Efficient asymmetric syntheses, determination of absolute configurations and biological activities of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3- (phenylamino)propyl]piperazine as a novel potent dopamine uptake inhibitor in the central nervous system

Article abstract of DOI:10.1016/j.bmc.2004.02.041

An efficient asymmetric synthesis of the chiral N-(3-chloro-2- hydroxypropyl)anilines (2a and 2b) was achieved through the regioselective ring-opening reaction of chiral epichlorohydrin with aniline. This was applied to an asymmetric synthesis of the enantiomers of 1-[4,4-bis(4-fluorophenyl) butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 1 as a novel potent dopamine uptake inhibitor. Both enantiomers as trihydrochlorides, 4a·3HCl and 4b·3HCl, could be synthesized in good total yields and optical purities of 100% ee in three steps synthesis, respectively. The absolute configurations of 4a·3HCl and 4b·3HCl were determined using the modified Mosher's method with the related compounds, the intermediates (2a and 2b) and the free bases (4a and 4b). The analytical results indicated that 4a·3HCl and 4b·3HCl have the (S)- and (R)-configuration, respectively, and a series of reactions to provide them proceeded without the apparent influence on the stereochemistry at the chiral centers. In in vitro pharmacological evaluations, 4a·3HCl and 4b·3HCl showed potent dopamine transporter binding affinities, high dopamine, moderate serotonin, and weak norepinephrine uptake inhibitory activities, and 4a·3HCl exhibited a more potent and selective dopamine uptake inhibition over the serotonin or norepinephrine uptake inhibition as compared with 4b·3HCl. An ex vivo evaluation revealed that the oral administrations of both enantiomers at a dose of 30mg/kg in rats displayed apparent dopamine uptake inhibitory activities and 4a·3HCl had a stronger tendency to inhibit dopamine uptake compared with 4b·3HCl.

Full text of DOI:10.1016/j.bmc.2004.02.041

Bioorganic & Medicinal Chemistry 12 (2004) 3069–3078
Efficient asymmetric syntheses, determination of absolute
configurations and biological activities of 1-[4,4-bis(4-
fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]-
piperazine as a novel potent dopamine uptake inhibitor in
the central nervous system
Makoto Kimura,a,* Tomoko Masuda,a Koji Yamada,a Masaki Mitani,a Nobuo Kubota,a
Nobuyuki Kawakatsu,a Kenichi Kishii,a Masato Inazu,b Yuji Kiuchi,c Katsuji Oguchid
and Takayuki Namikia,*
aPOLA Chemical Industries, Inc., Pharmaceutical R&D Laboratories, 560 Kashio-cho, Totsuka-ku, Yokohama,
Kanagawa 244-0812, Japan
bDepartment of Pharmacology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
cDepartment of Pathophysiology, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku,
Tokyo 142-8555, Japan
dDepartment of Pharmacology, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Received 14 November 2003; accepted 25 February 2004
Available online 27 April 2004
Abstract—An efficient asymmetric synthesis of the chiral N-(3-chloro-2-hydroxypropyl)anilines (2a and 2b) was achieved through
the regioselective ring-opening reaction of chiral epichlorohydrin with aniline. This was applied to an asymmetric synthesis of the
enantiomers of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 1 as a novel potent dopamine
uptake inhibitor. Both enantiomers as trihydrochlorides, 4aÆ3HCl and 4bÆ3HCl, could be synthesized in good total yields and optical
purities of 100% ee in three steps synthesis, respectively. The absolute configurations of 4aÆ3HCl and 4bÆ3HCl were determined using
the modified Mosher’s method with the related compounds, the intermediates (2a and 2b) and the free bases (4a and 4b). The
analytical results indicated that 4aÆ3HCl and 4bÆ3HCl have the (S)- and (R)-configuration, respectively, and a series of reactions to
provide them proceeded without the apparent influence on the stereochemistry at the chiral centers. In in vitro pharmacological
evaluations, 4aÆ3HCl and 4bÆ3HCl showed potent dopamine transporter binding affinities, high dopamine, moderate serotonin, and
weak norepinephrine uptake inhibitory activities, and 4aÆ3HCl exhibited a more potent and selective dopamine uptake inhibition
over the serotonin or norepinephrine uptake inhibition as compared with 4bÆ3HCl. An ex vivo evaluation revealed that the oral
administrations of both enantiomers at a dose of 30 mg/kg in rats displayed apparent dopamine uptake inhibitory activities and
4aÆ3HCl had a stronger tendency to inhibit dopamine uptake compared with 4bÆ3HCl.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
porter (DAT) located in the dopaminergic nerve
terminals.1 Dopaminergic neurotransmission has been
reported to be closely associated with the CNS disorders
such as Parkinson’s disease,2 depression,3 or cocaine
abuse,4;5 so the DAT has been suggested as one target
for research into new treatments of these CNS disorders.
Dopamine (DA) is a neurotransmitter involved in many
biological processes in the central nervous system (CNS)
and the dopaminergic neurotransmission is terminated
by a reuptake mechanism through the dopamine trans-
In our previous study,6;7 we reported that novel diphenyl
piperazine derivatives represented by 1-[4,4-bis(4-fluoro-
phenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piper-
azine 1 (Fig. 1) showed apparent binding affinities for
Keywords: Dopamine uptake inhibitory activity; Diphenyl piperazine
derivative; Asymmetric synthesis; Absolute configuration.
* Corresponding authors. Tel.: +81-45-826-7264; fax: +81-45-826-72-
49; e-mail addresses: m-kimura@pola.co.jp; t-namiki@pola.co.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.041

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M. Kimura et al. / Bioorg. Med. Chem. 12 (2004) 3069–3078
H
OH
N
)
N (CH2
3
N
N
N
O
F
F
F
F
1
GBR12909
Figure 1.
the DAT in rat striatal membranes and some of them
were approximately equivalent in activity to GBR129098
(Fig. 1) known as a potent DA uptake inhibitor. In
addition, from a pharmacological study using in vivo
brain microdialysis, compound 1 was found to increase
extracellular DA levels in rat striatum significantly and
dose dependently, which was much greater than that by
GBR12909. These findings indicated that these novel
diphenyl piperazine derivatives including compound 1
could serve as new DA uptake inhibitory molecules
different from GBR12909 analogues. Moreover, it was
suggested that these compounds might show great
promise for the treatments of the CNS disorders caused
by the depletion of DA or related to the DAT such as
Parkinson’s disease,2 depression,3 or cocaine addiction,9
and also help to characterize biological and pharmaco-
logical profiles of the DAT.10
quantities sufficient for their biological and pharmaco-
logical studies. Since compound 1 has a b-phenylamino-
2-propanol moiety containing an asymmetric carbon in
its structure, it was considered that an efficient con-
struction of this moiety in its optically active and pure
form would play an important role in the asymmetric
synthesis of the enantiomers of 1. The ring opening of
epoxides with amines is a simple and useful procedure
for the synthesis of b-amino alcohols, and various pro-
cedures involving the direct reaction of epoxides with
amines and the procedures using additives such as metal
or metal catalyst have been reported.12–18 Among them,
we focused on the ring-opening reaction of epichloro-
hydrin with aniline for the efficient construction of the
b-phenylamino-2-propanol moiety required for the
subsequent reaction with 1-[4,4-bis(4-fluorophenyl)-
butyl]piperazine, and selected commercially available
chiral epichlorohydrin as the chiral synthon. However,
all three carbons of epichlorohydrin have the potential
to be attacked by amines depending on the reaction
conditions, and this may cause a decrease in the yield
and optical purity of the desirable chiral b-amino-2-
propanol derivatives. Therefore, the ring-opening reac-
tion of chiral epichlorohydrin with aniline was necessary
to be regioselective and provide the optically pure
product. So, we examined in detail this ring-opening
reaction under neutral conditions without additives.
However, all of these compounds are racemates with
respect to the asymmetric carbons in the b-amino-2-
propanol moieties. Since it is generally accepted that
enantiomers exhibit different biological properties,11 it is
very important and interesting to synthesize the enanti-
omers of these compounds, determine their absolute
configurations, and compare their biological and phar-
macological activities as a DA uptake inhibitor.
In this paper, we established an efficient asymmetric
synthesis of the enantiomers of compound 1 as a repre-
sentative of these novel diphenyl piperazine derivatives
and determined their absolute configurations at the
secondary alcohol moiety along with those of their
important intermediates using the modified Mosher’s
method. As in vitro pharmacological evaluations, bind-
ing affinities for the DAT and uptake inhibitory activities
for DA of both enantiomers were evaluated, and in order
to evaluate the selectivity for DA uptake inhibition, their
uptake inhibitory activities for serotonin (5-HT) and
norepinephrine (NE) were also examined. Moreover, for
a preliminary in vivo evaluation, ex vivo DA uptake
inhibitory activities of both enantiomers were measured.
The ring-opening reaction of (S)-(+)-epichlorohydrin
with aniline was undertaken under various reaction
conditions (Scheme 1), and the results are shown in
Table 1. The treatment of (S)-(+)-epichlorohydrin with
1.0 equiv of aniline in ethanol (EtOH) at room temper-
ature for 65 h produced the desirable chiral N-(3-chloro-
2-hydroxypropyl)aniline 2a in a 54% yield and 97.3% ee
(entry 1), together with N,N-bis(3-chloro-2-hydroxy-
propyl)aniline 3a in a 7% yield as a major by-product
that was separable by silica gel column chromatography.
Cl
*
*
OH
H
a
OH
OH
N
Cl
2. Results and discussion
+
NH2
N
*
Cl
2.1. Efficient synthesis of optically active 1-[4,4-bis(4-
fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)prop-
yl]piperazine
2a : from (S)-epichlorohydrin
2b : from (R)-epichlorohydrin
3a : from (S)-epichlorohydrin
An efficient and scalable asymmetric synthesis of the
enantiomers of compound 1 was required to obtain
Scheme 1. Reagents and conditions: (a) (S)-(+)- or (R)-())-epichlo-
rohydrin, EtOH.

M. Kimura et al. / Bioorg. Med. Chem. 12 (2004) 3069–3078
Table 1. Preparations of chiral N-(3-chloro-2-hydroxypropyl)anilines 2a and 2b
3071
Entry
Epichlorohydrin
(configuration)
Conditions
Product
Yield (%)
eea (%)
Aniline (equiv)
Solvent
Temp.
Time (h)
1
2
3
4
5
6
7
(S)
(S)
(S)
(S)
(S)
(S)
(R)
1.0
1.5
2.0
2.0
2.0
2.0
2.0
EtOH
EtOH
EtOH
EtOH
EtOH
––
rt
65
65
65
9
2a
2a
2a
2a
2a
2a
2b
54
60
65
72
89
52
89
97.3
97.8
97.9
96.9
96.7
94.9
96.9
rt
rt
40 ꢁC
Reflux
Reflux
Reflux
2.5
6
EtOH
2.5
a Enantiomeric excesses were determined by chiral HPLC analysis.
No remarkable decrease in the optical purity of 2a was
observed by chiral HPLC analysis, showing that the
optical purity of 2a was almost retained considering that
of chiral epichlorohydrin (98.8% ee) as the starting
material. To suppress the production of 3a and increase
that of 2a, (S)-(+)-epichlorohydrin was treated with 1.5
or 2.0 equiv of aniline in EtOH at room temperature
(entries 2 and 3). The use of excess aniline simulta-
neously induced an increase in 2a production and sup-
pression in 3a, and the production of 3a was reduced to
a minimum (0.9% yield) when 2.0 equiv of aniline were
used. Reaction temperature is closely related to reaction
time and product yield and is an important factor for
efficient synthesis. Therefore, we next examined the
effect of the reaction temperature on this reaction (entries
4 and 5). The results showed that increasing the reaction
temperature was effective in reducing the reaction time
and increasing the yield of 2a without the apparent
influence on the regioselectivity and optical purity.
Eventually, under reflux conditions, 2a could be pre-
pared in an 89% yield and 96.7% ee (entry 5). Since the
ring-opening reaction of racemic epichlorohydrin with
found to be applicable to the asymmetric synthesis of
b-amino- or b-thio-2-propanol derivatives using various
aliphatic and aromatic amines or thiols (data not
shown).
Next, we attempted to apply this regioselective ring-
opening reaction of chiral epichlorohydrin with aniline
to the asymmetric synthesis of both enantiomers of 1
(Scheme 2). As described above, the chiral N-(3-chloro-
2-hydroxypropyl)anilines, 2a and 2b, as the important
intermediates were prepared from chiral epichlorohyd-
rins and aniline under reflux conditions in EtOH. The
subsequent alkylation of 1-[4,4-bis(4-fluorophenyl)-
butyl]piperazine with 2a or 2b in the presence of
potassium carbonate and potassium iodide in EtOH
under reflux gave the desirable diphenylbutyl piperazine
derivatives 4a or 4b in 90% or 92% yield after purifica-
tion by silica gel column chromatography, respectively,
and both compounds showed an optical purity greater
than 98.5% ee. These findings indicate that this alkyl-
ation proceeded in high yield and excellent optical
purity, showing no occurrence of racemization in the b-
amino-2-propanol moiety. Subsequently, the treatment
of the resultant diphenyl piperazine derivatives 4a and
4b with hydrogen chloride in EtOH followed by
recrystallization gave the corresponding trihydrochlo-
rides, 4aÆ3HCl and 4bÆ3HCl, in high yields and optical
purities of 100% ee, respectively.
aniline in the absence of solvent has been reported,12
a
similar reaction using (S)-(+)-epichlorohydrin was
attempted (entry 6). However, this reaction without
solvent under heated conditions resulted in a significant
decrease of the yield of 2a with an increase of 3a and a
decrease of the optical purity of 2a responsible for the
production of 2b. Using the same procedure as in entry
5, the ring-opening reaction of (R)-())-epichlorohydrin
with aniline gave the other enantiomer 2b in an 89%
yield and 96.9% ee (entry 7). Thus, the above results
indicate that the ring opening of chiral epichlorohydrin
Thus, the total yields of 4aÆ3HCl and 4bÆ3HCl after three
steps synthesis starting from chiral epichlorohydrin were
66% and 77%, respectively. Furthermore, the efficient
procedure to provide 4aÆ3HCl and 4bÆ3HCl established
herein has also been found to be applicable to their
asymmetric syntheses on a kilogram scale by a minor
modification (data not shown). Both enantiomers of 1
with aniline proceeded in
a highly regioselective
manner with high optical purity. Moreover, this ring-
opening reaction of chiral epichlorohydrin was also
H
OH
H
OH
N
a
N
Cl
b
c
.
4a 3HCl
NH2
*
N
N
*
.
4b 3HCl
F
F
4a : from 2a
4b : from 2b
2a
2b
Scheme 2. Reagents and conditions: (a) (S)-(+)- or (R)-())-epichlorohydrin, EtOH, reflux, 89%; (b) 1-[4,4-bis(4-fluorophenyl)butyl]piperazine,
K2CO3, KI, EtOH, reflux 90–92%; (c) 7.0 N HCl in EtOH, 82–94%.

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M. Kimura et al. / Bioorg. Med. Chem. 12 (2004) 3069–3078
obtained as trihydrochlorides, 4aÆ3HCl and 4bÆ3HCl,
were used for the pharmacological evaluations.
(S)-MTPA esters 5–12 were prepared from the corre-
sponding secondary alcohols, 2a, 2b, 4a, and 4b, and
(S)- or (R)-MTPA chloride in the presence of N,N-
dimethylaminopyridine and triethylamine in dichlo-
romethane (Schemes 3 and 4).22;23 The 1H NMR spectra
obtained from the MTPA esters could be registered, and
the chemical shifts for as many protons as possible with
respect to the obtained MTPA esters could be assigned.
The chemical shift differences (Dd ¼ dS À dR) are calcu-
lated and summarized in Figures 3 and 4.
2.2. Determination of absolute configuration using the
modified Mosher’s method
The absolute configurations of organic compounds can
be determined by a few direct physical methods
including X-ray crystallography, circular dichroism, or
optical rotatory dispersion, but they have some limita-
tions. On the other hand, there are popular indirect
chemical methods such as Mosher’s method, which can
also predict the absolute configurations of organic sub-
stances. In 1973, Dale and Mosher proposed that a
With respect to the chemical shift differences obtained
from the 2a MTPA esters, 5 and 6, the Dd values for the
methylene protons adjacent to the chlorine atom located
on the right side of the MTPA plane were all positive,
while those for methylene protons adjacent to the aniline
moiety and aromatic protons at the ortho-position
located on the left side of the MTPA plane were all
negative. When these results are applied to the config-
urational correlation model in Figure 2, 2a is shown to
have the (S)-configuration at the secondary carbinol
stereogenic center. For the chemical shift differences
method
using
2-methoxy-2-phenyl-2-(trifluoro-
methyl)acetic acid (MTPA) esters19 could be used to
determine the absolute configurations of many chiral
alcohols and amines with various auxiliaries.20;21 The
robustness of the method was demonstrated by modi-
fying the original Mosher’s method to expand the range
of adaptive compounds that allowed for the absolute
configuration determination of many different organic
compounds including natural products.22–24
OH
OMTPA
H
H
N
Cl
a
N
Cl
The absolute configurations of four compounds, the
important intermediates (2a and 2b) and the free bases
(4a and 4b), containing the b-amino-2-propanol moiety
were determined using the modified Mosher’s method to
elucidate those of 4aÆ3HCl and 4bÆ3HCl. The basic
concept of Mosher’s method as well as a configurational
correlation model based on the method for elucidating
the absolute configuration at the secondary carbinol
stereogenic center are shown in Figure 2.19;22 The (R)- or
*
*
5 : from 2a and (R)-MTPA chloride
6 : from 2a and (S)-MTPA chloride
7 : from 2b and (R)-MTPA chloride
8 : from 2b and (S)-MTPA chloride
2a
2b
Scheme 3. Reagents and conditions: (a) (S)- or (R)-MTPA chloride,
DMAP, Et3N, CH2Cl2, room temperature, 90–94%.
(Ph)
δR2 lowered (Ph effect)
δR1 lowered (Ph effect)
(OMe)
Ph
---(R)-MTPA ester
---(S)-MTPA ester
OMe
R2
R1
O
(S) CF3
H
O
MTPA plane
OMTPA
∆δ > 0
∆δ =δ(S)-MTPA - δ(R)-MTPA
R1
R2
∆δ < 0
H
Figure 2. Preferred conformation for MTPA esters proposed by Mosher and a model to determine the absolute configuration of secondary alcohols.
H
H
OH
N
N
OR
a
*
N
N
*
N
N
F
F
R=MTPA
F
F
9 : from 4a and (R)-MTPA chloride
10 : from 4a and (S)-MTPA chloride
11 : from 4b and (R)-MTPA chloride
12 : from 4b and (S)-MTPA chloride
4a
4b
Scheme 4. Reagents and conditions: (a) (S)- or (R)-MTPA chloride, DMAP, Et3N, CH2Cl2, room temperature, 89–94%.

M. Kimura et al. / Bioorg. Med. Chem. 12 (2004) 3069–3078
3073
obtained from the 2b MTPA esters, 7 and 8, the signs of
the Dd values on both sides of the MTPA plane were
opposite to those of the 2a MTPA esters indicating that
the absolute configuration of 2b is the (R)-configuration.
rat striatal membranes,25 and the results are shown in
Table 2. Both compounds showed the same potent DAT
binding affinities with IC50 values of 2.00 nM, which
were equivalent in potency to the racemate 1 as a tri-
hydrochloride. These results indicate that their absolute
configurations have little influence on the binding
affinity for the GBR12935 binding site at the DAT.
In the case of the 4a MTPA esters, 9 and 10, the Dd
values for the methylene protons, amine proton, and
aromatic protons at the ortho-position on the aniline
moiety side located on the left side of the MTPA plane
were all negative. On the other hand, the Dd value for
one methylene proton on the piperazine moiety side
located on the right side of the MTPA plane was posi-
tive, and that for another methylene proton was almost
zero, possibly because it was located near the MTPA
plane. For the 4b MTPA esters, 11 and 12, the contrary
chemical shift differences to those of the MTPA esters of
4a were observed. These findings indicate that 4a and 4b
have the (S)-configuration and (R)-configuration,
respectively, and the final compounds, 4aÆ3HCl and
4bÆ3HCl, also have these corresponding configurations.
2.4. Uptake inhibitory activities for monoamines, DA,
5-HT, and NE
As described in the DAT binding study, both enantio-
mers possessed equipotent binding affinities for the
DAT. Subsequently, to examine the difference in DA
uptake inhibition, DA uptake inhibitory activities of
4aÆ3HCl and 4bÆ3HCl were measured using radiolabeled
DA in rat striatal homogenates.26 In addition, to eval-
uate the selectivity for DA uptake inhibition relative to
other monoamine uptake inhibitions, 5-HT and NE
uptake inhibitory activities with them were measured
using the corresponding radiolabeled monoamines in
rat cerebral cortex homogenates.26 The results of these
uptake inhibitory activities for DA, 5-HT, and NE are
shown in Table 2.
From the results described above, it is indicated that a
series of reactions to provide 4aÆ3HCl and 4bÆ3HCl
starting from the ring opening of chiral epichlorohydrin
with aniline proceeded without the apparent influence
on the stereochemistry at the chiral centers.
With respect to the DA uptake inhibitory activity, both
enantiomers, 4aÆ3HCl and 4bÆ3HCl, showed potent
activities with IC50 values of 2.6 and 6.7 nM, respec-
tively. Thus, both enantiomers showed distinct DA
uptake inhibitory activities and 4aÆ3HCl with the (S)-
configuration was 2.6-fold more active than 4bÆ3HCl
with the (R)-configuration. These results indicate that
their absolute configurations apparently influence the
DA uptake inhibitory activity, although both com-
pounds show equipotent binding affinities for the DAT.
Since [3H]GBR12935 has been known to bind to a
piperazine acceptor site in addition to the DAT,27 the
difference in the results between both enantiomers
observed in the DAT binding affinity and DA uptake
inhibitory activity may be due to the different assay
conditions with the radioligands having the different
character.
2.3. DAT binding studies
Both enantiomers, 4aÆ3HCl and 4bÆ3HCl, were evalu-
ated for DAT binding affinity using [3H]GBR12935 in
OR
H
OR
H
H
H
Cl
N
N
Cl
+0.116
+0.020
+0.086
-0.043
-0.067
+0.090
-0.098
-0.093
R=MTPA
R=MTPA
δΗ(5) - δH(6)
(7) - δH(8)
δΗ
Figure 3. 1H NMR chemical shift differences between (S)- and (R)-
MTPA esters 5, 6, 7, and 8 derived from 2a and 2b, respectively,
expressed in ppm.
Concerning the 5-HT uptake inhibitory activity,
4aÆ3HCl and 4bÆ3HCl showed moderate activities with
IC50 values of 214.5 and 481.0 nM, respectively.
+0.624(NH)
RO HN
-0.325(NH)
+0.080
NH OR
-0.081
H
N
N
N
N
H
+0.097
+0.156
-0.043
-0.121
F
F
- 0.074
+0.002
+0.073
- 0.007
R=MTPA
R=MTPA
F
F
δΗ(9) - δH(10)
δΗ(11) - δH(12)
Figure 4. 1H NMR chemical shift differences between (S)- and (R)-MTPA esters 9, 10, 11, and 12 derived from 4a and 4b, respectively, expressed in
ppm.

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M. Kimura et al. / Bioorg. Med. Chem. 12 (2004) 3069–3078
Table 2. DAT binding affinities and monoamine uptake inhibitory activities of the racemate and the enantiomers
Compd
Configuration
DAT binding affinity
IC50 (nM)a
Monoamine uptake inhibitory activity IC50 (nM)a
[3H]DA
[3H]5-HT
[3H]NE
1aÆ3HCl
4aÆ3HCl
4bÆ3HCl
(R,S)
(S)
(R)
2.00 0.29
2.00 0.10
2.00 0.53
NTb
NTb
NTb
2.6 1.0
6.7 2.7
214.5 10.8
481.0 193.0
1817.0 883.9
2058.4 240.4
a IC50 values represent the concentrations inhibiting 50% of specific bindings and were calculated by nonlinear regression fitting. Each value
represents the mean SE from three experiments conducted in duplicate.
b Not tested.
Compound 4aÆ3HCl possessed an approximately 2.2-
fold more potent 5-HT uptake inhibitory activity than
4bÆ3HCl, which shows that their absolute configurations
have an apparent influence on potency. In comparison
between their DA and 5-HT uptake inhibitions, the
selectivities for DA to 5-HT of 4aÆ3HCl and 4bÆ3HCl
were 83- and 72-fold, respectively, showing that 4aÆ3HCl
has a more selective uptake inhibitory activity for DA
over 5-HT compared with 4bÆ3HCl.
ties for the DAT, and potent and selective DA uptake
inhibitory activities. Then, for a preliminary in vivo
evaluation, we attempted to examine and compare the
inhibition of DA uptake by both enantiomers in an
ex vivo study. The changes in DA uptake inhibition in
rat striatum for 6 h after the oral administrations of
4aÆ3HCl and 4bÆ3HCl at a dose of 30 mg/kg were mea-
sured, and the results are shown in Figure 5.
The oral administrations of 4aÆ3HCl and 4bÆ3HCl
induced eminent DA uptake inhibitions. The inhibitions
by both enantiomers reached maximum levels quickly,
and maintained high inhibition levels for 6 h, although
their activities gradually decreased. Compound 4aÆ3HCl
had a stronger tendency to inhibit DA uptake than
4bÆ3HCl, exhibiting a significant difference at 2 and 3 h
after following administrations, which may reflect their
observed in vitro DA uptake inhibitory activities.
However, the difference in the ex vivo DA uptake
inhibitory activity between both compounds was not
large compared with that in the in vitro DA uptake
inhibitory activity, which appeared due to the differences
in their pharmacokinetic properties such as absorption,
metabolism, protein binding, or distribution through
blood–brain barrier.
With respect to the NE uptake inhibitory activity,
4aÆ3HCl and 4bÆ3HCl possessed weak activities with
IC50 values of 1817.0 and 2058.4 nM, respectively, and
the difference in the NE uptake inhibition between both
enantiomers was slight. On the other hand, the ratios of
uptake inhibition for NE to DA with 4aÆ3HCl and
4bÆ3HCl were 699 and 307, respectively, showing that
the differences in uptake inhibitory activities of both
compounds between DA and NE were greater than two
orders of magnitude. Again, 4aÆ3HCl possessed an
approximately 2.3-fold more selective DA uptake
inhibitory activity as compared with 4bÆ3HCl.
The above results for monoamine uptake inhibitory
activity indicated that both enantiomers possessed high
DA, moderate 5-HT, and weak NE uptake inhibitory
activities. Compound 4aÆ3HCl was more potent in all
three monoamine uptake inhibitory activities compared
with 4bÆ3HCl. Furthermore, 4aÆ3HCl exhibited an
approximately 80- or 700-fold selective DA uptake
inhibitory activity over the 5-HT or NE uptake inhibitory
activity, and these selectivities were greater compared
with 70- or 300-fold selectivity of 4bÆ3HCl. Thus, their
absolute configurations were shown to have an apparent
tendency to influence the DA, 5-HT, and NE uptake
inhibitory activities with the selectivity for DA uptake
inhibition. These results indicate that 4aÆ3HCl with the
(S)-configuration is preferable over 4bÆ3HCl with the (R)-
configuration for the DA uptake inhibitory activity and
selectivity, which may be very useful for obtaining
knowledge about the structure and biological and phar-
macological profiles of the DAT. Furthermore, from an
in vitro selectivity study by means of other 49 neuro-
transmitter receptors, ion channels, and transporters,
4aÆ3HCl has also been found to display the highest
affinity and selectivity for the DAT (data not shown).
Thus, the above results combined with the in vitro DA
uptake inhibitory activity and selectivity indicate that
100
80
*
**
60
40
20
0
1
2
3
4
6
Time after administration (hr)
Figure 5. Effects of 4aÆ3HCl and 4bÆ3HCl on the DA uptake in the rat
striatal synaptosomes ex vivo. Compound 4aÆ3HCl (j: 30 mg/kg),
4bÆ3HCl (Ã: 30 mg/kg), or vehicle (saline) was administered orally, and
the uptake of 100 nM [3H]DA was measured for 5 min. The results are
expressed as % inhibition of specific uptake in vehicle control. Each
value represents the mean with SEM of four rats. *p < 0.05, **p < 0.01:
significantly different from 4aÆ3HCl (t-test).
2.5. Ex vivo DA uptake inhibitory activities
As described in the in vitro study, both enantiomers,
4aÆ3HCl and 4bÆ3HCl, exhibited notable binding affini-

M. Kimura et al. / Bioorg. Med. Chem. 12 (2004) 3069–3078
3075
both enantiomers are potent and selective DA uptake
inhibitors in the CNS.
analyses were performed by Yanaco CHN CORDER
MT-5 (C, H, N) and Flask Combustion (Cl). Column
chromatography were performed on silica gel (BW-200,
Fuji Silisia Chemical, Ltd, 100–200 mesh).
3. Conclusion
4.1. Determination of optical purity
We established an efficient asymmetric synthesis of the
chiral N-(3-chloro-2-hydoroxypropyl)anilines (2a and
2b) through the regioselective ring-opening reaction of
chiral epichlorohydrin with aniline and applied this
reaction to an asymmetric synthesis of the enantiomers
of 1 as a novel potent dopamine uptake inhibitor. Both
enantiomers of 1 as trihydrochlorides, 4aÆ3HCl and
4bÆ3HCl, could be synthesized in good total yields and
optical purities of 100% ee in three steps synthesis,
respectively. The absolute configurations of 4aÆ3HCl
and 4bÆ3HCl were determined using the modified
Mosher’s method with the related compounds, the inter-
mediates (2a and 2b) and the free bases (4a and 4b). The
analytical results indicated that 4aÆ3HCl and 4bÆ3HCl
have the (S)- and (R)-configuration, respectively, and a
series of reactions to provide them proceeded without
the apparent influence on the stereochemistry at the
chiral centers. In in vitro pharmacological evaluations,
both enantiomers possessed high DA uptake inhibitory
activities and 4aÆ3HCl exhibited a 2.6-fold more potent
DA uptake inhibitory activity compared with 4bÆ3HCl,
although both compounds showed equipotent DAT
binding affinities. In comparison between their DA,
5-HT, and NE uptake inhibitory activities, both enantio-
mers showed notably selective DA uptake inhibitions
over the 5-HT or NE uptake inhibition, and 4aÆ3HCl
was more selective than 4bÆ3HCl. In an ex vivo evalua-
tion, the oral administrations of both enantiomers at a
dose of 30 mg/kg in rats displayed apparent DA uptake
inhibitions, with 4aÆ3HCl having a stronger tendency to
inhibit DA uptake compared with 4bÆ3HCl. Thus, all the
above data suggest that 4aÆ3HCl and 4bÆ3HCl have
excellent potential as effective DA uptake inhibitors for
the treatment of diseases in the CNS such as Parkinson’s
disease, depression, or cocaine addiction, and also useful
tools for the characterization of the structure and bio-
logical and pharmacological profiles of the DAT. And
now, we conduct these studies using animal models.
HPLC analyses on optical purity of the intermediates,
2a and 2b, the free bases, 4a and 4b, and their corre-
sponding trihydrochlorides, 4aÆ3HCl and 4bÆ3HCl, were
performed on a Daisel Chiralcel OD-R column (4.6 mm
i.d. · 250 mm) using a JASCO system equipped with a
UV detector [eluent (0.5 M NaClO4, HClO4: pH 2.0)/
CH3CN ¼ 70:30; flow rate, 0.8 mL/min; UV at 245 nm;
temperature, 24 ꢁC]. Their optical purities were calcu-
lated based on the peak area ratios of (R)- and (S)-iso-
mers.
4.1.1. (S)-())-N-(3-Chloro-2-hydroxypropyl)aniline (2a)
and (S)-N,N-bis(3-chloro-2-hydroxypropyl)aniline (3a).
These compounds were prepared under various condi-
tions as described in Table 1. Herein, the preparation
under an optimum condition (entry 5) is described. A
mixture of (S)-(+)-epichlorohydrin (1.92 g, 20.8 mmol)
and aniline (3.87 g, 41.6 mmol) in EtOH (21 mL) was
heated under reflux for 2.5 h. The mixture was cooled to
room temperature and concentrated in vacuo. The res-
idue was separated by silica gel column chromatography
(CHCl3 as an eluent) to give 2a (3.43 g, 89%, 96.7% ee)
as white crystals and 3a (0.05 g, 0.9%) as a colorless oil.
Compound 2a. Mp 35–37 ꢁC. IR (KBr) cmÀ1: 3264,
1
1606, 1242. H NMR (CDCl3) d 3.24 (1H, dd, J ¼ 7:8,
14.0 Hz), 3.39 (1H, dd, J ¼ 7:8, 14.0 Hz), 3.62–3.72 (2H,
m), 3.98–4.11 (1H, m), 6.64 (2H, d, J ¼ 7:8 Hz), 6.73
(1H, t, J ¼ 7:8 Hz), 7.16 (2H, t, J ¼ 7:8 Hz). HRFAB-
MS calcd for C9H13ClNO [M+H]þ: 186.0686. Found:
25
186.0684. ½aꢀ )11.0 (c 1.01, CHCl3).
D
1
Compound 3a. H NMR (CDCl3) d 3.24–3.42 (4H, m),
3.63–3.79 (4H, m), 3.98–4.13 (2H, m), 6.61–7.24 (5H,
m). FAB-MS m=z 278 [M+H]þ.
4.1.2. (R)-(+)-N-(3-Chloro-2-hydroxypropyl)aniline (2b).
This compound was prepared from (R)-())-epichloro-
hydrin and aniline in 89% yield and 96.9% ee according
to the procedure described for 2a. White crystals. Mp
4. Experimental
1
35–37 ꢁC. IR (KBr) cmÀ1: 3264, 1606, 1242. H NMR
€
All melting points were determined using a Buchi
micromelting point apparatus without correction. IR
spectra were measured with a Nicolet FT-IR 205 spec-
(CDCl3) d 3.24 (1H, dd, J ¼ 7:8, 14.0 Hz), 3.39 (1H, dd,
J ¼ 7:8, 14.0 Hz), 3.62–3.72 (2H, m), 3.98–4.11 (1H, m),
6.64 (2H, d, J ¼ 7:8 Hz), 6.73 (1H, t, J ¼ 7:8 Hz), 7.16
1
trometer. H NMR spectra were recorded on a JEOL
(2H, t, J ¼ 7:8 Hz). HRFAB-MS calcd for C9H13ClNO3
25
GSX spectrometer (270 MHz). Chemical shifts were
reported in ppm (d) values, based on tetramethylsilane as
an internal standard. The following abbreviations were
used: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet,
m ¼ multiplet, dd ¼ double doublet, dt ¼ double triplet,
br s ¼ broad singlet. MS spectra were recorded using a
JEOL SX-102 mass spectrometer. Optical rotations were
measured on a JASCO DIP-370 apparatus. Elemental
[M+H]þ: 186.0686. Found: 186.0682. ½aꢀ +10.0 (c 0.98,
D
CHCl3).
4.1.3. (S)-(+)-1-[4,4-Bis(4-fluorophenyl)butyl]-4-[2-hydr-
oxy-3-(phenylamino)propyl]piperazine (4a). A mixture
of 2a (1.00 g, 5.39 mmol), 1-[4,4-bis(4-fluorophen-
yl)butyl]piperazine (1.78 g, 5.39 mmol), potassium

3076
M. Kimura et al. / Bioorg. Med. Chem. 12 (2004) 3069–3078
carbonate (0.89 g, 6.44 mmol), and potassium iodide
(0.09 g, 0.542 mmol) in EtOH (20 mL) was heated under
reflux for 4 h. After removal of the insoluble materials by
filtration, the filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatogra-
phy (CHCl3/CH3OH ¼ 99:1as an eluent) to give 4a
(2.33 g, 90%, 98.7% ee) as white crystals. Mp 99–100ꢁC.
4.2. General procedure for syntheses of MTPA esters22;23
A mixture of each secondary alcohol (2a, 2b, 4a, or 4b),
N,N-dimethylaminopyridine (1.2 equiv), triethylamine
(3.0 equiv), and (R)- or (S)-MTPA chloride (1.2 equiv) in
dichloromethane was stirred at room temperature under
a nitrogen atmosphere. After removal of the solvent, the
residue was purified by silica gel column chromatogra-
phy (CHCl3/CH3OH ¼ 99:1as an eluent) to obtain the
corresponding MTPA esters.
1
IR (KBr) cmÀ1: 3329, 1603, 1222. H NMR (CDCl3) d
1.48–1.59 (2H, m), 1.98–2.10 (2H, m), 2.28–2.64 (10H,
m), 2.65–2.72 (2H, m), 3.06 (1H, dd, J ¼ 5:9, 12.9 Hz),
3.25 (1H, dd, J ¼ 4:1, 12.9 Hz), 3.86 (1H, t, J ¼ 7:8 Hz),
3.89–4.02 (1H, m), 6.63 (2H, d, J ¼ 7:6 Hz), 6.92–7.00
(4H, m), 7.01(1H, t, J ¼ 7:6 Hz), 7.23–7.34 (6H, m).
4.2.1. (S)-MTPA ester (5) from 2a and (R)-MTPA
chloride. A colorless oil. Yield 93%. IR (KBr) cmÀ1
:
HRFAB-MS calcd for C29H36F2N3O [M+H]þ: 480.2826.
25
Found: 480.2830. ½aꢀ +12.2 (c 1.02, CHCl3).
1752, 1604, 1510, 1170. 1H NMR (CDCl3) d 3.44 (2H, d,
J ¼ 5:9 Hz), 3.63 (3H, s), 3.77 (1H, dd, J ¼ 5:4,
12.4 Hz), 3.84 (1H, dd, J ¼ 4:3, 12.4 Hz), 5.39–5.48 (1H,
m), 6.54 (2H, d, J ¼ 7:6 Hz), 6.76 (1H, t, J ¼ 7:3 Hz),
7.18 (2H, t, J ¼ 7:3 Hz), 7.37 (3H, d, J ¼ 7:3 Hz), 7.56
(2H, d, J ¼ 7:8 Hz). HRFAB-MS calcd for
C1295H20ClF3NO3 [M+H]þ: 402.1084. Found: 402.1087.
D
4.1.4. (R)-())-1-[4,4-Bis(4-fluorophenyl)butyl]-4-[2-hydr-
oxy-3-(phenylamino)propyl]piperazine (4b). This com-
pound was prepared from 2b and 1-[4,4-bis(4-
fluorophenyl)butyl]piperazine in 92% yield and 98.8% ee
according to the procedure described for 4a. White
crystals. Mp 99–100 ꢁC. IR (KBr) cmÀ1: 3329, 1603,
½aꢀ )68.0 (c 1.01, CHCl3).
D
1
1222. H NMR (CDCl3) d 1.48–1.59 (2H, m), 1.98–2.10
(2H, m), 2.28–2.64 (10H, m), 2.65–2.72 (2H, m), 3.06
(1H, dd, J ¼ 5:9, 12.9 Hz), 3.25 (1H, dd, J ¼ 4:1,
12.9 Hz), 3.86 (1H, t, J ¼ 7:8 Hz), 3.89–4.02 (1H, m),
6.63 (2H, d, J ¼ 7:6 Hz), 6.92–7.00 (4H, m), 7.01(1H, t,
4.2.2. (R)-MTPA ester (6) from 2a and (S)-MTPA
chloride. A colorless oil. Yield 94%. IR (KBr) cmÀ1
:
1752, 1604, 1510, 1170. 1H NMR (CDCl3) d 3.52 (3H, s),
3.54 (2H, d, J ¼ 7:3 Hz), 3.75 (2H, dd, J ¼ 1:4, 5.1Hz),
5.37–5.47 (1H, m), 6.64 (2H, d, J ¼ 7:8 Hz), 6.77 (1H, t,
J ¼ 7:3 Hz), 7.20 (2H, t, J ¼ 7:3 Hz), 7.39 (3H, d,
J ¼ 7:3 Hz), 7.54 (2H, d, J ¼ 7:8 Hz). HRFAB-MS
J ¼ 7:6 Hz), 7.23–7.34 (6H, m). HRFAB-MS calcd for
25
D
C29H36F2N3O [M+H]þ: 480.2826. Found: 480.2835. ½aꢀ
)12.5 (c 1.03, CHCl3).
calcd for C19H20ClF3NO3 [M+H]þ: 402.1084. Found:
25
402.1092. ½aꢀ +5.3 (c 1.02, CHCl3).
D
4.1.5. (S)-())-1-[4,4-Bis(4-fluorophenyl)butyl]-4-[2-hydr-
oxy-3-(phenylamino)propyl]piperazine trihydrochloride
.
(4a 3HCl). A solution of 7.0 N HCl/EtOH (2.0 mL) was
4.2.3. (S)-MTPA ester (7) from 2b and (R)-MTPA
added dropwise to a solution of 4a (2.00 g, 4.17 mmol) in
EtOH (10 mL) under ice bath cooling and the mixture
was stirred at room temperature for 1h. The resultant
precipitates were collected by filtration and recrystallized
from EtOH to give 4aÆ3HCl (2.26 g, 82%, 100% ee) as
white crystals. Mp 232–233 ꢁC. IR (KBr) cmÀ1: 3318,
1602, 1508, 1223. 1H NMR (DMSO-d6) d 1.52–1.68 (2H,
m), 2.03–2.09 (2H, m), 2.24–2.94 (12H, m), 3.11–3.32
(2H, m), 4.02 (1H, t, J ¼ 7:8 Hz), 4.19–4.32 (1H, m), 6.83
(2H, d, J ¼ 7:3 Hz), 7.09–7.34 (7H, m), 7.43–7.71(4H,
m). Anal. Calcd for C29H35F2N3O Æ 3HCl: C, 59.14; H,
chloride. A colorless oil. Yield 90%. IR (KBr) cmÀ1
:
1750, 1604, 1509, 1171. 1H NMR (CDCl3) d 3.52 (3H, s),
3.53 (2H, d, J ¼ 5:5 Hz), 3.73 (2H, dd, J ¼ 1:5, 5.5 Hz),
5.36–5.47 (1H, m), 6.63 (2H, d, J ¼ 7:9 Hz), 6.76 (1H, t,
J ¼ 7:3 Hz), 7.11–7.32 (2H, m), 7.33–7.51 (3H, m), 7.53–
7.61(2H, m). HRFAB-MS calcd for C 19H20ClF3NO3
25
[M+H]þ: 402.1084. Found: 402.1082. ½aꢀ )5.8 (c 0.98,
D
CHCl3).
4.2.4. (R)-MTPA ester (8) from 2b and (S)-MTPA
6.50; N, 7.13; Cl, 18.06. Found: C, 59.41; H, 6.55; N,
chloride. A colorless oil. Yield 91%. IR (KBr) cmÀ1
:
25
D
7.08; Cl, 17.99. ½aꢀ )7.6 (c 0.99, CH3OH).
1751, 1604, 1509, 1170. 1H NMR (CDCl3) d 3.44 (2H, d,
J ¼ 5:9 Hz), 3.63 (3H, s), 3.77 (1H, dd, J ¼ 5:4,
12.4 Hz), 3.80 (1H, dd, J ¼ 4:3, 12.4 Hz), 5.38–5.49 (1H,
m), 6.51(2H, dd, J ¼ 1:0, 8.9 Hz), 6.76 (1H, dt, J ¼ 1:0,
7.4 Hz), 7.18 (2H, t, J ¼ 7:3 Hz), 7.37 (3H, d,
J ¼ 7:3 Hz), 7.56 (2H, d, J ¼ 7:8 Hz). HRFAB-MS
4.1.6. (R)-(+)-1-[4,4-Bis(4-fluorophenyl)butyl]-4-[2-hy-
droxy-3-(phenylamino)propyl]piperazine trihydrochloride
.
(4b 3HCl). This compound was prepared from 4b in 94%
yield and 100% ee according to the procedure described
for 4aÆ3HCl. White crystals. Mp 232–233 ꢁC. IR (KBr)
calcd for C19H20ClF3NO3 [M+H]þ: 402.1084. Found:
25
402.1079. ½aꢀ +66.7 (c 1.03, CHCl3).
D
1
cmÀ1: 3318, 1602, 1508, 1223. H NMR (DMSO-d6) d
1.52–1.68 (2H, m), 2.04–2.09 (2H, m), 2.24–2.94 (12H,
m), 3.12–3.32 (2H, m), 4.02 (1H, t, J ¼ 7:8 Hz), 4.19–4.32
(1H, m), 6.83 (2H, d, J ¼ 7:3 Hz), 7.09–7.34 (7H, m),
7.43–7.71(4H, m). Anal. Calcd for C 29H35F2N3O Æ 3HCl:
4.2.5. (S)-MTPA ester (9) from 4a and (R)-MTPA
chloride. A colorless oil. Yield 90%. IR (KBr) cmÀ1
:
1749, 1603, 1507, 1224, 1160. 1H NMR (CDCl3) d 1.42–
1.53 (2H, m), 1.93–2.11 (2H, m), 2.32–2.64 (10H, m),
2.49 (1H, dd, J ¼ 4:6, 13.5 Hz), 2.68 (1H, dd, J ¼ 8:4,
C, 59.14; H, 6.50; N, 7.13; Cl, 18.06. Found: 59.21; H,
25
D
6.52; N, 7.11; Cl, 18.01. ½aꢀ +8.1( c 1.02, CH3OH).

M. Kimura et al. / Bioorg. Med. Chem. 12 (2004) 3069–3078
3077
13.5 Hz), 3.23 (1H, dd, J ¼ 6:5, 13.8 Hz), 3.40 (1H, dd,
J ¼ 4:6, 13.8 Hz), 3.69 (3H, s), 3.72 (1H, br s), 3.86 (1H,
t, J ¼ 8:1Hz), 5.43–5.51 (1H, m), 6.52 (2H, d,
J ¼ 7:6 Hz), 6.71(1H, t, J ¼ 8:1Hz), 6.91–7.03 (3H, m),
7.12–7.24 (7H, m), 7.34–7.41(3H, m), 7.53–7.61(2H,
[3H]GBR12935 (53 Ci/mmol) was purchased from Du
Pont-NEN (Boston, MA, USA). The assay was termi-
nated by filtration through Whatman GB/F glass fiber
filtermats, presoaked with 0.1% bovine serum albumin
solution, with a Brandel Cell Harvester (Gaithersberg,
MD, USA). Filters were assayed for radioactivity with
Packard Tris-Carb Liquid Scintillation Counter (Meri-
den, CT, USA) in 4 mL Aquasol-2.
m). HRFAB-MS calcd for C39H43F5N3O3 [M+H]þ:
25
D
696.3225. Found: 696.3223. ½aꢀ )19.5 (c 1.01, CHCl3).
4.2.6. (R)-MTPA ester (10) from 4a and (S)-MTPA
chloride. A colorless oil. Yield 94%. IR (KBr) cmÀ1
:
4.4. [3H]DA, [3H]5-HT, and [3H]NE uptake assays
1748, 1604, 1508, 1224, 1160. 1H NMR (CDCl3) d 1.38–
1.51 (2H, m), 1.89–2.13 (2H, m), 2.22–2.73 (10H, m),
2.50 (1H, dd, J ¼ 5:4, 13.5 Hz), 2.61 (1H, dd, J ¼ 7:0,
13.5 Hz), 3.35 (1H, dd, J ¼ 4:1, 9.7 Hz), 3.44 (1H, dd,
J ¼ 4:9, 9.7 Hz), 3.57 (3H, s), 3.86 (1H, t, J ¼ 7:8 Hz),
4.04 (1H, br s), 5.21–5.43 (1H, m), 6.60 (2H, d,
J ¼ 7:3 Hz), 6.73 (1H, t, J ¼ 7:3 Hz), 6.92–7.03 (3H, m),
7.13–7.20 (7H, m), 7.34–7.42 (3H, m), 7.53–7.64 (2H,
Male Wistar rats (6–7 weeks) were used for the mono-
amine uptake assays. The [3H]DA, [3H]5-HT, and
[3H]NE uptake assays were proceeded according to the
published procedure.26 Briefly, synaptosomes were pre-
pared by homogenization of rat striatum (for [3H]DA
uptake assay) or cerebral cortex (for [3H]5-HT and
[3H]NE uptake assays). The uptake assays were initiated
by the addition of 100 lL of [3H]-monoamines to poly-
styrene tubes prefilled with 890 lL of synaptosomes in
Krebs–Henseleit buffer, which contained 121 mM NaCl,
25 mM NaHCO3, 11.1 mM glucose, 4.7 mM KCl,
1.4 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4,
130 lM EDTAÆ2Na, 10 lM nialamide, 0.2% ascorbic
acid, and 10 lL of test compounds (final concentration
range: 10À11–10À5 M), which were diluted with dimethyl
sulfoxide (final dimethyl sulfoxide concentration was
less than 0.1%), and they were incubated for 5 min at
37 ꢁC. After they were cooled in an ice bath, 100 lL of
[3H]-monoamines (100 nM final concentration) were
added and they were incubated for 5 min (for [3H]DA
uptake assay) or 10 min (for [3H]5-HT and [3H]NE
uptake assays), respectively. The assays were terminated
by filtration through Whatman GB/F glass fiber filter-
mats, presoaked with saline, with a Brandel Cell Har-
vester (Gaithersberg, MD, USA). Filters were assayed
for radioactivity with Packard Tris-Carb Liquid Scin-
tillation Counter (Meriden, CT, USA) in 10 mL Aqua-
sol-2.
m). HRFAB-MS calcd for C39H43F5N3O3 [M+H]þ:
25
D
696.3225. Found: 696.3231. ½aꢀ +15.2 (c 0.99, CHCl3).
4.2.7. (S)-MTPA ester (11) from 4b and (R)-MTPA
chloride. A colorless oil. Yield 89%. IR (KBr) cmÀ1
:
1748, 1604, 1508, 1225, 1159. 1H NMR (CDCl3) d 1.32–
1.51 (2H, m), 1.92–2.13 (2H, m), 2.33–2.64 (10H, m),
2.49 (1H, dd, J ¼ 5:4, 13.3 Hz), 2.61 (1H, dd, J ¼ 6:4,
13.3 Hz), 3.39 (1H, dd, J ¼ 7:9, 13.9 Hz), 3.50 (1H, dd,
J ¼ 5:0, 13.3 Hz), 3.52 (3H, s), 3.89 (1H, t, J ¼ 7:6 Hz),
4.04 (1H, br s), 5.32–5.54 (1H, m), 6.60 (2H, d,
J ¼ 7:6 Hz), 6.73 (1H, t, J ¼ 7:6 Hz), 6.92–7.03 (3H, m),
7.14–7.21(7H, m), 7.43–7.51(3H, m), 7.64–7.72 (2H,
m). HRFAB-MS calcd for C39H43F5N3O3 [M+H]þ:
25
D
696.3225. Found: 696.3229. ½aꢀ )14.2 (c 1.03, CHCl3).
4.2.8. (R)-MTPA ester (12) from 4b and (S)-MTPA
chloride. A colorless oil. Yield 89%. IR (KBr) cmÀ1
:
1
1748, 1604, 1507, 1225. H NMR (CDCl3) d 1.33–1.48
(2H, m), 1.92–2.13 (2H, m), 2.27–2.54 (8H, m), 2.49 (1H,
dd, J ¼ 4:3, 13.2 Hz), 2.62–2.84 (2H, m), 2.68 (1H, dd,
J ¼ 8:4, 13.2 Hz), 3.24 (1H, dd, J ¼ 5:9, 12.4 Hz), 3.41
(1H, dd, J ¼ 4:9, 12.4 Hz), 3.42 (1H, br s), 3.60 (3H, s),
3.72 (1H, t, J ¼ 6:5 Hz), 5.42–5.63 (1H, m), 6.52 (2H, d,
J ¼ 7:3 Hz), 6.71(1H, t, J ¼ 7:3 Hz), 6.93–7.02 (3H, m),
7.14–7.23 (7H, m), 7.38–7.54 (3H, m), 7.61–7.73 (2H,
4.5. Ex vivo DA uptake inhibition studies
Male Sprague-Dawley rats were fasted overnight. Test
compounds at 30 mg/kg or vehicle (saline) were admin-
istered orally. At 1, 2, 3, 4, and 6 h after the adminis-
trations, rats were decapitated. Their brains were
removed to an ice-cooled dish for dissection of the
striatum and they were weighted. The tissue was
homogenized in 20 vol of ice-cold 0.32 M sucrose with a
Teflon/glass homogenizer. Homogenates were centri-
fuged at 700g for 10 min, with retention of the super-
natant followed by centrifugation at 17,500g for 20 min
at 4 ꢁC. The resulting pellet was re-suspended in 0.32 M
sucrose, and then ice-cold Krebs–Henseleit buffer
(121 mM NaCl, 25 mM NaHCO3, 11.1 mM glucose,
4.7 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 1.2 mM
KH2PO4, 1 30lM EDTAÆ2Na, 10 lM nialamide, 0.2%
ascorbic acid) bubbled with 95% O2 and 5% CO2 was
added to prepare synaptosomes. DA uptake experi-
ments were conducted in assay tubes containing 900 lL
of synaptosomes and 100 lL of [3H]DA (100 nM final
m). HRFAB-MS calcd for C39H43F5N3O3 [M+H]þ:
25
D
696.3225. Found: 696.3218. ½aꢀ +21.4 (c 1.02, CHCl3).
4.3. DAT binding studies
Male Wistar rats (6–7 weeks) were used for the DAT
binding studies. Binding assay for the DAT was deter-
mined according to the published procedure.25 Briefly,
the rat striatal membranes were incubated with
[3H]GBR12935 (1 nM final concentration) and test
compounds (final concentration range: 10À11–10À5 M),
which were diluted with dimethyl sulfoxide (final
dimethyl sulfoxide concentration was less than 0.1%),
for 60 min at 4 ꢁC in 50 mM Tris–citrate (pH 7.4) buffer
containing 120 mM NaCl and 4 mM MgCl2.

3078
M. Kimura et al. / Bioorg. Med. Chem. 12 (2004) 3069–3078
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nated after 5 min by rapid filtration through a Brandel
Cell Harvester (Gaithersberg, MD, USA) and the mix-
ture was rinsed with 3 · 5 mL of ice-cold Krebs–Hense-
leit buffer through Whatman GB/F glass fiber filters that
had been presoaked in saline for at least 1h. Filter mats
were allowed to air dry and then placed in scintillation
vials containing 10 mL Aquasol-2. Radioactivity was
determined by Packard Tris-Carb Liquid Scintillation
Counter (Meriden, CT, USA). Specific uptake was
determined by subtracting the accumulation at 4 ꢁC
from that at 37 ꢁC. The results are expressed as
% inhibition of specific uptake in vehicle control. Each
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