 64.44; H, 5.59; N, 5.18. Found: C, 64.37; H,
5.60; N, 5.12. Anal. Calcd for C27H29N2O2F2Cl (HCl salt): C,
66.59; H, 6.00; N, 5.75. Found: C, 66.47; H, 6.03; N, 5.72.
Biologica l Meth od s. [125I]RTI-55 bin d in g a ssa 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 cell lines, transfected with NET, DAT, and SERT trans-
porters,35,36 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 (PBS), and 10 mL of lysis
buffer (2 mM HEPES, 1 mM EDTA) was added. After 10 min,
cells were scraped from plates, poured into centrifuge tubes,
and centrifuged for 20 min at 30000g. The supernatant was
removed, and the pellet was resuspended in 20-32 mL of 0.32
M sucrose, depending on the density of binding sites in a given
cell line (i.e. resuspension volumes which bound e10% of the
total radioactivity). The pellets were 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 was used as the
assay diluent. The membranes were preincubated with drug
for 10 min prior to addition of [125I]RTI-55. The assay tubes
Ack n ow led gm en t. This work was supported in part
by a Benedict Cassen Postdoctoral Fellowship (S.W.C.)
sponsored by the Education and Research Council of
the Society of Nuclear Medicine. The in vitro assays
were performed by Dr. A. J anowsky or the Oregon
Health Sciences University under a contract from the
Cocaine Treatment Discovery Program of the National
Institute on Drug Abuse, Medication Development Divi-
sion (Contract No. NO1DA-7-8071).
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