Structure-Activity Relationship Studies on a Series of 3α-[Bis(4-fluorophenyl)methoxy]tropanes and 3α-[Bis(4-fluorophenyl)methylamino]tropanes As Novel Atypical Dopamine Transporter (DAT) Inhibitors for the Treatment of Cocaine Use Disorders

Article abstract of DOI:10.1021/acs.jmedchem.7b01454

The development of medications to treat cocaine use disorders has thus far defied success, leaving this patient population without pharmacotherapeutic options. As the dopamine transporter (DAT) plays a prominent role in the reinforcing effects of cocaine that can lead to addiction, atypical DAT inhibitors have been developed that prevent cocaine from binding to DAT, but they themselves are not cocaine-like. Herein, a series of novel DAT inhibitors were synthesized, and based on its pharmacological profile, the lead compound 10a was evaluated in phase I metabolic stability studies in mouse liver microsomes and compared to cocaine in locomotor activity and drug discrimination paradigms in mice. A molecular dynamic simulation study supported the hypothesis that atypical DAT inhibitors have similar binding poses at DAT in a conformation that differs from that of cocaine. Such differences may ultimately contribute to their unique behavioral profiles and potential for development as cocaine use disorder therapeutics.

Full text of DOI:10.1021/acs.jmedchem.7b01454

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Structure−Activity Relationship Studies on a Series of 3α-[Bis(4-
fluorophenyl)methoxy]tropanes and 3α-[Bis(4-
fluorophenyl)methylamino]tropanes As Novel Atypical Dopamine
Transporter (DAT) Inhibitors for the Treatment of Cocaine Use
Disorders
Mu-Fa Zou,† Jianjing Cao,† Ara M. Abramyan,† Theresa Kopajtic,‡ Claudio Zanettini,† Daryl A. Guthrie,†
Rana Rais,§ Barbara S. Slusher,§ Lei Shi,† Claus J. Loland,∥ and Amy Hauck Newman*,†
†Molecular Targets and Medications Discovery Branch, National Institute on Drug AbuseIntramural Research Program, National
Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, United States
‡Psychobiology Section, Molecular Neuropsychiatry Research Branch, National Institute on Drug AbuseIntramural Research
Program, National Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland 21224, United States
§Department of Neurology, Johns Hopkins Drug Discovery, The Johns Hopkins University School of Medicine, 855 North Wolfe
Street, Baltimore, Maryland 21205, United States
∥Molecular Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark
S
* Supporting Information
ABSTRACT: The development of medications to treat cocaine use
disorders has thus far defied success, leaving this patient population
without pharmacotherapeutic options. As the dopamine transporter
(DAT) plays a prominent role in the reinforcing effects of cocaine
that can lead to addiction, atypical DAT inhibitors have been
developed that prevent cocaine from binding to DAT, but they
themselves are not cocaine-like. Herein, a series of novel DAT
inhibitors were synthesized, and based on its pharmacological
profile, the lead compound 10a was evaluated in phase I metabolic
stability studies in mouse liver microsomes and compared to cocaine
in locomotor activity and drug discrimination paradigms in mice. A
molecular dynamic simulation study supported the hypothesis that
atypical DAT inhibitors have similar binding poses at DAT in a
conformation that differs from that of cocaine. Such differences may ultimately contribute to their unique behavioral profiles and
potential for development as cocaine use disorder therapeutics.
modality will cure all. Hence, efforts to develop medications to
treat this family of disorders must be tailored to the specific
substance or substances of abuse, comorbidities with other
neuropsychiatric illnesses, and ultimately individual treatment
needs, which significantly increases the challenge.
Cocaine (1, Figure 1) is a psychostimulant and a highly
addictive drug of abuse. In 2015, an estimated 1.8 million
people aged 12 and older were current cocaine users.5
However, to date, no medication to treat cocaine abuse has
proven successful.6 Cocaine binds to the dopamine transporter
(DAT) and inhibits the reuptake of dopamine from the synapse
into presynaptic neurons, resulting in an increase in
extracellular dopamine levels.7−9 The blockade of DAT has
been proposed to be the primary mechanism underlying
INTRODUCTION
■
In 2016, the U.S. Surgeon General’s report on alcohol, drugs,
and health in America was entitled “Facing Addiction in
America”.1 Even the title of this report commands a change in
perspective from the misperception of addiction being a
problem of inner cities and weak willed individuals to a severe
public health matter that affects families and communities,
without socioeconomic or moral bounds. Substance use
disorder is a chronic medical condition, taking its toll on our
public health care and judicial systems in an economically
unsustainable way. As more than 20 million Americans suffer
from substance use disorders, certainly, the development of
prevention and treatment options as alternatives to incarcer-
ation is the appropriate and ethical solution to this escalating
global problem. Although the recent opioid epidemic2−4 has
redefined public perception of drug dependence, like cancer,
addiction is not a single illness for which one treatment
Received: September 29, 2017
© XXXX American Chemical Society
A
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structural requirements for potency and selectivity at DAT
within this class of drugs.13,23 Most 3-aryl analogues of cocaine
that have been evaluated in animal models of psychostimulant
abuse have demonstrated behavioral effects and predicted abuse
liability similar to cocaine. However, modification at both the 1-
and 2-positions have resulted in several DAT inhibitors that do
not share cocaine’s behavioral profile in animal models,24−28
demonstrating that structural modifications on the tropane
structure can affect both binding affinity to DAT and in vivo
activity.
In comparison to cocaine and its 3-aryl analogues, another
class of tropane-based DAT inhibitors, based on 3α-
(diphenylmethoxy)tropane (benztropine, 2, Figure 1), bind to
DAT with high affinity but typically do not produce cocaine-
like behavioral effects in animals.28,29 We have previously
reported that replacement of the oxygen atom in the
benztropines with a nitrogen atom results in the formation of
a class of benztropinamines (e.g., compound 3, Figure 1) that
bind to DAT with high affinity and selectivity.30 The
benztropinamines show similar SAR to the benztropines in
binding to DAT as well as the other monoamine transporters
such as the norepinephrine transporter and SERT.
Figure 1. Chemical structures of cocaine and other tropane-based
DAT inhibitors.
cocaine’s psychostimulant effects and abuse liability.10,11 As
such, DAT has been the target for the development of potential
medications to treat cocaine addiction for nearly three decades,
however, thus far none of the clinically available DAT inhibitors
tested in this patient population has been FDA approved for
treatment.6,12−14
We and others have shown that a substituent in the 2-
position in the benztropine class of compounds is not necessary
for binding to DAT with high affinity, in contrast to the
cocaine-like structures.31−35 However, if there is a substituent in
the 2-position, high affinity binding to DAT is conferred when
this substituent is in the β-configuration; the S-enantiomer
being the eutomer (e.g., compound 4, Figure 1), in contrast to
cocaine, in which the R-enantiomer binds with highest DAT
affinity.36 Importantly, numerous studies have revealed that the
benztropine-based dopamine uptake inhibitors not only have
distinct structural requirements from cocaine and its 3-aryl
analogues for binding to DAT but also exhibit different
behavioral profiles in animal models of cocaine abuse.29,37−48
Indeed, these studies suggest that structurally divergent
tropane-based compounds may bind differently to DAT,
affected by subtle structural changes, and that the consequence
DAT belongs to the neurotransmitter:sodium symporter
family. X-ray crystal structures of its homologues, such as
bacterial homologue LeuT,15 drosophila DAT,16−18 and the
human serotonin transporter (hSERT),19 as well as computa-
tional models,20,21 have provided a clearer picture of how the
inhibitors bind and the molecular mechanisms underlying the
transport of substrate.22 Significant effort has been directed
toward the elucidation of the molecular structure and function
of DAT as well as characterization of the binding sites of
cocaine and other dopamine uptake inhibitors.21 However, the
molecular-level details of transport inhibition by abused drugs
as well as clinically therapeutic DAT inhibitors such as
methylphenidate remains unknown, and this includes how
chronic use affects dopamine homeostasis over time.
Structure−activity relationship (SAR) studies on cocaine and
its 3-aryl analogues have proven useful in the identification of
a
Scheme 1. Synthesis of N-Substituted Benztropines and Benztropinamines
a
Reagents and conditions: (a) chlorobis(4-fluorophenyl)methane, CH3CN, NaHCO3, KI, overnight; (b) (i) ACE-Cl, 1,2-dichloroethane, reflux, 3 h,
(ii) MeOH, reflux overnight; (c) (i) haloacetamide, CH3CN, NaHCO3, KI, overnight or acid chloride, 10% NaHCO3 aqueous solution, CHCl3, rt,
20 min, (ii) LAH, THF.
B
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a
Scheme 2. Synthesis of N-Substituted-2β-substituted Benztropines
a
Reagents and conditions: (a) RBr, DMF, 65−70 °C, overnight; (b) NH2NH2, EtOH, reflux, 2 h; (c) 12e, LAH, THF, rt, overnight; (d) (COCl)2,
DMSO, Et3N, CH2Cl2, −78 °C, 1 h; (e) CH3PPh3Br, n-BuLi, THF, rt, overnight.
a
Scheme 3. Synthesis of 2β-Isoxazol Derivatives of Benztropine
a
Reagents and conditions: (a) n-BuLi, THF, rt, overnight; (b) HCl (3N), THF, reflux, 4 h.
of conformational changes at the transporter may be related to
their in vivo activity profiles.21,49,50
discriminate cocaine from saline.27,51 Notably, 5 and 6 have
similar binding affinities at DAT, SERT, and NET. As
compound 5 displays an atypical behavioral profile but is
reported to bind DAT in a conformation similar to cocaine, we
prepared compounds 5, 6, and several 2β-isoxazol-substituted
compounds that are hybrids of benztropines 2, 4, and the
phenyltropane 5 to further investigate the effects of this
substituent on the pharmacological profile of these molecules.
Subsequent evaluation of a subset of DAT-selective analogues
was conducted to determine their potency in inhibiting
[3H]DA uptake at WT human DAT (hDAT) or [3H]-
WIN35,428 binding at WT hDAT or the Y156F mutant
Herein, we report an extension of SAR in this class of atypical
DAT inhibitors wherein compounds based on benztropinamine
and N-substituted or 2-substituted benztropines were synthe-
sized and evaluated for binding affinities at DAT, SERT, and
NET. Previous studies have shown that the isoxazol phenyl-
tropane derivative 5 (Figure 1), an analogue of cocaine, fails to
stimulate locomotor activity in mice and does not substitute in
rats trained to discriminate cocaine from saline.27,51 In contrast,
6 (Figure 1), a constitutional isomer of 5, does stimulate
locomotion and substitutes for cocaine in rats trained to
C
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Table 1. In Vitro Binding Data on 2-Substituted or Unsubstituted 3α-[Bis(4-fluorophenyl]methoxy)tropanes or 3α-[Bis(4-
a
fluorophenyl]methylamino)tropanes
e
DAT Ki SEM SERT Ki SEM NET Ki SEM
hERG
compd
A
R1
R2
(nM)
(nM)
(nM)
(μM)
hERG:DAT
10a
10b
10c
10d
10e
12a
12b
12c
13a
15
O
CH2CH2N(CH3)2
CH2CH2N(CH3)2
(CH2)2N(CH2)5
(CH2)2N(CH2)5
R-(2-amino-3-methyl)butyl
c-Pr-CH2
H
H
H
H
H
12.0 1.54
14.3 0.824
13.2 1.72
10.6 1.47
21.0 0.634
12.0 1.78
26.1 1.55
53.8 3.22
17.2 0.958
13.5 1.82
13.2 1.50
5.59 0.619
9.71 0.92
21.5 2.31
24.6 1.78
29.2 3.24
3150 258
12000 1400
1460 141
3780 549
5400 673
3730 334
746 57.6
1370 106
5580 674
5100 664
3870 135
4600 680
1350 151
2640 27.6
1420 116
490 56.4
933 23.6
4010 573
370 53.2
612 87.8
2350 269
555 73.3
1450 109
4470 224
862 88.7
5200 598
2130 160
1420 125
1490 190
2920 209
1640 153
7350 934
8.23
5.48
5.15
NT
NT
NT
14
686
383
390
NH
O
NH
NH
O
CO2CH2CH3
O
3-ethylindole
CO2CH2CH3
536
345
O
R-(2-amino-3-methyl)butyl
CH2CH2NH2
CO2CH2CH3
NT
NT
NT
4.56
NT
1.14
2.35
3.96
NT
O
CO2CH2CH3
O
CH2CH2N(CH3)2
R-(2-amino-3-methyl)butyl
CH2CH2NH2
CHCH2
b
b
20 (GA2-50)
21 (GA2-99)
O
H
H
H
H
H
H
O
c
22 (JHW007)
O
(CH2)3CH3
113
109
161
d
23 (PG466)
N
(CH2)3CH3
b
24 (JHW013)
O
c-Pr-CH2
b
25 (GA1-69)
O
3-ethylindole
a
Each Ki value represents data from at least three independent experiments, each performed in triplicate. Ki values were analyzed by PRISM. Binding
assays are described in detail in Experimental Methods. Binding data previously published70 using the same methods as described in the
b
Experimental Methods section and included for comparison. Binding data previously published35 using the same methods as described in the
c
Experimental Methods section and included for comparison. Binding data previously published30 using the same methods as described in the
d
e
Experimental Methods section and included for comparison. Data from PDSP: NIMH Psychoactive Drug Screening Program using methods
described.71
a
Table 2. In Vitro Binding Data of 2-Isoxazole-3α-[bis(4-fluorophenyl]methoxy)tropanes
compd
type
X
R
DAT Ki SEM (nM)
SERT Ki SEM (nM)
NET Ki SEM (nM)
1, cocaine
98.1 6.58
8.55 1.50
10.7 1.31
60.8 8.86
14.4 0.90
93.5 11.0
66.6 2.64
69.2 9.12
293 30.0
29100 6330
23500 2180
3830 486
1320 173
4410 536
756 73.5
2,120 314
887 75.1
1170 112
1100 106
413 55.8
2710 394
1580 220
1640 232
b
5
A
A
B
B
B
B
B
Me
Cl
Cl
b
6
CH3
H
( )-19a
(S)-(+)-19a
( )-19b
( )-19c
H
CH3
Cl
( )-19d
F
2990 436
a
Each Ki value represents data from at least three independent experiments, each performed in triplicate. Ki values were analyzed by PRISM. Binding
assays are described in detail in Experimental Methods. Compounds were published previously and included for comparison.27,51
b
transiently expressed in COS7 cells. The Y156F mutation has
previously been shown to adversely affect binding affinities of
the atypical DAT inhibitors while having no effect on the
binding affinity of cocaine.52
CHEMISTRY
■
The aim of this first series of compounds was to extend SAR to
further explore the addition of a terminal amine group in
proximity to the bridgehead nitrogen of the benztropine and
benztropinamine derivatives unsubstituted in the 2-position.
D
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The synthesis began with the reductive amination53 of
tropinone to give the corresponding amine 730 (Scheme 1).
Coupling of 7 with chlorobis(4-fluorophenyl)methane gave the
benztropineamine 8.30 Subsequent N-demethylation with 1-
chloroethyl chloroformate (ACE-Cl) using the Oloffson
procedure gave the corresponding N-nor compound, 9b.
Note that 9a54 was previously reported and used to make the
corresponding N-substituted benztropines. N-Alkylation and
reduction of the intermediate amide using LAH gave
benztropines (10a and 10c from 9a) and benztropineamines
(10b, 10d, and 10e from 9b) with various substitutions at the
bridgehead nitrogen position.
In Scheme 2, ( )-N-nor-2β-carboethoxy-3α-[bis(4-
fluorophenyl)methoxy]tropane (11)34,55 was alkylated to give
the respective ( )-N-substituted analogues (12a−e). Depro-
tection of the phthalimide protected amine in 12d with
hydrazine resulted in the formation of amine 13a. Reduction of
12e with LAH gave the alcohol 13b. Swern oxidation of 13b
formed the aldehyde 14, which was transformed to the olefin
product 15 under Wittig reaction conditions.
In Scheme 3, the 2-isoxazole analogues (19a−d) were
prepared using a similar strategy described for 551 wherein the
oximes 17a−d were treated with 2 equiv of n-butyl lithium (n-
BuLi) and then reacted with ( )-2β-carboethoxy-3α-bis[(4-
fluorophenyl)methoxy]tropane (16)34,35,55 to give 18a−d.
Cyclization to the isoxazole analogues 19a−d was achieved
by refluxing in THF with aqueous hydrochloric acid (3 N HCl).
Both intermediates 18a−d and final products 19a−d appeared
to be unstable under these reaction conditions and started to
decompose after a short time while stirring at reflux, resulting in
low isolated yields of 19a−d. (+)-19a was prepared from
(+)-16 using the same procedure.
affinity either (e.g., 10a vs 15). Interestingly, NET binding was
significantly decreased with this substitution from Ki = 933 to
5100 nM, resulting in compound 15 being one of the most
DAT-selective analogues in the series.
None of the three series of tropane-based DAT uptake
inhibitors demonstrated high binding affinity at SERT or NET.
In all three sets, the selectivity for DAT over SERT was
consistently higher than that of DAT over NET except 12b and
12c, which showed higher DAT selectivity over NET than that
of DAT over SERT (for 12b, NET/DAT = 56 > SERT/DAT =
29; for 12c, NET/DAT = 83 > SERT/DAT = 25). Compounds
10a−c and 12b were tested for hERG channel activity and
compared to 20, 22, 23, and 24. Cardiotoxicity, in the form of
drug-induced QT interval prolongation poses a significant
liability for drug development. This off-target activity is
primarily due to the inhibition of cardiac hERG K+ currents.56
Typically, a >30-fold selectivity for the drug target over hERG
channel inhibition (IC50) is considered sufficient to move
forward with development. In this series, all compounds tested
showed hERG:DAT ratios of >300, with 10a having the highest
ratio of 686.
In Table 2, the 2-isoxazole substituent, an excellent
bioisostere for the methyl ester in previously described 3-
aryltropane analogues,51 was explored in the benztropine class
of compounds. This substituent was well tolerated in the
benztropine series as well, with all analogues 19a−d showing
moderately high DAT affinities (Ki range = 61−94 nM).
Notably, S-(+)-19a demonstrated the highest DAT affinity (Ki
= 14 nM) in the series, comparable to the 3- aryltropane
analogues 5 and 6.51 Fluoro- or chloro- substitution on the 4-
position in the ( )-2β-phenylisoxazol-analogues did not affect
DAT binding affinity. In general, none of the compounds
demonstrated high binding affinities at NET or SERT.
However, compared to the 2-isoxazol-3α-phenyltropane
compounds 5 and 6, the 2-isoxazol-3α-[bis(4-fluorophenyl)-
methoxy]tropane compounds were less selective for DAT over
NET and SERT.
Molecular Pharmacology and Mutagenesis Studies.
To assess the effect of these substituents specifically on DAT
function, a subset of analogues (10a, 10b, 12b, 12c, (+)-19a,
and ( )-19a) was tested for their potency to inhibit [3H]DA
uptake in COS7 cells transiently expressing human DAT. The
analogues were added in a range of concentrations, typically
from 0.1 nM to 100000 nM, followed by a fixed concentration
of [3H]DA to allow transport. The reaction was stopped after 5
min, and the amount of [3H]DA taken up by the COS7 cells
was determined by scintillation counting and plotted as a
function of the concentration of added analogue. All analogues
from this subset inhibited [3H]DA uptake in the Ki range of
10−1300 nM (Table 3). In this series, compounds 10a (Figure
2A) and 10b emerge as among the DA uptake inhibitors with
highest the potency (Ki = 47 [32;68] nM and 40 [32;49] nM,
respectively; mean [SEM interval], n = 4 and 3). Accordingly,
their inhibition potency is >4 times higher than what we
observe for cocaine (Ki = 198 [113;347] nM; mean [SEM
interval], n = 3).
RESULTS AND DISCUSSION
■
SAR at DAT, SERT, and NET. All final compounds (10a−e,
12a−c, 13a, 15 and 19a−d) were evaluated for binding at
DAT, SERT, and NET in rat brain membranes. Methods for
the binding assays are described in the Experimental Methods
section. The results of these binding studies are summarized in
Tables 1 and 2 and compared to several previously reported
and known DAT inhibitors 1, 5, 6 and 20−25. For selected
compounds, human ether-a-gogo related gene (hERG) channel
data are also included.
Changes in DAT binding affinity and selectivity as a result of
modifications to the benztropine structure were evaluated. In
general, these novel analogues displayed low nanomolar DAT
binding affinities and were selective for DAT over NET and/or
SERT. We have previously shown that compounds in which the
3-position ether linkage is replaced with a secondary amine to
give N-substituted benztropinamine analogues show similar
binding affinities at DAT in comparison to their benztropine
counterparts (e.g., 22 vs 23).30 In the present study, we
observed the same trend as 10e vs 20 have DAT Ki values = 21
and 13 nM, respectively. Likewise, 10b vs 10a and 10d vs 10c
have similar Ki values. Hence, this substitution has no
demonstrable effect on DAT binding or selectivity. When the
2-position was substituted with β-COOEt, DAT binding
affinities were also not considerably affected (e.g., 12a vs 24,
12b vs 25, 12c vs 20, or 13a vs 21) as previously reported with
other analogues34,35,55 and in contrast to the cocaine class of
molecules in which the 2-position substituent is essential for
high affinity binding to DAT.36 Moreover, adding the 2β-
ethenyl substituent did not significantly affect DAT binding
Although all DAT inhibitors by definition block DA uptake,
they seem to do this by stabilizing DAT in different
conformations. There is compelling evidence from biochemis-
try, molecular dynamics simulations, and structural biology21,49
that cocaine has a binding preference to the outward facing
conformation of DAT. Here, the binding site centrally located
halfway through the membrane and overlapping with the
E
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a
Table 3. [3H]DA Uptake Inhibition for Selected Analogues
DAT inhibitors are also competitive to dopamine binding but
induce a different protein conformation in which the binding
site is not as exposed to the outside.49,50 Thus, far, no X-ray
crystal structure of DAT in complex with an atypical DAT
inhibitor has been reported. However, compelling biochemical
and computational evidence have proposed that, in contrast to
cocaine, the binding of an atypical inhibitor to DAT is stabilized
by the formation of a hydrogen bond between the OH-group of
Tyr156 in TM3 and Asp79 in TM1.21,49,50,57,58 The two
residues are located right “above” the ligand binding site and
the H-bond formation could induce a closure of the
extracellular gate, excluding access to the binding site from
the extracellular side. In support of this hypothesis, we have
previously shown that the affinity of atypical DAT inhibitors,
such as the benztropine 22 or (R)-modafinil, are adversely
affected by the removal of the OH-group in Tyr156
(Y156F).21,57 In contrast, the affinity for cocaine is not affected
by the Y156F mutation, suggesting that the OH-group does not
contribute to cocaine binding. Accordingly, we wanted to assess
whether or not compounds 10a, 10b, 12b, 12c, and (+)- and/
or ( )-19a demonstrated an atypical DAT inhibitor binding
pose by evaluating their binding affinities in DAT wild-type
(WT) and the Y156F mutant (Figure 2B and Table 4). The
results were compared to the effects of compounds 5, 6, 20, 23,
and 25 (Table 4). Inhibition of [3H]WIN35,428 binding on
COS7 cells transiently expressing WT DAT or Y156F was
determined. In contrast to cocaine (Y156F:WT affinity ratio =
1.4),52 all the analogues showed a decrease in binding affinity
for Y156F relative to DAT WT and hence Y156F:WT affinity
ratios >2, with some appreciably higher. Remarkably, the
benztropinamines 10b and 23 showed Y156F:WT affinity
ratios of 20 and 15, respectively, suggesting pronounced
perturbation of their binding site by the mutation. For
comparison, the classical atypical DAT inhibitor, 22, had a
Y156F:WT affinity ratio of 5.5.52 Moreover, both compounds 5
and 6 showed Y156F:WT affinity ratios of 9.0 and 4.0,
respectively.
compd
DAT WT Ki (nM)
n
dopamine (kM)
999 [891;1120]
198 [113;347]
9.7 [6.0;16]
25
3
5
6
4
3
5
4
3
3
4
3
3
1
5
6
16 [11;25]
10a
47 [32;68]
10b
12b
12c
40 [32;49]
944 [401;2220]
1050 [714;1540]
212 [162;278]
1300 [962;1760]
133 [96;185]
161 [132;198]
62 [49;79]
S-(+)-19a
( )-19a
20
22
23
a
Inhibition potencies (Ki values) of dopamine, cocaine, and indicated
atypical inhibitors assessed as inhibition of [3H]DA uptake as depicted
for 10a in Figure 2A. Experiments are performed on COS7 cells
transiently expressing DAT WT. The Ki values were calculated from
the IC50 values from the nonlinear regression analysis (Prism 6.0,
GraphPad) using the equation Ki = IC50/(1 + (L/Kd)), where Kd is the
affinity for WIN 35,428 and L is the concentration of added [3H]WIN
35,428. Data are shown as mean [SEM interval]. All data are
performed in triplicate.
Preclinical Evaluation of Compound 10a in Mice.
Mouse Microsomal Stability Assay. On the basis of its ease of
synthesis, DAT affinity (Ki = 12.0 nM), high hERG:DAT ratio
(686), and Y156F:WT affinity ratio = 6.3, we chose compound
10a as a lead molecule in this series for further evaluation. A
phase I metabolic stability assay was conducted with 10a in
mouse liver microsomes. Compound 10a was found to be
remarkably stable, with 88% remaining following 1 h incubation
in mouse liver microsomes, fortified with NADPH, suggesting
it is highly stable to cytochrome P450 dependent metabolism
(Figure 3). As a result of this promising profile, further
behavioral testing was conducted in mice.
Locomotor Activity in Mice after Administration of
Cocaine or 10a. In this model, as expected, cocaine dose
dependently increased locomotion at doses of 20 and 40 mg/
kg, producing a maximum of ≈300% of the activity counts
compared to administration of vehicle (Veh vs 20 mg/kg, q =
2.98, p < 0.05; Veh vs 40 mg/kg, q = 3.06, p < 0.05; Figure 4).
Compound 10a was tested up to a dose (56 mg/kg) that
produced a marked disruption of behavior indicated by
suppression of locomotion to 14% of vehicle performance
(Veh vs 56 mg/kg, q = 2.97, p < 0.05; Figure 4). None of the
doses of 10a significantly increased activity counts more than
vehicle (Veh vs 3 mg/kg, q = 0.162, ns; Veh vs 10 mg/kg, q =
0.175, ns; Veh vs 30 mg/kg, q = 2.27, ns; Figure 4). The
Figure 2. Pharmacological characterization and assessment of binding
conformation of compound 10a. (A) Inhibition of [3H]dopamine
uptake by compound 10a on COS7 cells transiently expressing DAT
WT. Dotted line shows uptake inhibition by cocaine as determined
previously.52 (B) Inhibition of [3H]WIN 35,428 binding by 10a on
COS7 cells transiently expressing DAT WT (black squares) or DAT
Y156F (blue circles). The observed change in IC50 value by the Y156F
mutation is 6.3-fold, suggesting a perturbation of the binding site for
10a by removal of the OH-group on Tyr156. Gray lines (WT, dotted;
Y156F, solid) show regression analysis for comparison of similar
binding experiments using cocaine performed previously,52 showing
no significant difference in IC50 values. The observed IC50 values for
10a are basis on the calculated Ki values shown in Table 3. Data are
means SEM of 3−4 experiments performed in triplicate.
binding site for dopamine17,21 is readily accessible from the
extracellular environment. In contrast, binding of the atypical
F
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a
Table 4. Assessment of DAT Inhibitor Binding Properties for Selected Analogues
compd
DAT WT Ki (nM)
n
DAT Y156F Ki (nM)
n
Y156F:WT affinity ratio
b
5
6
27 [21;35]
35 [21;54]
5
5
3
4
4
4
3
3
3
4
4
242 [162;362]
140 [129;152]
63 [57;69]
3
4
4
3
4
4
4
4
5
3
3
9.0
4.0
6.3
20
b
10a
10 [7.6;14]
10b
16 [9.7;28]
202 [148;274]
1080 [913;1270]
1560 [1200;2030]
183 [114;293]
3490 [2790;4360]
307 [257;367]
355 [266;474]
1700 [1050;2730]
12b
147 [93;234]
221 [140;349]
28 [17;47]
7.3
7.1
6.5
2.4
3.8
15
12c
S-(+)-19a
( )-19a
1480 [1390;1590]
81 [69;94]
b
20
b
23
24 [14;40]
b
25
253 [163;394]
6.7
a
́
The affinity (Ki value) for the compounds binding to DAT WT or the Y156F mutant is determined by their ability to block the binding of
[3H]WIN 35,428 to COS7 cells transiently expressing DAT WT or Y156F as depicted for 10a in Figure 2B. Data were analyzed by nonlinear
regression analysis using Prism 6.0 (GraphPad) and are shown as mean [SEM interval]. The Ki values were calculated from the IC50 values using the
equation Ki = IC50/(1 + (L/Kd)), where Kd is the affinity for WIN 35,428 and L is the concentration of added [3H]WIN 35,428. The IC50 values
b
were calculated from means of pIC50 values and SEM interval from pIC50 SEM. All data are performed in triplicate. These compounds have been
previously reported.27,30,51,54
Effect of Administration of 10a in Mice Discriminating
Cocaine from Saline. To further evaluate for possible cocaine-
like behavioral effects, compound 10a was tested in a drug
discrimination paradigm in which mice were trained to
discriminate between injections (ip) of cocaine 10 mg/kg and
saline. Cocaine dose dependently increased responding on the
cocaine-designated lever with an ED50 of 3.5 mg/kg (95% CI,
2−5.1) and a maximum of >80%. In contrast, compound 10a
administered 5 or 30 min before the start of the session failed
to substitute for cocaine and occasioned a maximum of 23%
(95% CI, −10.8−56.2, ns) cocaine lever responding (Figure
5A). None of the doses of cocaine or 10a decreased the overall
group response rate below 50% of control performance,
indicating that the doses tested did not markedely interfere
with the execution of the behavioral task (Figure 5B). Of note,
Figure 3. Phase I metabolic stability of 10a in mouse liver microsomes.
10a was incubated in mouse liver microsomes with and without
the dose of 56 mg/kg of 10a was not evaluated in the drug
discrimination paradigm as it was previously shown to
substantially suppress locomotor activity (Figure 4). The
results of both in vivo tests in mice support an atypical
behavioral profile for compound 10a as has been reported for
several previously described benztropine analogues.40−52
Computational Studies on 10a. On the basis of the
behavioral data coupled with its Y156F:WT affinity ratio of 6.3,
we went on to examine the binding pose of 10a at DAT. A
molecular dynamics (MD) simulation study was conducted to
test our hypothesis that the atypical DAT inhibitors have
similar binding poses at DAT to one another but bind to a
conformation that differs from that of cocaine. We have further
hypothesized that such differences may ultimately contribute to
their unique behavioral profiles and potential for development
as cocaine abuse therapeutics.49,57
Previously, we have characterized the isomeric preference of
the classic atypical DAT inhibitor, 22, in its binding to hDAT
with MD simulations in combination with Markov state model
analysis.58 We have shown the hydrogen attached to the
nitrogen in the tropane ring (tNH) of 22 prefers the equatorial
form in transitioning hDAT toward an inward-facing
conformation.58 With the assumption that 10a has a similar
tNH isomeric preference, based on the stabilized binding pose
of 22 in an inward-occluded conformation of hDAT, we
selected a similar binding pose of 10a in hDAT from our
NADPH (negative control), and percent remaining over time was
measured via LC/MS/MS. 10a showed complete stability under all
conditions. Data are presented as mean SEM (n = 3 per time point).
Figure 4. Effect of cocaine (5−40 mg/kg; open circles) and 10a (3−
56 mg/kg; open squares) on the locomotor activity of mice during 1 h
session. Ordinates: locomotor activity expressed in counts/h. Abscissa:
dose in mg/kg. Each point represents the mean ( SEM) of six
animals. Note that 10a failed to produce a significant increase in
locomotor activity. Asterisks indicate a significant difference from
locomotion obtained after corresponding vehicle administration
(Dunnet’s test; p < 0.05).
absence of locomotor stimulating effects by 10a indicates a
pharmacological profile atypical for DAT inhibitors.
G
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docking results and carried out MD simulations of the hDAT/
10a complex (Figure 6).
As expected, our simulation results show that these two
compounds share a large number of common binding residues
in the central binding site of DAT (Figure 6B). The central
ligand binding site can be divided into three subsites, A, B, and
C,59 in which the tropane ring and the alkyl chain of 10a and
22 are located in subsite A, whereas the two fluorophenyl rings
occupy subsites B and C. Specifically, in subsite A, the charged
amine of the tropane ring forms a salt bridge interaction with
Asp79, while the alkyl chain forms a hydrophobic−aromatic
interaction with Phe320. One of the fluorophenyl rings is in
hydrophobic contact with residues from TM3 and TM8 in
subsite B, whereas the other fluorophenyl ring interacts with
nonpolar residues from TM3, TM6, and TM10 in subsite C.
Interestingly, in comparison to the previous reported binding
poses of the benztropine analogues in an outward-facing DAT
model,50 our current models of the hDAT/2258 and hDAT/
10a complexes reveal deeper binding positions of these two
compounds, which likely correlate with the induced conforma-
tional transition of hDAT from the outward-facing to inward-
facing state: specifically the interacting residues in subsites A
and B remain largely identical, while the deeper binding
positions in the inward-facing state result in interactions with
residues located more intracellularly on TM3 and TM6 in
subsite C. However, even in their deeper binding positions, the
resulting poses are still consistent with previous mutagenesis
study50 that indicated Asn157 in TM3 is critical in interacting
with one of the phenyl ring’s fluorine substituent, whereas
Ala480 is in close vicinity to the other fluorine, although the
terminal N(CH3)2 of 10a appears to shift its pose toward
Asn157 (Figure 6B,C).
Figure 5. Effects of doses of cocaine (0.3−20 mg/kg; open circles)
and 10a (0.3−30 mg/kg) given 5 min (open square) or 30 min (filled
triangles) in mice trained to discriminate cocaine (10 mg/kg, ip) from
vehicle. (A) Ordinates: percentage of responses emitted on the
cocaine lever. (B) Ordinates: response rate expressed as percentage of
response rate after saline administration. Abscissa: dose in mg/kg.
Each point represents the mean ( SEM) of six animals. Asterisks
indicate value significant different from 0 (i.e., 95% CI do not include
0). Note that 10a occasioned less than 25% cocaine lever response.
Figure 6. Molecular modeling the binding pose of 10a. (A) Side view of the hDAT/10a complex. (B) Interaction frequencies of the binding site
residues (heavy atoms of which are within 5 Å of ligand heavy atoms) for the last 300 ns of each trajectory averaged for each condition. (C) A
zoomed-in view of 22 and 10a binding poses, showing the hDAT residues Asn157 and Ala480 that have been previously mutated. The results in C
are colored in the same way as in B.
H
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spectra (CDCl3, 77.2, or DMSO-d6, 39.5). Gas chromatography−mass
spectrometry (GC/MS) data were acquired (where obtainable) using
an Agilent Technologies (Santa Clara, CA) 6890N GC equipped with
an HP-5MS column (cross-linked 5% PH ME siloxane, 30 m × 0.25
mm i.d. × 0.25 μm film thickness) and a 5973 mass-selective ion
detector in electron-impact mode. Ultrapure grade helium was used as
the carrier gas at a flow rate of 1.2 mL/min. The injection port and
transfer line temperatures were 250 and 280 °C, respectively, and the
oven temperature gradient used was as follows: the initial temperature
(100 °C) was held for 3 min and then increased to 295 °C at 15 °C/
min over 13 min, and finally maintained at 295 °C for 10 min.
Combustion analysis was performed by Atlantic Microlab, Inc.
(Norcross, GA), and the results agree within 0.5% of calculated
values. Melting point determination was conducted using a Thomas−
Hoover melting point apparatus and are uncorrected. On the basis of
NMR and combustion data, all final compounds are >95% pure. The
eluting solvent system CMA refers to CHCl3/CH3OH/NH4OH in the
percentage of methanol indicated where NH4OH is 1%.
CONCLUSION
■
In summary, novel dopamine uptake inhibitors based on N-
substituted or 2β-substituted benztropine or benztropinamines
were synthesized. Binding evaluation revealed that substitution
at the tropane nitrogen resulted in retention of high DAT
binding affinity (Ki range = 10.6−53.8 nM) and selectivity over
SERT and NET and that replacing the 3-position O with a N
had no effect on the transporter binding profile of these
compounds. Replacement of the 2β-carboalkoxy group with the
2β-isoxazole in the benztropine series resulted in a slight
decrease in DAT binding affinity in the racemic group of
analogues, however, S-(+)-19a demonstrated DAT affinity in
the same range as the N-substituted analogues, as well as the
previously reported isoxazole analogues from Carroll and
colleagues (5 and 6). Interestingly, replacement of the 2β-
carboalkoxy group with an ethenyl group gave the most DAT
selective analogue in the series. These data extend our
previously reported SAR and confirm that the benztropines
and the 3-aryl derivatives of cocaine appear to access different
binding poses to DAT despite very similar binding affinities and
structural similarity.
On the basis of its ease of synthesis, DAT affinity (Ki = 12.0
nM), high hERG:DAT ratio (686), and Y156F:WT affinity
ratio = 6.3, we chose compound 10a as a lead molecule in this
series for further evaluation. Compound 10a showed metabolic
stability in mouse liver microsomes, with 88% remaining after 1
h, suggesting resistance to CYP dependent oxidation. Unlike
cocaine, compound 10a did not significantly increase
locomotor activity and did not generalize to the cocaine
discriminative stimulus in mice trained to discriminate 10 mg/
kg of cocaine from saline at either a 5 or 30 min pretreatment
time. These data suggest that despite being a potent DA uptake
inhibitor (Ki = 47 nM), compound 10a exhibits a profile that is
consistent with an atypical DAT inhibitor. The Y156F mutant
data strongly support its binding to DAT as different from that
of cocaine, and the molecular dynamic simulation studies
suggest that it binds similarly to the classic atypical DAT
inhibitor, compound 22. Of course, further evaluation for off-
target binding affinities, pharmacokinetics, and in additional
models of psychostimulant abuse will be required to validate
the potential for development of 10a as a cocaine abuse
medication. Nevertheless, these data further support the
atypical DAT hypothesis as a class of compounds that may
have potential as pharmacotherapeutics to treat psychostimu-
lant use disorders.
8-Methyl-8-azabicyclo[3.2.1]octan-3-amine Hydrochloride (7).30
To a solution of 8-methyl-8-azabicyclo[3.2.1]octan-3-one (1.0 g, 7.2
mmol) in MeOH (20 mL) was added ammonium formate (4.2 g, 67
mmol) and H2O (2.1 mL), followed by 10% Pd/C (0.9 g, 0.8 mmol).
The reaction mixture was stirred at rt overnight. The catalyst was
filtered over Celite, and the filtrate was concentrated under reduced
pressure. The residue was dissolved in EtOH (16 mL), and conc HCl
(1.25 mL) was added dropwise to give the product as a white
1
precipitate (1.2 g, 78% yield). H NMR (400 MHz, DMSO-d6) δ
10.99 (1H, br s), 8.42 (1H, s), 3.70−3.81 (2H, br s), 2.67 (1H, br s),
2.48−2.69 (5H, m), 2.02−2.25 (6H, m). GC-MS (EI) 141 (M + 1).
N-(Bis(4-fluorophenyl)methyl)-3,8-dimethyl-8-azabicyclo[3.2.1]-
octan-3-amine (8).30 A mixture of 7 (1.1 g, 5.0 mmol), chlorobis(4-
fluorophenyl)methane, NaHCO3 (2.4 g, 29 mmol), and KI (0.10 g, 0.6
mmol) in CH3CN (100 mL) was stirred at reflux overnight at 120 °C
in a sealed pressure bottle. After cooling to rt, the reaction mixture was
filtered and the filtrate was evaporated to dryness. H2O (100 mL) was
added to the residue, followed by extraction with CH2Cl2 (3 × 100
mL). The combined organic layers were dried over MgSO4. The
solvent was removed, and the crude product was purified by flash
column chromatography (90:10:1 CH2Cl2:MeOH:NH4OH) to give
the product as a brown solid (1.3 g, 76% yield). GC-MS (EI) 341 (M
− 1).
N-(Bis(4-fluorophenyl)methyl)-8-azabicyclo[3.2.1]octan-3-amine
(9b). 1-Chloroethyl chloroformate (ACE-Cl, 1.6 mL, 15 mmol) was
added to the mixture of 8 (1.3 g, 3.8 mmol) and Na2CO3 (1.6 g, 15
mmol) in 1,2-dichloroethane (20 mL). The reaction mixture was
stirred at reflux for 3 h and filtered. The filtrate was concentrated
under reduced pressure, followed by addition of MeOH (20 mL) and
stirring at reflux overnight to give the crude product, which was
converted to the free base and purified by flash column
chromatography (8:2:0.1, CHCl3:EtOH:NH4OH) to give the product
1
as a yellow oil (0.80 g, 64% yield). H NMR (400 MHz, CD3OD) δ
7.18−7.23 (4H, m), 6.94−7.00 (4H, m), 4.84−4.86 (1H, m), 3.65−
3.68 (1H, s), 3.18 (1H, m), 2.77 (1H, m), 2.22−2.24 (1H, m), 2.03−
2.11 (3H, m), 1.92−1.97 (2H, m), 1.58−1.68 (2H, m), 1.38−1.39
(1H, m). GC-MS (EI) 327 (M − 1).
EXPERIMENTAL METHODS
■
Synthesis. Reaction conditions and yields were not optimized.
Anhydrous solvents were purchased from Aldrich and were used
without further purification except for THF, which was freshly distilled
from sodium benzophenone ketyl. All other chemicals and reagents
were purchased from Sigma-Aldrich Co. LLC, Combi-Blocks, TCI
America, OChem Incorporation, Acros Organics, Ark Pharm, and Alfa
Aesar. Unless otherwise stated, amine final products either remained as
free bases or were converted into oxalate salts, typically by treating the
free base in isopropanol with 1:1 molar ratio of oxalic acid in acetone.
Some of the oxalate salts were recrystallized from hot methanol or a
methanol−acetone solvent mixture. Spectroscopic data and yields refer
to the free base. Flash chromatography was performed using silica gel
2-(3-(Bis(4-fluorophenyl)methoxy)-8-azabicyclo[3.2.1]octan-8-
yl)-N,N-dimethylethanamine Oxalate (10a; JJC7-043). A mixture of
9a54 (0.36 g, 1.1 mmol), 2-chloro-N,N-dimethylacetamide (0.13 g, 1.1
mmol), NaHCO3 (0.48 g, 5.7 mmol), and KI (50 mg, 0.30 mmol) in
CH3CN (20 mL) was stirred at reflux overnight at 120 °C in a sealed
pressure bottle. After cooling to rt, the reaction mixture was filtered
and the filtrate was evaporated to dryness. H2O (50 mL) was added to
the residue, followed by extraction with CH2Cl2 (3 × 50 mL). The
combined organic layers were dried over MgSO4. The solvent was
removed, and the crude product was purified by flash column
chromatography (90:10:1 CH2Cl2:MeOH:NH4OH) to give the amide
as a brown solid (0.35 g, 92%). 1H NMR (400 MHz, CDCl3) δ 7.25−
7.28 (4H, m), 6.99−7.03 (4H, m), 5.40 (1H, s), 3.83 (4H, m), 3.68−
3.70 (1H, m), 3.10 (1H, s), 2.95−2.97 (1H, s), 2.34−2.39 (4H, m),
2.19−2.21 (2H, m), 2.01−2.04 (2H, m). GC-MS (EI) 413 (M − 1).
(EMD Chemicals, Inc.; 230−400 mesh, 60 Å). H and 13C NMR
1
spectra were acquired using a Varian Mercury Plus 400 spectrometer
at 400 and 100 MHz, respectively. Chemical shifts are reported in
parts-per-million (ppm) and referenced according to deuterated
solvent for 1H spectra (CDCl3, 7.26, or DMSO-d6, 2.50) and 13C
I
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Reduction of the amide to the amine, using the method described for
10e, gave the product as a yellow oil (0.30 g, 89%), which was
converted to the oxalate salt in acetone and recrystallized from hot i-
The amide (0.40 g, 0.94 mmol) was dissolved in THF (2.8 mL) and
added dropwise to a mixture of LAH (0.07 g, 1.9 mmol) in THF (1
mL) at 0 °C and stirred overnight at rt. The mixture was quenched
with H2O (0.4 mL), 15% NaOH (0.4 mL), and H2O (1.2 mL)
successively at 0 °C. The resulting mixture was filtered and washed
with THF and evaporated to dryness. The crude product was purified
by flash column chromatography (eluting with 10% CMA) to give the
product as a brown oil (350 mg, 90%). The free base was converted to
the oxalate salt in acetone and recrystallized from hot i-PrOH to give
the product as a white solid; mp 119−121 °C; [α]2D4 +19.54 (MeOH, c
1
PrOH to give the product as an orange solid; mp 165−167 °C. H
NMR (400 MHz, CD3OD) δ 7.38−7.42 (4H, m), 7.03−7.08 (4H, m),
5.59 (1H, s), 3.71 (1H, m), 3.46−3.55 (2H, m), 3.31 (4H, m), 2.92
(6H, s), 2.48−2.50 (2H, m), 2.33−2.37 (2H, m), 2.21−2.24 (4H, m).
13C NMR (100 MHz, CD3OD) δ 160.98, 138.14, 128.34, 128.25,
114.96, 114.74, 79.89, 79.65, 71.44, 69.45, 67.00, 61.71, 52.02, 50.25,
46.93, 43.24, 43.10, 30.52, 23.95. GC-MS (EI) 400 (M). Anal.
(C24H30F2N2O·2C2H2O4·2.5H2O) C, H, N.
1
0.65). H NMR (400 MHz, CD3OD) δ 7.38−7.42 (4H, m), 7.06−
7.10 (4H, m), 5.19 (1H, s), 3.43 (1H, m), 2.3.1 (1H, m), 2.94−3.03
(2H, m), 1.82−2.45 (10H, m), 1.01−1.04 (6H, m, 2 × CH3). 13C
NMR (100 MHz, CD3OD) δ 163.49, 163.43, 161.00, 129.30, 129.21,
115.19, 114.97, 63.33, 61.89, 58.12, 53.37, 48.29, 46.94, 45.77, 29.66,
23.83, 16.68, 16.44. Anal. (C25H33F2N3·3C2H2O4·0.5H2O·0.5i-PrOH)
C, H, N.
N-(Bis(4-fluorophenyl)methyl)-8-(2-(dimethylamino)ethyl)-8-
azabicyclo[3.2.1]octan-3-amine Oxalate (10b). Compound 10b was
prepared as described for 10a from 9b (0.36 g, 1.1 mmol) to give the
product as a yellow oil (0.28 g, 64% yield over two steps), which was
converted to the oxalate salt in acetone and recrystallized from hot
1
MeOH to give the product as a white solid; mp 152−155 °C. H
NMR (400 MHz, CD3OD) δ 7.30−7.37 (4H, m), 7.02−7.08 (4H, m),
4.99−5.09 (1H, m), 3.76−3.78 (2H, m), 3.50−3.52 (1H, m), 3.29−
3.35 (4H, m), 2.92−2.97 (6H, m), 2.30−2.54 (4H, m), 2.21−2.24
(2H, m), 1.94−2.08 (2H, m). 13C NMR (100 MHz,) δ129.19, 129.15,
129.11, 129.07, 114.98, 114.76, 68.13, 63.32, 61.94, 61.40, 52.35,
46.94, 46.76, 45.54, 45.27, 42.88, 42.81, 42.57, 30.31, 25.54, 24.84,
24.56, 23.83. GC-MS (EI) 397 (M − 2). Anal. (C24H31F2N3·3C2H2O4·
3H2O) C, H, N.
General Procedure for the Preparation of 12a−e from
(
)-11. To a solution of N-nor-2β-carboethoxy-3α-[bis(4-
fluorophenyl)methoxy]tropane ( )-1134,35,55 in DMF (5 mL/1.0
mmol of ( )-11) was added the bromoalkane (1.2 equiv) and K2CO3
(2.0 equiv). The mixture was heated to 65 °C and stirred at this
temperature overnight. The mixture was then filtered and the filtrate
was concentrated. The residue was diluted with H2O (10 mL/1.0
mmol of ( )-11), basified with NaHCO3 to pH 9, and extracted with
CHCl3 (×3). The combined organic layers were dried (MgSO4) and
concentrated. The residue was purified by column chromatography
(eluting with CMA) to give the product.
3-(Bis(4-fluorophenyl)methoxy)-8-(2-(piperidin-1-yl)ethyl)-8-
azabicyclo[3.2.1]octane Oxalate (10c). Compound 10c was prepared
as described for 10a from 9a (0.33 g, 1.0 mmol) and 2-bromo-1-
(piperidin-1-yl)ethanone (0.21 g, 1.0 mmol) to give the product as a
yellow oil (0.36 g, 82% yield over two steps), which was converted to
the oxalate salt and recrystallized from hot MeOH to give the product
(
)-N-Cyclopropylmethyl-2β-carboethoxy-3α-[bis(4-
fluorophenyl)methoxy]tropane (12a). Compound 12a was prepared
from ( )-11 (245 mg, 0.61 mmol) and 1-bromomethylcyclopropane
1
1
as a white solid; mp 183−184 °C. H NMR (400 MHz, CD3OD) δ
in 84% yield. H NMR (400 MHz, CDCl3) δ 7.32−7.21 (m, 4H),
7.37−7.41 (4H, m), 7.03−7.08 (4H, m), 5.59 (1H, s), 3.67 (1H, m),
2.46−2.50 (2H, m), 3.52−3.53 (4H, m), 3.23−3.31 (6H, m), 2.23−
2.33 (6H, m), 1.82−1.85 (4H, m), 1.62−1.63 (2H, m). 13C NMR
(100 MHz, CD3OD) δ 164.77, 163.42, 161.00, 138.14, 138.11 128.33,
128.25, 114.97, 114.75, 79.64, 66.90, 53.53, 51.16, 23.90, 22.73, 21.14.
GC-MS (EI) 440 (M). Anal. (C27H34F2N2O·2C2H2O4·1.25H2O) C,
H, N.
7.06−6.93 (m, 4H), 5.34 (s, 1H), 4.18−4.04 (m, 2H), 4.00 (d, J = 5.2
Hz, 1H), 3.95 (d, J = 4.8 Hz, 1H), 3.20 (br s, 1H), 2.70 (s, 1H), 2.35
(dd, J = 5.2, 12.4 Hz, 1H), 2.18−1.62 (m, 8H), 1.23 (t, J = 7.0 Hz,
3H), 0.84−0.72 (m, 1H), 0.53−0.44 (m, 1H), 0.44−0.34 (m, 1H),
0.12−0.00 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 172.63, 163.26,
160.82, 138.54, 138.41, 128.40, 128.33, 115.42, 115.20, 80.31, 70.88,
60.44, 60.37, 60.18, 58.05, 51.86, 36.21, 25.87, 24.67, 14.20, 10.29,
4.37, 2.16. Anal. (C27H31F2NO3) C, H, N.
N-(Bis(4-fluorophenyl)methyl)-8-(2-(piperidin-1-yl)ethyl)-8-
azabicyclo[3.2.1]octan-3-amine Oxalate (10d). Compound 10d was
prepared as described for 10b from 9b (0.33 g, 1.0 mmol) and 2-
bromo-1-(piperidin-1-yl)ethan-1-one (206 mg, 1.0 mmol) to give the
product as a yellow oil (0.33 g, 75% yield over two steps), which was
converted to the oxalate salt in acetone and recrystallized from hot
(
)-N-(2-(1H-Indol-3-yl))ethyl-2β-carboethoxy-3α-[bis(4-
fluorophenyl)methoxy]tropane (12b). Compound 12b was prepared
from ( )-11 (265 mg, 0.66 mmol) and 2-bromoethylindole in 90%
yield. 1H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H), 7.57 (d, J = 8.4 Hz,
1H), 7.40 (d, J = 8.0 Hz, 1H), 7.30−7.24 (m, 4H), 7.17 (m, 1H), 7.09
(m, 1H), 7.04−6.96 (m, 5H), 5.36 (s, 1H), 4.10−3.98 (m, 3H), 3.23
(br s, 1H), 2.91−2.79 (m, 2H), 2.75 (s, 1H), 2.62−2.53 (m, 2H),
2.24−1.77 (m, 6H), 1.20 (t, J = 7.2 Hz, 3H). Anal. (C33H34F2N2O3) C,
H, N.
1
MeOH to give the product as a white solid; mp 191−192 °C. H
NMR (400 MHz, CD3OD) δ 7.32−7.38 (4H, m), 7.03−7.07 (4H, m),
5.09 (1H, s), 3.43−3.51 (4H, m), 3.19−3.34 (6H, m), 2.97 (1H, br s),
2.54−2.56 (2H, m), 2.21−2.31 (4H, m), 1.99−2.03 (2H, m), 1.81−
1.84 (4H, m), 1.63 (2H, br s). 13C NMR (100 MHz, CD3OD) δ
163.33, 163.23, 160.80, 138.14, 129.20, 129.12, 114.97, 114.76, 61.94,
61.49, 53.48, 51.23, 45.02, 32.65, 24.65, 22.68, 21.12. GC-MS (EI) 437
(M − 2). Anal. (C27H35F2N3·3C2H2O4·1.5H2O) C, H, N.
( )-N-(2-Amino-3-methylbutyl)-2β-carboethoxy-3α-[bis(4-
fluorophenyl)methoxy]tropane (12c). Compound 12c was prepared
from ( )-11 (310 mg, 0.77 mmol) and 2-(Fmoc-amino)-3-
methylbutyl bromide in 73% yield. 1H NMR (400 MHz, CDCl3)
δ7.38−7.20 (4H, m), 7.06−6.95 (4H, m), 5.33 (1H, s), 4.20−4.04 (m,
2H), 3.97 (m, 1H), 3.43 (1H, m), 3.03−2.94 (2H, m), 2.18−1.62
(10H, m), 1.24 (t, J = 7.0 Hz, 3H), 1.01−1.04 (m, 8H), ppm. Anal.
(C28H36F2N2O3·H2O) C, H, N.
( )-N-(2-(1,3-Dioxoisoindolin-2-yl)ethyl)-2β-carboethoxy-3α-[bis-
(4-fluorophenyl)methoxy]-tropane (12d). Compound 12d was
prepared from ( )-11 and 2-bromoethylphthalimide in 89% yield.
1H NMR (400 MHz, CDCl3) δ 7.83−7.80 (m, 2H), 7.70−7.67 (m,
R-8-(2-Amino-3-methylbutyl)-N-(bis(4-fluorophenyl)methyl)-8-
azabicyclo[3.2.1]octan-3-amine Oxalate (10e). To Fmoc-L-valine
(0.50 g, 1.5 mmol) in CH2Cl2 (10 mL) was added SOCl2 (1 mL). The
reaction mixture was stirred at reflux for 2 h. The solvent was removed,
followed by crystallization from hexane to give Fmoc-L-valine-Cl,
which was added to a mixture of 9b (0.50 g, 1.5 mmol), 10% NaHCO3
aqueous solution (10 mL), and CHCl3 (10 mL). The reaction mixture
was stirred at rt for 20 min. The solvent was removed, and H2O (50
mL) was added to the residue, followed by extraction with CH2Cl2 (3
× 50 mL). The combined organic layers were dried over MgSO4. The
solvent was removed, and the crude product was purified by flash
column chromatography (90:10:1, CH2Cl2:MeOH:NH4OH) to give
2H), 7.27−7.20 (m, 4H), 7.06−6.90 (m, 4H), 5.31 (s, 1H), 3.96 (m,
1H), 3.85−3.72 (m, 1H), 3.70−3.46 (m, 4H), 3.26 (br s, 1H), 2.62 (s,
1H), 2.57−2.43 (m, 2H), 2.20−1.60 (m, 6H), 1.06 (t, J = 7.2 Hz, 3H).
( )-N-[(N,N-Dimethylamino)-2-oxoethyl]-2β-carboxyethyl-3α-
[bis(4-fluorophenyl)methoxy]-tropane (12e). Compound 12e was
prepared from ( )-11 (355 mg, 0.88 mmol) and 2-bromo-N,N-
dimethylacetamide (176 mg, 1.06 mmol) and purified by column
1
the amide as a brown solid (400 mg, 64%). H NMR (400 MHz,
CDCl3) δ 7.19−7.25 (4H, m), 6.97−7.02 (4H, m), 5.30 (1H, m),
3.25−3.28 (1H, m), 2.83−2.91 (1H, m), 1.61−2.32 (10H, m), 0.90−
0.95 (6H, m, 2 × CH3). Note: deprotection of the Fmoc group
occurred during the reaction, based on the NMR data.
1
chromatography (eluting with 5% CMA) in 99% yield (425 mg). H
NMR (400 MHz, CDCl3) δ 7.28−7.20 (m, 4H), 7.02−6.97 (m, 4H),
J
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5.33 (s, 1H), 4.16−4.06 (m, 1H), 3.98−3.92 (m, 2H), 3.69 (m, 1H),
3.07 (m, 1H), 3.07−3.05 (m, 2H), 3.05 (s, 3H), 2.89 (s, 3H), 2.68 (m,
1H), 2.23−2.14 (m, 1H), 2.12−1.92 (m, 4H), 1.80−1.74 (m, 1H),
1.21 (t, J = 7.4 Hz, 3H). GC-MS (EI) m/z 441 (M-OEt), 414 (M-
CONMe2).
General Procedure for the Synthesis of 19a−d from 16. n-
BuLi (2.0 M in hexane, 2.6 equiv) was added dropwise to the solution
of oxime 17 (1.3 equiv) in dry THF at 0 °C under argon. The mixture
was stirred for 2 h at rt after the addition. The reaction mixture was
then cooled to 0 °C, and 1634,35,55 (1 equiv) in THF was added. The
mixture was allowed to warm to rt overnight. The mixture was diluted
with H2O, and the two layers were separated. The aqueous layer was
extracted with CHCl3 (×3). The combined organic layers were dried
(MgSO4) and concentrated to give the crude product 18 in almost
quantitative yield, which was used in the next step without further
purification.
Crude 18 was dissolved in THF (10 mL/1 mmol 18) and 3 N HCl
(2.5 equiv) was added. The mixture was heated to reflux for 4 h before
it was cooled to rt. The reaction mixture was then basified with 2N
NaHCO3 solution to pH 9. The two layers were separated, and the
aqueous layer was extracted with CHCl3 (×3). The combined organic
layers were dried (MgSO4) and concentrated. The residue was purified
by column chromatography (5% CMA) to give the pure products
19a−d.
( )-N-(2-Amino)ethyl-2β-carboxyethyl-3α-[bis(4-fluorophenyl)-
methoxy]-tropane (13a). To a solution of 12d (242 mg, 0.42 mmol)
in EtOH (6 mL) was added NH2NH2 (27 mg, 0.84 mmol), and the
mixture was heated to reflux for 2 h. The mixture was then cooled to
rt, and the white precipitate was filtered. The filtrate was concentrated.
The residue was purified by column chromatography (eluting with
1
15% CMA) to afford the pure product in 75% yield. H NMR (400
MHz, CDCl3) δ 7.30−7.20 (m, 4H), 7.05−6.96 (m, 4H), 5.33 (s, 1H),
3.97 (m, 1H), 3.87−3.75 (m, 1H), 3.70−3.45 (m, 2H), 3.23 (br s,
1H), 2.68−2.45 (m, 5H), 2.18−1.57 (m, 6H), 1.07 (t, J = 7.2 Hz, 3H).
13C NMR (100 MHz, CDCl3) δ 172.11, 163.48, 161.04, 138.68,
138.53, 128.63, 126.60, 115.60, 115.40, 80.43, 70.73, 63.14, 60.20,
58.81, 52.15, 51.67, 37.25, 36.01, 25.75, 25.70. Anal. (C25H30F2N2O3·
0.5H2O) C, H, N.
( )-N-(N,N-Dimethylamino)ethyl 2β-Hydroxymethyl-3α-[bis(4-
fluorophenyl)methoxy]tropane (13b). To a suspension of LAH
(125 mg, 3.92 mmol) in dry THF (1 mL) was added dropwise the
solution of N-[(N,N-dimethylamino)-2-oxoethyl] 2β-carboxyethyl-3α-
[bis(4-fluorophenyl)methoxy]-tropane (12e) (400 mg, 0.82 mmol) in
THF (4 mL) at 0 °C. The reaction mixture was allowed to warm to rt
after the addition and stirred overnight. The mixture was then cooled
to 0 °C, and H2O (0.1 mL) was added slowly, followed by addition of
NaOH (6N, 0.3 mL) at rt. The white solid that formed was filtered off,
and the filtrate was dried (K2CO3) and concentrated to give the
product 13b (320 mg, 90%), which was pure by TLC and was used for
the next step without further purification. 1H NMR (400 MHz,
CDCl3) δ 7.27−7.21 (m, 4H), 7.02−6.95 (m, 4H), 5.34 (s, 1H), 3.68
(dd, J = 3.4, 10.4 Hz, 1H), 3.48 (dd, J = 6.0, 10.8 Hz, 1H), 3.42−3.35
(m, 2H), 3.15 (m, 1H), 2.52−2.44 (m, 1H), 2.43−2.37 (m, 2H),
2.34−2.26 (m, 1H), 2.22 (s, 6H), 2.14−1.80 (m, 7H).
( )-2β-(3-Phenylisoxazol-5-yl)-3α-[bis(4-fluorophenyl)methoxy]-
tropane (19a). Compound 19a was prepared from ( )-16 and 17a as
a white solid in 18% yield; mp 158.5−160.5 °C. 1H NMR (400 MHz,
CDCl3) δ 7.80−7.77 (m, 2H), 7.46−7.41 (m, 3H), 7.35−7.25 (m,
4H), 7.05−6.97 (m, 4H), 6.52 (s, 1H), 5.50 (s, 1H), 3.74 (d, J = 5.2
Hz, 1H), 3.41 (br s, 1H), 3.29 (s, 1H), 3.16 (m, 1H), 2.26 (s, 3H),
2.18−1.64 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 162.30, 138.08,
129.73, 129.40, 128.77, 128.75, 128.70, 128.19, 128.12, 126.78, 115.53,
115.48, 115.31, 115.27, 99.86, 80.28, 71.94, 63.48, 60.57, 45.24, 41.84,
36.56, 25.63, 24.98. Anal. (C30H28F2N2O2) C, H, N.
S-(+)-2β-(3-Phenylisoxazol-5-yl)-3α-[bis(4-fluorophenyl)-
methoxy]tropane (S-(+)-19a). S-(+)-19a was prepared from S-(+)-16
and 17a in 15% yield; mp 159−160.5 °C; [α]2D5 11.1° (CHCl3, c 0.6).
NMR spectra were identical to that of the racemate. Anal.
(C30H28F2N2O2) C, H, N.
( )-2β-[3-(4-Methylphenyl)isoxazol-5-yl]-3α-[bis(4-fluorophenyl)-
methoxy]tropane (19b). Compound 19b was prepared from ( )-16
and 17b as a white solid in 21% yield; mp 164−167 °C. 1H NMR (400
MHz, CDCl3) δ 7.67 (d, J = 7.2 Hz, 2H), 7.36−7.20 (m, 6H), 7.03−
6.97 (m, 4H), 6.48 (s, 1H), 5.49 (s, 1H), 3.73 (d, J = 5.4 Hz, 1H), 3.40
(br s, 1H), 3.28 (s, 1H), 3.14 (m, 1H), 2.38 (s, 3H), 2.26 (s, 3H),
2.16−1.80 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 174.48, 163.43
(d, J = 15.2 Hz), 162.25, 160.98 (d, J = 14.4 Hz), 139.79, 138.10,
138.07, 137.99, 137.95, 129.44, 128.78, 128.70, 128.11, 128.19, 126.66,
126.52, 115.52, 115.47, 115.30, 115.26, 99.74, 80.25, 71.94, 63.46,
60.59, 45.24, 41.84, 36.58, 25.63, 24.99, 21.40. Anal. (C31H30F2N2O2)
C, H, N.
( )-2β-[3-(4-Chlorophenyl)isoxazol-5-yl]-3α-[bis(4-fluorophenyl)-
methoxy]tropane (19c). Compound 19c was prepared from ( )-16
and 17c as a white solid in 12% yield. 1H NMR (400 MHz, CDCl3) δ
7.72 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H), 7.33−7.28 (m, 4H),
7.04−6.98 (m, 4H), 6.50 (s, 1H), 5.49 (s, 1H), 3.72 (d, J = 4.2 Hz,
1H), 3.40 (br s, 1H), 3.29 (s, 1H), 3.16 (m, 1H), 2.27 (s, 3H), 2.20−
1.80 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 174.91, 163.37 (d, J =
13.7 Hz), 161.33, 160.92 (d, J = 13.6 Hz), 138.00, 137.97, 137.90,
137.87, 135.73, 129.04, 128.75, 128.66, 128.20, 128.12, 128.06, 127.89,
115.54, 115.50, 115.33, 115.29, 99.78, 80.31, 71.84, 63.46, 60.57,
45.22, 41.81, 36.46, 25.61, 24.94. Anal. (C30H27ClF2N2O2) C, H, N.
( )-2β-[3-(4-Fluorophenyl)isoxazol-5-yl]-3α-[bis(4-fluorophenyl)-
methoxy]tropane (19d). Compound 19d was prepared from ( )-16
and 17d as a white solid in 12% yield. 1H NMR (400 MHz, CDCl3) δ
7.76 (m, 2H), 7.40−7.26 (m, 6H), 7.04−6.98 (m, 4H), 6.50 (s, 1H),
5.49 (s, 1H), 3.72 (d, J = 4.2 Hz, 1H), 3.40 (br s, 1H), 3.29 (s, 1H),
3.16 (m, 1H), 2.27 (s, 3H), 2.20−1.80 (m, 6H). 13C NMR (100 MHz,
CDCl3) δ 174.91, 164.89, 163.29, 162.41, 161.38, 160.99, 138.05,
137.93, 128.75, 128.72, 128.68, 128.63, 128.20, 125.64, 125.61, 115.95,
115.73, 115.53, 115.49, 115.31, 115.28, 99.74, 80.28, 71.91, 63.47,
60.56, 45.23, 41.86, 36.54, 25.61, 24.97. Anal. (C30H27F3N2O2) C, H,
N.
(
)-N-(N,N-Dimethylamino)ethyl 2β-Formyl-3α-[bis(4-
fluorophenyl)methoxy]tropane (14). To a solution of (COCl)2 (23
μL, 0.26 mmol) in dry CH2Cl2 (1 mL) at −78 °C was added slowly a
solution of DMSO (38 mg, 0.49 mmol) in CH2Cl2 (1 mL) under
argon. After 30 min, a solution of alcohol (13b) (95 mg, 0.22 mmol)
in dry CH2Cl2 was added and the reaction mixture was stirred for 1 h
at −78 °C. Et3N (0.14 mL, 0.99 mmol) was added, and the mixture
was allowed to warm to rt, then diluted with H2O and extracted with
CHCl3. The combined organic layers were dried (MgSO4) and
concentrated to give the crude product 9 (90 mg, 95%), which was
used in the next step without purification. 1H NMR (400 MHz,
CDCl3) δ 9.56 (s, 1H), 7.27−7.20 (m, 4H), 7.02−6.95 (m, 4H), 5.34
(s, 1H), 3.97 (m, 1H), 3.68 (m, 1H), 3.14 (m, 1H), 2.50 (m, 1H),
2.43−2.27 (m, 3H), 2.24−2.16 (m, 2H), 2.21 (s, 6H), 2.11−1.78 (m,
5H).
(
)-N-(N,N-Dimethylamino ethyl 2β-Ethenyl-3α-[bis(4-
fluorophenyl)methoxy]tropane(15). n-BuLi (0.18 mL, 1.36 M in
hexane, 0.25 mmol) was added dropwise to the suspension of
methytriphenylphosphonium bromide (90 mg, 0.25 mmol) in dry
THF (1 mL) at 0 °C, under argon. The resulting yellow-orange
solution was stirred for 30 min before the ice−H2O bath was removed.
The crude aldehyde 14 (90 mg, 0.21 mmol) in dry THF (1 mL) was
then added, and the solution was stirred at rt overnight. The reaction
mixture was then diluted with H2O (5 mL) and extracted with CHCl3
(3 × 10 mL). The combined organic layers were dried (MgSO4) and
concentrated. The residue was purified by preparative TLC (eluting
1
with 10% CMA) to give the product 15 (41 mg) in 46% yield. H
NMR (400 MHz, CDCl3) δ 7.29−7.23 (m, 4H), 7.16−6.96 (m, 4H),
6.00 (ddd, J = 8.2, 10.2, 17.2 Hz, 1H), 5.36 (s, 1H), 4.93−4.83 (m,
2H), 3.27 (d, J = 4.4 Hz, 1H), 3.13 (m, 1H), 3.04 (m, 1H), 2.44 (m,
1H), 2.37 (m, 3H), 2.24 (s, 6H), 2.08−1.82 (m, 6H), 1.75−1.70 (m,
1H). 13C NMR (100 MHz, CDCl3) δ 128.49, 128.41, 128.35, 128.26,
115.41, 115.37, 115.20, 115.16, 114.81, 80.01, 73.92, 65.11, 60.02,
50.38, 44.70, 35.63, 25.60, 25.22. GC-MS (EI) m/z 426 (M+). Anal.
(C26H32F2N2O) C, H, N.
Radioligand Binding Assays. DAT Binding Assay. Striata were
dissected from male Sprague−Dawley rat brains (supplied on ice from
K
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Journal of Medicinal Chemistry
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Bioreclamation (Hicksville, NY) and prepared by homogenizing
tissues in 20 volumes (w/v) of ice-cold modified sucrose phosphate
buffer (0.32 M sucrose, 7.74 mM Na2HPO4, 2.26 mM NaH2PO4, pH
adjusted to 7.4) using a Brinkman Polytron (setting 6 for 20 s) and
centrifuged at 20000 rpm for 10 min at 4 °C. The resulting pellet was
resuspended in buffer, recentrifuged, and suspended in buffer again to
a concentration of 10 mg/mL, original wet weight (OWW).
Experiments were conducted in assay tubes containing 0.5 mL sucrose
phosphate buffer, 0.5 nM [3H]WIN 35,428 (Kd value = 5.53, specific
activity = 84 Ci/mmol; PerkinElmer Life Sciences, Waltham, MA), 1.0
mg of tissue OWW, and various concentrations of inhibitor. The
reaction was started with the addition of tissue, and tubes were
incubated for 120 min on ice. Nonspecific binding was determined
using 100 μM cocaine HCl.
SERT Binding Assay. Membranes from frozen brain stem dissected
from male Sprague−Dawley rat brains (supplied on ice from
Bioreclamation, Hicksville, NY) were homogenized in 20 volumes
(w/v) of 50 mM Tris buffer (120 mM NaCl and 5 mM KCl, adjusted
to pH 7.4) at 25 °C using a Brinkman Polytron (at setting 6 for 20 s).
The tissue was centrifuged at 20000 rpm for 10 min at 4 °C. The
resulting pellet was suspended in buffer and centrifuged again. The
final pellet was resuspended in cold buffer to a concentration of 15
mg/mL OWW. Experiments were conducted in assay tubes containing
0.5 mL of buffer, 1.4 nM [3H]citalopram (Kd value = 1.94 nM, specific
activity = 83 Ci/mmol; PerkinElmer Life Sciences, Waltham, MA), 1.5
mg of brain stem tissue, and various concentrations of inhibitor. The
reaction was started with the addition of the tissue, and the tubes were
incubated for 60 min at rt. Nonspecific binding was determined using
10 μM fluoxetine.
pcDNA3 (Invitrogen, Carlsbad, CA). The Y156F mutation was
introduced using QuickChange (adapted from Stratagene, La Jolla,
CA) and confirmed by restriction enzyme mapping and DNA
sequencing. DAT WT and Y156F cDNA containing plasmids were
amplified by transformation into XL1 blue competent cells
(Stratagene) and grown in LB media overnight at 37 °C in an orbital
incubator (Infors) at 200 rpm. Plasmids were harvested using the maxi
prep kit (Qiagen) according to the manufacturer’s manual.
Cell Culture and Transfection. COS7 cells were grown in
Dulbecco’s Modified Eagle’s Medium 041 01885 supplemented with
10% fetal calf serum, 2 mM L-glutamine, and 0.01 mg/mL gentamicin
at 37 °C in 10% CO2. DAT WT and Y156F were transiently
transfected into COS7 cells with Lipo2000 (Invitrogen) according to
manufacturer’s manual using a cDNA:Lipo2000 ratio of 1:2.
[3H]DA Uptake Experiments. Uptake assays were performed on
intact COS7 cells essentially as described61 using 3,4-[Ring-2,5,6-3H]-
dihydroxyphenylethylamine ([3H]DA) (30−60 Ci/mmol) (Perki-
nElmer). Briefly, transfected COS7 cells were plated in 24-well dishes
(105 cells/well) coated with polyornithine (Sigma) to achieve an
uptake level of no more than 10% of total added [3H]DA. The uptake
assays were carried out 2 days after transfection in uptake buffer (UB)
(25 mM HEPES, 130 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 1.2 mM
MgSO4, 1 mM L-ascorbic acid, 5 mM D-glucose, and 1 μM of the
catechol-O-methyltransferase inhibitor Ro 41-0960 (Sigma), pH 7.4).
Prior to the experiment, the cells were washed once in 500 μL of UB,
and the nonlabeled compound was added to the cells in the indicated
concentrations in a total volume of 500 μL. The assay was initiated by
the addition of 6−10 nM [3H]DA. Nonspecific uptake was determined
with 1 μM nomifensine (Sigma-Aldrich). After 5 min of incubation at
rt, the cells were washed twice with 500 μL of ice-cold UB, lysed in
250 μL of (24-well) 1% SDS, and left for >30 min at 37 °C on gentle
shaking. All samples were transferred to 24-well counting plates
(PerkinElmer, Waltham, MA), and 500 μL (24-well) of Opti-phase Hi
Safe 3 scintillation fluid (PerkinElmer) was added followed by
counting of the plates in a Wallac Tri-Lux β-scintillation counter
(PerkinElmer). All experiments were carried out with 12 determi-
nations of DA or inhibitor concentrations ranging from 1 nM to 1 mM
performed in triplicates.
NET Binding Assay. Membranes from frozen frontal cortex
dissected from male Sprague−Dawley rat brains (supplied on ice
from Bioreclamation, Hicksville, NY) were homogenized in 20
volumes (w/v) of 50 mM Tris buffer (120 mM NaCl and 5 mM
KCl, adjusted to pH 7.4) at 25 °C using a Brinkman Polytron (at
setting 6 for 20 s). The tissue was centrifuged at 20000 rpm for 10 min
at 4 °C. The resulting pellet was suspended in buffer and centrifuged
again. The final pellet was resuspended in cold buffer to a
concentration of 80 mg/mL OWW. Experiments were conducted in
assay tubes containing 0.5 mL of buffer, 0.5 nM [3H]nisoxetine (Kd
value = 1.0 nM, specific activity = 82 Ci/mmol; PerkinElmer Life
Sciences, Waltham, MA), 8 mg of frontal cortex tissue, and various
concentrations of inhibitor. The reaction was started with the addition
of the tissue, and the tubes were incubated for 180 min at 0−4 °C.
Nonspecific binding was determined using 1 μM desipramine.
The solvent used to dissolve the various analogues of benztropine
was typically methanol and was present at a final concentration of 5%.
Extensive studies previously in this and other laboratories determined
that methanol has no effect on binding at DAT and SERT. However,
there is an effect of methanol on binding at NET and therefore
methanol concentration was controlled in all tubes in that assay. When
compounds were not soluble in methanol we used either ethanol or
DMSO at final concentrations of no greater than 5 or 6%, respectively.
Previous studies found no effect of either of these solvents at these
concentrations on binding at any of the sites. For all three monoamine
transporter binding assays, incubations were terminated by rapid
filtration through Whatman GF/B filters, presoaked in 0.3% (SERT)
or 0.05% (DAT, NET) polyethylenimine, using a Brandel R48 filtering
manifold (Brandel Instruments Gaithersburg, Maryland). The filters
were washed twice with 5 mL of cold buffer and transferred to
scintillation vials. Cytoscint (MP Biomedicals, OH) (3.0 mL) was
added, and the vials were counted the next day using a Beckman 6000
liquid scintillation counter (Beckman Coulter Instruments, Fullerton,
California) or a Tri-Carb 2910-B liquid scintillation counter
(PerkinElmer Life Sciences, MA). The Ki values for the benztropine
derivatives were obtained using nonlinear least-squares regression
(using GraphPad Prism Software, San Diego, CA) of the displacement
data giving IC50 values, from which affinities (Ki values) were
calculated using the Cheng−Prusoff equation.60
[3H]WIN35,428 Binding Experiments. Binding assays were carried
out essentially as described for the uptake experiments on whole cells
only using [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane ([3H]-
WIN35,428) (76−87 Ci/mmol) (PerkinElmer). Previous to the
binding experiment, cells were washed once in ice cold UB and, after
the addition of unlabeled compound in the indicated concentrations
and [3H]WIN35,428, the reactions were incubated at 5 °C until
equilibrium were obtained (>90 min). All experiments were carried
out with 12 determinations with inhibitor concentration range from 1
nM to 1 mM, performed in triplicate.
Mouse Microsomal Stability Assay. Phase I metabolic stability
assay was conducted in mouse liver microsomes. For phase I
metabolism, the reaction was carried out with 100 mM potassium
phosphate buffer, pH 7.4, in the presence of NADPH regenerating
system (1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3 mM
MgCl2, 0.4 U/mL glucose-6-phosphate dehydrogenase, 50 μM sodium
citrate). Reactions in triplicate were initiated by addition of the liver
microsomes to the incubation mixture (compound final concentration
was 10 μM; 0.5 mg/mL microsomes). Negative controls in the
absence cofactors were carried for both to determine the specific
cofactor free degradation. Compound disappearance was monitored
via LC/MS/MS as described previously.53 Briefly, chromatographic
analysis was performed using an Accela ultra high-performance system
consisting of an analytical pump and an autosampler coupled with
TSQ Vantage mass spectrometer (Thermo Fisher Scientific Inc.,
Waltham MA). Separation of the analyte from potentially interfering
material was achieved at ambient temperature using Agilent Eclipse
Plus column (100 mm × 2.1 mm i.d.) packed with a 1.8 μm C18
stationary phase. The mobile phase used was composed of 0.1% formic
acid in acetonitrile and 0.1% formic acid in H2O with gradient elution,
starting with 10% (organic), linearly increasing to 99% up to 2.5 min,
maintaining at 99% (2.5−3.5 min), and reequlibrating to 10% by 4.5
Molecular Pharmacology. Site-Directed Mutagenesis. Synthetic
cDNA encoding the human DAT (synDAT) were subcloned into
L
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Journal of Medicinal Chemistry
Article
min. The total run time for each analyte was 5.0 min. The mass
transitions used for compound 10a was 401.256 > 356.219 and that for
internal standard is 423.110 > 207.060.
molecular modeling and simulations of hDAT in complex with 22, we
found that the equatorial tNH isomer of 22 is the more stable isomer
in the binding site of hDAT.58 Assuming that the equatorial tNH
isomer is also the stable form of 10a in the binding site, we docked it
into the central binding site of the inward-occluded hDAT model
using the induced-fit docking (IFD) protocol61 implemented in the
Schrodinger suite (release 2016-4). We selected a 10a binding pose
that has the lowest docking score among the poses similar to that of 22
in our established hDAT/22 model.58
Desmond MD systems (D. E. Shaw Research, New York, NY) with
OPLS3 force field64 was used for the MD simulations. hDAT was
placed into explicit 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
lipid bilayer (POPC) using the orientation of dDAT/nortriptyline
structure (PDB 4M48)65 from the Orientation of Proteins in
Membranes database.66 Simple point charge (SPC) water model67
was used to solvate the system, charges were neutralized, and 0.15 M
NaCl was added. The total system size was ∼130000 atoms. The
NPγT ensemble was used with constant temperature (310 K)
maintained with Langevin dynamics, 1 atm constant pressure achieved
with the hybrid Nose−Hoover Langevin piston method68 on an
anisotropic flexible periodic cell, and a constant surface tension (x−y
plane). The system was initially minimized and equilibrated with
restraints on the ligand heavy atoms and protein backbone atoms,
followed by production runs at 310 K with all atoms unrestrained.
Four independent trajectories for each hDAT/22 and hDAT/10a
conditions were collected with the aggregated simulation lengths of 5.7
μs for hDAT/22 and 6.3 μs for hDAT/10a.
Locomotor Activity Studies in Mice. Ambulatory activity of
Male Swiss Webster mice (Taconic Farms) was studied in 40 cm3 clear
acrylic chambers. The acrylic chambers were placed inside monitors
(Omnitech Electronics, Columbus, OH) that were equipped with
light-sensitive detectors spaced 2.5 cm apart along two perpendicular
walls. Mounted on the opposing walls were infrared light sources that
were directed at the detectors. Counts of horizontal activity were
registered each time the subject interrupted a single beam and
cumulated to obtain the total of the session (1 h). Cocaine or 10a was
administered intraperitoneally (ip) at a volume of 1 mL/kg
immediately before the start of the session. No habituation to the
experimental chambers was provided to the animals before testing.
Each dose of the test compound was studied in six mice, and mice
were used only once. Drug effects were evaluated by analysis of
variance (ANOVA) and subsequent Dunnett’s multiple comparison
test.
Drug Discrimination Studies. Experimental details are essentially
identical to those described previously.57 Sessions were conducted
with male Swiss Webster mice (Taconic Farms) placed in 29.2 × 24.2
× 21 cm3 operant-conditioning chambers (modified ENV-001; MED
Associates, St. Albans, VT) containing two response keys (levers
requiring a downward force of 0.4 N) with pairs of green and yellow
light-emitting diodes above each. A dispenser delivered 45 mg food
pellets (BioServ, Frenchtown, NJ) to a tray located between the
response keys, and a light was mounted near the ceiling to provide
overall illumination. The chambers were located inside sound
attenuated boxes that contained a white noise generator. Mice were
initially trained with food reinforcement to press both levers and
eventually trained to press one after cocaine (10 mg/kg, ip) and the
other after saline (ip) injection. All responses produced audible clicks
of a relay mounted behind the front wall of the chamber. The ratio of
responses to food pellets (fixed ratio or FR) was gradually increased
until, under the final conditions, the completion of 10 consecutive
responses on the cocaine- or saline-appropriate lever produced food.
Incorrect responses reset the FR response requirement. The right-
versus left-assignments of cocaine and saline keys were counter-
balanced among subjects. Subjects were injected and placed in
chambers. Sessions started after a 5 min time-out period, during which
lights were off and responses had no consequences other than the
audible click. After the time-out, the house light was turned on until
the completion of the 10-response requirement and the presentation
of food. Sessions ended after 20 food presentations or 15 min,
whichever occurred first, and were conducted 5 days per week, with
cocaine (C) or saline (S) sessions alternating according with a
CSSCSC schedule. Testing was initiated after subjects met the criteria
of at least 85% cocaine- or saline-appropriate responding on four
consecutive sessions (two sessions of each) over the entire session, and
the first FR of the session. Test sessions were identical to training
sessions with the exceptions that cocaine (0.3−20 mg/kg) or 10a
(0.1−30 mg/kg) were administered before the starting of the session
and that 10 consecutive responses on either lever were reinforced.
Pretreatment time for cocaine was 5 min while for 10a was either 5 or
30 min. Straight lines were fitted to the linear portion of the dose−
effect curve that included one data point below 20%, one data point
above 80%, and all data points in between. Slope and intercepts were
then used to derive the dose of compounds that produced 50% of
cocaine lever responding (ED50) and relative confidence intervals
(CI). The increase in % cocaine lever response produced by doses of
test compounds was not considered significant if the relative 95% CI
include 0.
The binding site residues were determined to be those within 5.0 Å
of ligand heavy atoms within the last 300 ns of trajectories. The
analysis was performed using MDTraj69 in combination with in-house
Python scripts.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b01454.
Microanalysis data (PDF)
Representative hDAT model in complex with compound
and 22 (PDB)
Representative hDAT model in complex with compound
and 10a (PDB)
SMILES molecular formula strings data (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: (443) 740-2887. Fax: (443) 740-2111. E-mail:
anewman@intra.nida.nih.gov.
■
ORCID
Rana Rais: 0000-0003-4059-2453
Barbara S. Slusher: 0000-0001-9814-4157
Amy Hauck Newman: 0000-0001-9065-4072
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Lone Rosenquist is thanked for excellent technical assistance.
Support for this research was provided to A.H.N., M.F.Z., J.C.,
T.K., J.K., A.M.A., D.A.G., and L.S. by the National Institute on
Drug AbuseIntramural Research Program (Z1A DA000389
21). C.J.L. is supported by the Danish Council for Independent
Research (0602-02100B and 4183-00581). Care of the animals
was in accordance with the guidelines of the National Institutes
of Health and the National Institute on Drug Abuse Intramural
Molecular Docking and Dynamics Simulations. The pKa
values of the two nitrogens in 10a were calculated using three
programs (Epik and Jaguar62 from Schrodinger suite (release 2016-4)
and Chemicalize),63 which predicted pKa values of 9.9−10.3 for the
tropane nitrogen and 5.5−6.0 for the nitrogen in the alkyl chain. Thus,
for our docking and MD simulations under pH 7.4, the tropane
nitrogen but not the alkyl chain nitrogen was protonated. In our recent
M
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Article
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ABBREVIATIONS USED
■
DA, dopamine; DAT, dopamine transporter; SERT, serotonin
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