Structure-activity relationships at monoamine transporters for a series of N-substituted 3α-(bis[4-fluorophenyl]methoxy)tropanes: Comparative molecular field analysis, synthesis, and pharmacological evaluation

Article abstract of DOI:10.1021/jm030646c

The development of structure-activity relationships (SAR) with divergent classes of monoamine transporter ligands and comparison of their effects in animal models of cocaine abuse have provided insight into the complex relationship among structure, binding profiles, and behavioral activity. Many 3α-(diphenylmethoxy)tropane (benztropine) analogues are potent dopamine uptake inhibitors but exhibit behavioral profiles that differ from those of cocaine and other compounds in this class. One of the most potent and dopamine transporter (DAT) selective N-substituted benztropine analogues (N-(4-phenyl-n-butyl)-3α-(bis[4-fluorophenyl]methoxy)tropane, 1c) is devoid of cocaine-like behaviors in rodent models but is also highly lipophilic (cLogD = 5.01), which compromises its water solubility and may adversely affect its pharmacokinetic properties. To further explore the SAR in this series and ultimately to design dopamine uptake inhibitors with favorable lipophilicities for drug development, a comparative molecular field analysis (CoMFA) was performed on a set of benztropine analogues previously synthesized in our laboratory. The CoMFA field analysis on the statistically significant (r ²_cv = 0.632; r²_ncv = 0.917) models provided valuable insight into the structural features required for optimal binding to the DAT, which was used to design a series of novel benztropine analogues with heteroatom substitutions at the tropane N-8. These compounds were evaluated for binding at DAT, serotonin (SERT) and norepinephrine (NET) transporters, and muscarinic M1 receptors in rat brain. Inhibition of [ ³H]DA uptake in synaptosomes was also evaluated. Most of the analogues showed high DAT affinity (12-50 nM), selectivity (10- to 120-fold), potent inhibition of dopamine uptake, and lower lipophilicities as predicted by cLogD values.

Full text of DOI:10.1021/jm030646c

3388
J. Med. Chem. 2004, 47, 3388-3398
Structure-Activity Relationships at Monoamine Transporters for a Series of
N-Substituted 3r-(Bis[4-fluorophenyl]methoxy)tropanes: Comparative
Molecular Field Analysis, Synthesis, and Pharmacological Evaluation
Santosh S. Kulkarni,† Peter Grundt,† Theresa Kopajtic,‡ Jonathan L. Katz,‡ and Amy Hauck Newman*,†
Medicinal Chemistry and Psychobiology Sections, National Institute on Drug AbusesIntramural Research Program,
National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, Maryland 21224
Received December 29, 2003
The development of structure-activity relationships (SAR) with divergent classes of monoamine
transporter ligands and comparison of their effects in animal models of cocaine abuse have
provided insight into the complex relationship among structure, binding profiles, and behavioral
activity. Many 3R-(diphenylmethoxy)tropane (benztropine) analogues are potent dopamine
uptake inhibitors but exhibit behavioral profiles that differ from those of cocaine and other
compounds in this class. One of the most potent and dopamine transporter (DAT) selective
N-substituted benztropine analogues (N-(4-phenyl-n-butyl)-3R-(bis[4-fluorophenyl]methoxy)-
tropane, 1c) is devoid of cocaine-like behaviors in rodent models but is also highly lipophilic
(cLogD ) 5.01), which compromises its water solubility and may adversely affect its
pharmacokinetic properties. To further explore the SAR in this series and ultimately to design
dopamine uptake inhibitors with favorable lipophilicities for drug development, a comparative
molecular field analysis (CoMFA) was performed on a set of benztropine analogues previously
synthesized in our laboratory. The CoMFA field analysis on the statistically significant (r2
)
cv
0.632; r2ncv ) 0.917) models provided valuable insight into the structural features required for
optimal binding to the DAT, which was used to design a series of novel benztropine analogues
with heteroatom substitutions at the tropane N-8. These compounds were evaluated for binding
at DAT, serotonin (SERT) and norepinephrine (NET) transporters, and muscarinic M1 receptors
in rat brain. Inhibition of [3H]DA uptake in synaptosomes was also evaluated. Most of the
analogues showed high DAT affinity (12-50 nM), selectivity (10- to 120-fold), potent inhibition
of dopamine uptake, and lower lipophilicities as predicted by cLogD values.
Introduction
cocaine but had relatively lower affinities for SERT and
NET than for DAT.16-18 Muscarinic receptor binding
affinities were significantly reduced when the N-methyl
group was replaced with substituents with greater steric
bulk, resulting in selective DAT ligands.19-21 Many of
these compounds potently blocked dopamine uptake in
vitro but did not produce stimulation of locomotor
activity or cocaine-like discriminative stimulus effects
in rodent models of cocaine abuse.22,23 Their distinctive
SAR, compared to the 3-aryltropane class of DAT
inhibitors, and results of photoaffinity labeling experi-
ments suggested that the benztropines bound to a
distinctive site or sites on the DAT.8 It has been
proposed that these distinctive interactions at the
protein level may contribute to the benztropine’s dis-
crepant behavioral profiles.18,24
In contrast, perhaps a more important consideration
in drug development is the pharmacokinetic properties
of the compounds that are related to their physical
properties, including lipophilicity.25 Even if these com-
pounds have potent and selective in vitro actions, poor
bioavailability or slow pharmacokinetics could signifi-
cantly contribute to altering or negating in vivo actions.
Recently, a pharmacokinetic and blood-brain barrier
penetration study on several of these N-substituted
benztropine analogues was conducted.26 This study
demonstrated that these compounds show high brain-
to-plasma ratios that are comparable to cocaine. How-
Cocaine is a potent psychomotor stimulant and drug
of abuse that binds with moderate affinity to all three
monoamine transporters. The primary mechanism by
which cocaine exerts its reinforcing and psychomotor
stimulant effects is by inhibiting dopamine reuptake
through blockade of the dopamine transporter (DAT)
and subsequent accumulation of dopamine in meso-
corticolimbic regions of the brain.1-4 Although roles for
the other monoamine transporters, serotonin (SERT)
and norepinephrine (NET), in the reinforcing actions of
cocaine are under intensive investigation, the DAT
remains a primary target for the development of cocaine-
abuse medications.5,6 Extensive SAR with structurally
different chemical classes of DAT inhibitors and the
development of photoaffinity ligands and other molec-
ular tools have provided insight into inhibitor binding
site characteristics of the DAT.7-10 The three-dimen-
sional quantitative structure-activity relationship (3D-
QSAR) based models have also been used to understand
the binding site topology around several series of
tropane-based DAT inhibitors.11-15
In general, the 3R(bis[4-fluorophenyl]methoxy)tro-
panes bound to the DAT with higher affinities than
* To whom correspondence should be addressed. E-mail: anewman@
intra.nida.nih.gov. Phone: (410) 550-6568, extension 114. Fax: (410)
550-6855.
† Medicinal Chemistry Section.
‡ Psychobiology Section.
10.1021/jm030646c This article not subject to U.S. Copyright. Published 2004 by the American Chemical Society
Published on Web 05/15/2004

Structure-Activity Relationships of Tropanes
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3389
Table 1. Summary of CoMFA Results
alignment
AFI
FFI
AFII
FFII
r2
0.632
5
0.468
4
0.654
5
0.441
4
cv
no. of comp
SEP
0.368
0.917
0.175
121.851
52.9
0.438
0.862
0.224
87.251
60.9
0.357
0.917
0.175
121.163
49.0
0.450
0.856
0.228
82.937
55.2
r2
ncv
SEE
F
steric
electrostatic
47.1
39.1
51.0
44.8
r2
0.522
0.924
0.023
0.418
0.894
0.029
0.351
0.930
0.020
0.555
0.886
0.029
pred
r2
a
bs
standard deviationa
a Bootstrapping analysis (100 samples).
Figure 1. Chemical structures of cocaine and the parent
benztropine analogues.
ever, they have long durations of action and other
distinctive pharmacokinetic properties, which may con-
tribute to their lack of cocaine-like behavioral actions.
Complete pharmacokinetic and behavioral characteriza-
tion of N-(4-phenyl-n-butyl)-3R-[bis(4-fluorophenyl)-
methoxy]tropane (1c, Figure 1; DAT Ki ) 8.51 nM), one
of the most potent and selective compounds in this
series,18,19 was hampered by its high lipophilicity (cLogD
) 5.01), which limited its water solubility.26,27 To design
a series of compounds that retained the desirable
binding profile of 1c and to reduce lipophilicity, a 3D-
QSAR based CoMFA model was derived from a series
of 76 benztropine analogues reported earlier16-21 to
ascertain the important regions of the molecules for
binding with the DAT and subsequently to identify
those regions where modifications could be introduced
to reduce lipophilicity without compromising DAT bind-
ing affinities. It was determined that the diphenyl ether
portion of the benztropine molecule was critical for
binding to the DAT and that significant modification
could not be made without severely reducing activity.
Conversely, a wide diversity of N-substituent modifica-
tions were considerably better tolerated and suggested
that a novel series of heteroatom N-substituted 3R-(bis-
[4-fluorophenyl]methoxy)tropanes could be designed to
have desired pharmacological and pharmacokinetic
properties. Thirteen novel N-substituted analogues were
synthesized and evaluated for DAT, SERT, NET, mus-
carinic M1 receptor binding, and inhibition of dopamine
uptake.
Figure 2. Plot of predicted vs observed activity of molecules
from training (9) and test (2) sets using alignment AFI.
the SAR of the training set of compounds,21 thus serving
as a validation set for the CoMFA models.
The results of the CoMFA studies are summarized
in Table 1. A rigid-body rms fitting of the non-hydrogen
atoms of the tropane ring (AFI) showed a correlative
and predictive model. A cross-validated r2 of 0.632 was
obtained with five components, and a non-cross-vali-
dated r2 of 0.917 was observed with this model. The
relative contributions of steric (52.9%) and electrostatic
(47.1%) interactions were almost equal. A plot depicting
the observed vs predicted activities of the molecules
used in deriving this model is shown in Figure 2.
The test set of compounds contained variation at both
the diphenylmethoxy and N-8 substitutions. The N
substituent was varied in both size and shape. When
the CoMFA model was used to predict activity of the
test set of compounds, a predictivity measure r2
of
pred
0.522 was obtained. Figure 2 depicts the plot of observed
vs predicted activities of the test set compounds.
Realignment of these compounds using rigid-body
field fit (FFI) on the steric and electrostatic fields of the
template molecule decreased the significance of the
models. This alignment showed an r2cv of 0.468 with four
components, and the non-cross-validated r2 was 0.862.
Molecular Modeling
A 3D-QSAR based CoMFA study was performed on a
structurally diverse set of 76 benztropine analogues,
using DAT binding affinities (see Supporting Informa-
tion for more details). This set of ligands included
modifications at the C-3 diphenylmethoxy group as well
as at the N-8 position. These analogues varied in DAT
affinities over about 3 log units. The test set of com-
pounds was designed and synthesized on the basis of
However, it had reduced external predictivity with r2
pred
of 0.418. The analysis of the CoMFA models with
alignment derived from superimposing the centroids of
the aromatic rings and the N-8 atom (AFII) showed a
high internal consistency with r2 of 0.654 with five
cv
components and non-cross-validated r2 of 0.917. How-

3390 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Kulkarni et al.
Figure 3. CoMFA SD X Coeff contour plots from analysis of alignment AFI. Sterically favored (70% contribution) and unfavored
(30% contribution) regions are shown as green and yellow contours. The positive charge favoring (70% contribution) and negative
charge favoring (30% contribution) regions are shown as blue and red contours, respectively. Compound 1c is shown as a capped
stick model in these regions.
ever, this model had reduced external predictivity
because its r2
was 0.351. When this alignment was
In the electrostatic contours, the blue contours de-
scribe regions where positively charged groups enhance
the activity and red contours describe regions where
negatively charged groups enhance activity. The elec-
trostatic contour can be seen primarily toward the
diphenylmethoxy group. The red and blue contours are
seen near the para position of one of the phenyl rings.
One blue contour can be seen around the N-8 substitu-
ent, but steric interactions seem to have major contribu-
tion to the activity in this position.
pred
used as an input for rigid-body field fit, it provided a
reasonable degree of internal predictivity. There was an
improvement in the predictivity of the test set com-
pounds, r2
of 0.555. Because the alignments in
pred
CoMFA ignore the entropic effects, it is difficult to
speculate about the binding modes of these compounds
based on the alignment rules.
The analysis of the CoMFA contour maps can provide
more insight into the SAR of these compounds with
DAT. The steric and electrostatic contour maps (Figure
3) were derived from the product of the standard
deviation associated with the CoMFA column and
coefficient (SD X coeff) at each lattice point. Since rigid-
body rms fitting of the non-hydrogen atoms of the
tropane ring (AFI) showed a correlative and predictive
model, contour graphs were interpreted with this model.
The green contours represent regions of high steric
tolerance, while yellow contours represent regions of
unfavorable steric effects. There are many sterically
favored green contours around the molecule. These
green contours were dominant in the diphenylmethoxy
terminus of the molecules as well as at the N-8 sub-
stituent. The large green contours in the diphenyl-
methoxy region indicate that increasing steric bulk in
these regions will improve DAT binding affinity. The
few yellow contours are buried in two large green
contours, suggesting that in this region the relative
orientation of the phenyl rings is important because
variation in the torsion angles due to different substit-
uents (especially at the 2′-position) in the phenyl ring
decreased DAT binding affinities.15,28 This also suggests
that the diphenylmethoxy group has a significant influ-
ence on the DAT affinity.
Previously described SAR and the CoMFA contour
graphs showed the importance of different structural
regions on the benztropine molecule. An appropriately
substituted (i.e., 4′,4′′-diF) diphenylmethoxy group is
required for high-affinity DAT binding. This region of
the binding site is restricted because only small halogen
substitution in the para or meta positions are well
tolerated. This effect is more prominent when the N-8
position is substituted with sterically bulky groups
(allyl, benzyl, propylphenyl, butylphenyl).21 Thus, it was
hypothesized that the N-8 position would provide an
avenue to vary the physicochemical properties of these
molecules without adversely affecting DAT binding
affinity. Thus, the N-8 of 3R-(bis[4-fluorophenyl]methoxy)-
tropane was substituted with moieties having varied
physicochemical properties.
Chemistry
In Scheme 1, compound 1a19 was alkylated with
2-bromoethanol to give compound 2, which was then
reacted with the appropriate arylalkyl halides to give
compounds 3a and 3b. Direct alkylation of 1a gave the
final compounds 4a-c. Compound 4d was obtained
after reacting compound 4c with morpholine. Com-
pounds 5a and 5b were synthesized after alkylation

Structure-Activity Relationships of Tropanes
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3391
Scheme 1a
a (a) 2-Bromoethanol, TEA, toluene; (b) R1-x (x ) Br, Cl), aqueous NaOH; (c) R2-Br, TEA, toluene, or R2-Cl, K2CO3, DMF; (d)
morpholine, K2CO3, DMF; (e) N-(2-bromoalkyl)phthalimide, K2CO3 or NaHCO3, DMF or acetonitrile; anhydrous hydrazine, EtOH; (f)
R3-Cl, aqueous NaHCO3, CHCl3; (g) LiAlH4, THF; (h) ethyl 3-bromopropionate, K2CO3, DMF; 4 N NaOH, EtOH; (i) aniline, DCC, HOBt,
DMF; (j) 4-(dimethylamino)butyric acid, hydrochloride, CDI, pyridine.

3392 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Kulkarni et al.
Table 2. Binding Data for the Novel N-Substituted Benztropines at DAT, SERT, NET, and M1 Receptor and Inhibition of Dopamine
Uptakea
compd
DAT
SERT
NET
M1
DAUIb
1b
1c
1d
3a
3b
4a
4b
4d
5a
5b
6a
6b
6c
8a
11.8 ( 1.3c
8.51 ( 1.2f
20.0 ( 2.8g
65.9 ( 5.0
48.5 ( 6.7
703 ( 100
42.6 ( 2.2
1620 ( 31
13.9 ( 1.7e
26.0 ( 1.6
13.1 ( 1.5
36.0 ( 2.9
12.6 ( 0.1
27.6 ( 1.9
181 ( 5.6
48 ( 5.5
3260 ( 110d
376 ( 52
610 ( 81d
2210 ( 240
2980 ( 180
3450 ( 440
3060 ( 346
12100 ( 1600
3650 ( 520
28100 ( 2800
1420 ( 130
NTh
11.6 ( 0.9e
13.8 ( 1.7d
576 ( 11e
39f
1640 ( 240
2090 ( 220
1300 ( 75
7730 ( 740
14300 ( 640
23000 ( 2300
4600 (680
555 ( 69
47.9 ( 5.2
613 ( 90
390 ( 51
9280 ( 1000
336 ( 47
15800 ( 2300
1250 ( 140
2370 ( 170
556 ( 49
1230 ( 150
394 ( 16
23.4 ( 3.0
18.7 ( 2.0
22.3 ( 2.6
227 ( 29
48.9 ( 6.6
659 ( 96
26.0 ( 2.9
NTh
597 ( 14
637 ( 49
6.5 ( 0.5
8.1 ( 1.2
14.2 ( 1.7
5.0 ( 0.1
NTh
1090 ( 81
572 ( 76
1130 ( 120
652 ( 62
1490 ( 82
558 ( 81
1420 ( 170
3280 ( 420
4140 ( 340
368 ( 49
8b
10
cocaine
NTh
499 ( 64
286 ( 38
NTh
NTh
285 ( 27
3300 ( 170
61400 ( 11000
236 ( 21
a All binding data are recorded in Ki ( SEM (nM); the methods used to obtain these data are described in Experimental Methods.
b The inhibition of dopamine uptake data is recorded as IC50 ( SEM (nM). c Data from ref 16. d Data from ref 33. e Data from ref 20.
f Data from ref 19. g Data from ref 21. h NT ) not tested.
Table 3. Binding Selectivities for the Novel N-Substituted
with appropriately N-substituted phthalimides followed
Benztropines
by treatment with anhydrous hydrazine. Compound 5a
compd
DAT/SERT
DAT/NET
DAT/M1
ClogDa
was then reacted with the respective acid chlorides
under Schotten-Baumann conditions to give com-
pounds 6a and 6c. Reduction of compound 6a with
lithium aluminum hydride (LiAlH4) gave compound 6b.
Compounds 8a and 8b were synthesized from the acids
7a and 7b, which were obtained by alkylation of
compounds 1a and 1e, respectively, with ethyl 3-bromo-
propionate followed by saponification. Amidation of 1a
with 4-(N-dimethylamino)butyric acid using 1,1′-car-
bonyldiimidazole (CDI) followed by reduction with
LiAlH4 gave compound 10. All compounds were con-
verted into HBr or oxalate salts for biological evaluation.
1b
1c
1d
3a
3b
4a
4b
4d
5a
5b
6a
6b
6c
8a
276
44
82
32
27
11
336
14
331
21
46
30
45
54
3
52
260
149
52
1
68
2
2.27
5.01
3.36
4.19
4.44
4.73
2.01
2.05
1.61
0.94
3.29
3.74
2.62
3.53
4.62
1.71
1.51
9
63
8
13
8
17
86
17
10
90
91
42
34
31
119
23
8
102
49
31
52
51
8b
10
cocaine
Structure-Activity Relationships
10
1
12
215
All compounds were tested for displacement of the
respective radioligands labeling the DAT, SERT, NET,
and muscarinic M1 receptors. Several compounds showed
high to moderate binding affinities at the DAT (Table
2). Substitution with the amide linker (6a, 6c, 8a, and
8b) reduced the conformational flexibility of the N
substituent in the molecules but retained high affinity
and selectivity for the DAT. In contrast, DAT binding
affinity for the bis[4-chlorophenyl] analogue 8b was
significantly reduced (Ki ) 181 nM), even compared to
the reduction observed previously when the N-(4-
phenyl)-n-butyl substituent replaced the N-methyl group
on 1d.21
Although the role of conformational flexibility in the
N-8 binding region is not known, in the case of the GBR
series of DAT inhibitors, introduction of a double bond
in the N substituent improves DAT binding affinity
(GBR 12909 IC50 ) 3.7 nM, GBR13069 IC50 ) 0.9 nM).29
It is interesting to note that the number of rotatable
bonds have been found to be an important determinant
of oral bioavailability,30 suggesting that substitution
with an amide linker should have favorable effects on
bioavailability. The order of amide substitution has little
effect on DAT activity because compounds 6a and 8a
are similarly active. Thus, addition of an amide group
in this region of the 3R-(bis[4-fluorophenyl]methoxy)-
tropane molecule successfully reduced the lipophilicity
a Calculated lipophilicity at pH 7.4 from ACD/LogD Suite,
release 7.00, version 7.05, Advanced Chemistry Development Inc.
Toronto, Canada.
by about 1.5 log units without adversely affecting DAT
binding affinity. This substitution also showed good
selectivity (see Table 3) over muscarinic M1 receptor
binding. For example, compound 8a shows 119-fold
DAT/M1 selectivity, which is in accordance with earlier
observations where large N-8 substituents increased
DAT selectivity.19 The reduction of the amide in com-
pound 6b resulted in ∼3-fold reduction in the DAT
binding affinity. Modification of the four-carbon-linked
phenyl ring as in 1c with a terminal ether-linked phenyl
ring produced a significant reduction in DAT affinity
(4a). However, moving the oxygen in the linking chain
(3b) and replacing the phenyl ring with a heteroaryl
group (3a) or OH (4b) improved DAT binding affinity
although not to the high affinity of 1c. Substitution of
the phenyl ring with the amino group (5b and 10)
showed a slight reduction in activity. However, substi-
tution with a large aliphatic group (morpholine, 4d),
which resulted in a significant reduction in lipophilicity,
caused a substantial reduction in DAT binding affinity,
suggesting that there is a limitation to the steric bulk
at the N-8 position that can be tolerated by the DAT in
this series of molecules.

Structure-Activity Relationships of Tropanes
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3393
linker between the terminal binding element and the
N-8 atom was varied with heteroatom substitution.
Most of these compounds showed good binding affinity
at DAT with excellent selectivity over SERT, NET, and
muscarinic receptors. These compounds also showed
potent inhibition of dopamine uptake with predicted
favorable physicochemical properties. Thus, modifica-
tion of the N-substituent allowed the retention of a
desired in vitro profile while reducing the lipophilicity.
Ongoing in vivo studies will provide additional insight
into the role of physicochemical properties on the
behavioral actions of the benztropine analogues and
may provide new leads toward a cocaine-abuse medica-
tion.
Experimental Methods
All chemicals and reagents were purchased from Aldrich
Chemical Co. or Lancaster Synthesis, Inc., unless otherwise
indicated and were used without further purification. All
melting points were determined on a Thomas-Hoover melting
point apparatus and are uncorrected. The 1H and 13C NMR
spectra were recorded on Bruker (Billerica, MA) AC-300 or a
Varian Mercury Plus 400 instrument. Samples were dissolved
in an appropriate deuterated solvent (CDCl3). Proton chemical
shifts are reported as parts per million (δ ppm) relative to
tetramethylsilane (Me4Si, 0.00 ppm), which was used as an
internal standard. Chemical shifts for 13C NMR spectra are
reported as δ relative to deuterated chloroform (CDCl3, 77.0
ppm). Infrared spectra were recorded as a neat film on NaCl
plates, unless otherwise indicated, with a Perkin-Elmer Spec-
trum RX I FT-IR system. Microanalyses were performed by
Atlantic Microlab, Inc. (Norcross, GA), and the results agree
within (0.4% of the calculated values. All column chromatog-
raphy was performed using silica gel (Aldrich, 230-400 mesh,
60A) and CHCl3/MeOH (9:1) as eluting solvent unless other-
wise indicated. The free base was converted into oxalate salts
by treating the free base in acetone or ethanol with 1.1 or 2.1
equiv of oxalic acid followed by precipitation with diethyl ether.
Similarly, HBr salts were prepared by treating the free base
with methanolic HBr followed by precipitation with diethyl
ether.
Synthesis. N-(2-Hydroxyethyl)-3r-[(bis(4′-fluorophen-
yl)methoxy]tropane (2). The HCl salt of 1a (1.60 g, 4.38
mmol) was first converted to its free base form by extracting
with CHCl3 (3 × 50 mL) from 10% (w/v) aqueous sodium
carbonate solution (50 mL), drying, and evaporating to an oil,
which was refluxed with 2-bromoethanol (1.80 g, 14.40 mmol)
and triethylamine (1.20 mL) in toluene (20 mL) for 3 h under
argon gas. The solvent was evaporated under reduced pres-
sure, and the residue obtained was extracted with CHCl3 (50
mL × 2), washed with water (50 mL × 2), and passed over
anhydrous Na2SO4. The organic layer was evaporated under
reduced pressure to give the product (yield, 1.40 g, 86%), which
was used for further reactions, without purification: 1H NMR
(300 MHz, CDCl3) δ 2.03-2.17 (m, 4H), 2.49-2.52 (m, 2H),
2.89-2.93 (m, 2H), 3.05-3.06 (m, 2H), 3.78 (m, 1H), 3.97 (m,
2H), 4.05-4.06 (m, 2H), 5.01 (broad s, 1H), 5.39 (s, 1H), 6.99-
7.04 (m, 4H), 7.22-7.27 (m, 4H); 13C NMR (75 MHz, CDCl3) δ
25.17, 34.56, 55.85, 57.18, 62.67, 67.58, 80.73, 115.84, 116.13,
128.61, 128.72, 137.80, 137.84, 161.00, 164.27.
Figure 4. Correlation between DAT binding affinity and
dopamine uptake inhibition for N-substituted benztropines.
All of these N-substituted analogues were tested for
the inhibition of DA uptake, and these data (Figure 4)
were positively correlated with DAT binding affinities
(r2 ) 0.5919, F1,11 ) 15.95, p ) 0.0021). A significant
and positive correlation is to be expected because ligand
binding to the DAT is the initial event in the inhibition
of DA uptake.
Although none of the N-substituted analogues showed
high-affinity binding to SERT, the polar aliphatic chain
(4b) showed particularly weak binding affinity, thus
resulting in the most DAT/SERT selective compound in
this series. All of the N-substituted analogues showed
low binding affinities at NET, though most were less
selective than 1c.19
Previous studies showed that optimal binding affinity
to DAT was obtained with the N-(4-phenyl)-n-butyl
substituent (1c). This compound also showed good DAT
selectivity over muscarinic M1 receptor binding.19 Ad-
ditional SAR confirmed that the four-atom spacer
between the N-8 and terminal phenyl ring gave optimal
DAT binding affinity.31 The results of the present study
demonstrated that substitution with polar linkers and
heteroatoms on the N-8 position could retain desirable
binding profiles of the previous series of compounds,
with reduced lipophilicities, which may prove to be
favorable for in vivo pharmacokinetic properties and
behavioral activity.
Conclusions
A CoMFA model was derived using a data set of 76
previously reported 3R-(bis[4-fluorophenyl]methoxy)-
tropanes. This CoMFA model showed a good correlation
with steric and electrostatic descriptors. On the basis
of these studies, the diphenyl ether moiety appears to
be vital for binding this class of compounds to the DAT.
It may be noteworthy that the diphenyl ring system has
been observed in many GPCR ligands, suggesting this
moiety is conferring a unique functionality or “privileged
structure” for binding with the transmembrane receptor
site on the DAT.32 In contrast, the N-8 position tolerates
varied substitutions, and thus, it could be varied to
modulate the physicochemical properties of these com-
pounds. As such, a novel series of N-8 substituted
benztropine analogues was prepared in which the
N-[2(Thiophen-2-ylmethoxy)ethyl]-3r-[(bis(4′fluoro-
phenyl)methoxy]tropane Oxalate (3a). Compound 2 (0.75
g, 2.00 mmol) was dissolved in acetone (3 mL), and 2-chloro-
methylthiophene (0.40 g, 3.02 mmol) [prepared from thiophene
2-methanol (5 g, 43.75 mmol) and thionyl chloride (6.25 g, 3.83
mL, 52.52 mmol) in THF (20 mL), with heating at 50 °C for
2 h, evaporation under vacuum gave an oily product, which
was used without purification] was added. The reaction
mixture was stirred at reflux for 20 h after addition of 50%
(w/v) aqueous NaOH (12 mL). The reaction was quenched by
dilution of the mixture with water (25 mL), and the mixture
was extracted with CHCl3 (50 mL × 2). The solvent was

3394 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Kulkarni et al.
evaporated under reduced pressure, and the residue was
purified by column chromatography to obtain 0.07 g (7%) of
free base of compound 3a, which was converted to the oxalate
DMF (10 mL) at 110 °C for 10 h. The reaction mixture was
cooled to room temperature, diluted with water (25 mL), and
extracted with ethyl acetate (50 mL × 2). After the organic
layer was washed with water (25 mL × 2), the volatiles were
evaporated under reduced pressure. The crude product was
purified by column chromatography. The free base was con-
verted into the oxalate salt (yield, 0.21 g, 19%): mp 120-122
°C; 1H NMR (300 MHz, CDCl3) δ 1.88-1.94 (m, 6H), 2.17-
2.19 (m, 2H), 2.47-2.50 (t, 4H), 2.55-2.58 (t, 2H), 3.59-3.66
(m, 5H), 3.69-3.72 (t, 4H), 4.20-4.23 (m, 4H), 5.38 (s, 1H),
6.97-7.03 (m, 4H), 7.24-7.29 (m, 4H); 13C NMR (75 MHz,
CDCl3) δ 28.23, 35.30, 53.17, 54.44, 58.60, 64.18, 67.29, 69.02,
69.90, 70.22, 80.24, 115.59, 115.88, 128.65, 128.76, 138.65,
138.69, 153.86, 160.84, 164.10. Anal. (C28H36F2N2O3‚1.5C2H2O4)
C, H, N.
1
salt: semisolid; H NMR (300 MHz, CDCl3) δ 1.78-2.12 (m,
8H), 2.61-2.65 (t, 2H), 3.27 (m, 2H), 3.53-3.56 (m, 1H), 3.62-
3.66 (m, 2H), 4.69 (s, 2H), 5.35 (s, 1H), 6.94-7.02 (m, 6H),
7.24-7.29 (m, 5H); 13C NMR (75 MHz, CDCl3) δ 26.53, 35.99,
52.24, 59.45, 59.48, 68.04, 69.56, 69.83, 79.85, 115.50, 115.78,
126.10, 126.65, 127.00, 128.68, 128.79, 138.97, 139.01, 141.59,
160.80, 164.10. Anal. (C27H29F2NO2S‚C2H2O4‚0.25H2O) C, H,
N.
N-(2-Benzyloxyethyl)-3r-[(bis(4′-fluorophenyl)meth-
oxy]tropane Hydrobromide (3b). Compound 3b was ob-
tained from 2 (1.00 g, 2.68 mmol) and benzyl bromide (0.48
mL, 4.04 mmol) as described for compound 3a by refluxing
for 4 h. The free base was converted into the HBr salt (yield,
N-(2-Aminoethyl)- 3r-[(bis(4′-fluorophenyl)methoxy]-
tropane Hydrobromide (5a).20 Compound 1a (3.00 g, 9.10
mmol) was reacted with N-(2-bromoethyl)phthalimide (2.54 g,
10 mmol) and anhydrous K2CO3 (1.88 g, 13.60 mmol) in DMF
(20 mL) for 2 h at 80 °C. The product was isolated by diluting
the reaction mixture with cold water (50 mL), extracted with
ethyl acetate (50 mL × 2), dried (MgSO4), and evaporated
under reduced pressure to give an oily product (4.00 g, 7.96
mmol), which was dissolved in anhydrous ethanol (30 mL).
Anhydrous hydrazine (0.60 mL, 19.10 mmol) was added to the
reaction flask, and the mixture was allowed to stir at reflux
for 2 h. The mixture was evaporated under reduced pressure,
and the residue was treated with 40% (w/v) KOH solution (100
mL), extracted with CHCl3 (50 mL × 2), dried over anhydrous
MgSO4, and evaporated under reduced pressure to obtain 2.10
g (61% from 1a) of compound 5a, which was converted to the
1
0.28 g, 22%): mp 123-126 °C; H NMR (300 MHz, CDCl3) δ
1.81-2.17 (m, 8H), 2.72 (m, 2H), 3.29 (m, 2H), 3.59 (m, 1H),
3.71 (m, 2H), 4.53 (s, 2H), 5.36 (s, 1H), 6.99-7.02 (m, 4H),
7.26-7.32 (m, 9H); 13C NMR (75 MHz, CDCl3) δ 26.37, 35.78,
52.23, 59.76, 69.55, 73.57, 79.94, 115.53, 115.82, 128.01,
128.67, 128.77, 138.62, 138.87, 160.81, 164.07. Anal. (C29H31F2-
NO2‚HBr) C, H, N.
N-(3-Phenoxypropyl)-3r-[(bis(4′-fluorophenyl)meth-
oxy]tropane Hydrobromide (4a). The free base of com-
pound 1a (1.00 g, 3.04 mmol) was refluxed with 3-phenoxy-
propyl bromide (1.96 g, 9.10 mmol) and triethylamine (1.00
mL) in toluene (20 mL) for 4 h under an inert atmosphere.
The solvent was evaporated under reduced pressure, and the
residue was dissolved in CHCl3 (50 mL). After repeated
washing with water (50 mL × 2), the organic layer was
evaporated under reduced pressure to obtain an oily product,
which was purified by column chromatography as clear oil.
The free base was converted to the HBr salt (yield, 0.39 g,
1
HBr salt: mp >230 °C; H NMR (300 MHz, CDCl3) δ 1.78-
1.91 (m, 8H), 2.04-2.11 (m, 2H), 2.36-2.40 (t, 2H), 2.69-2.73
(t, 2H), 3.12-3.13 (m, 2H), 3.51-3.54 (m, 1H), 5.36 (s, 1H),
6.96-7.03 (m, 4H), 7.25-7.29 (m, 4H); 13C NMR (75 MHz,
CDCl3) δ 26.67, 36.70, 41.19, 55.87, 58.96, 70.10, 79.68, 115.46,
115.74, 128.69, 128.80, 139.10, 139.12, 160.75, 164.01. Anal.
(C22H26F2N2O‚2HBr) C, H, N.
1
27%): mp 190-193 °C; H NMR (300 MHz, CDCl3) δ 1.82-
2.17 (m, 10H), 2.61 (m, 2H), 3.30 (m, 2H), 3.58 (m, 1H), 4.02-
4.06 (t, 2H), 5.37 (s, 1H), 6.87-7.02 (m, 7H), 7.24-7.29 (m,
6H); 13C NMR (75 MHz, CDCl3) δ 26.42, 28.55, 35.98, 49.46,
59.09, 66.38, 69.77, 79.92, 114.87, 115.53, 115.81, 121.04,
128.68, 128.79, 129.82, 138.86, 159.29, 160.81, 164.07.
N-(4-Amino-n-butyl)-3r-[(bis(4′-fluorophenyl)methoxy]-
tropane Oxalate (5b). A suspension of compound 1a (1.62
g, 4.93 mmol), N-(4-bromo-n-butyl)phthalimide (1.46 g, 5.20
mmol), and sodium bicarbonate (2.10 g, 25 mmol) in 10 mL of
anhydrous acetonitrile was heated under an atmosphere of
argon at 120 °C in a sealed tube overnight. The volatiles were
removed in vacuo, and the residue was purified by flash
chromatography (eluant CHCl3/CH3OH, gradient from 20:1 to
10:1) to give 1.89 g (72%) of the phthalimide derivative. A
solution of the intermediate phthalimide (1.74 g, 3.30 mmol)
and hydrazine (0.20 mL, 6.40 mmol) in 50 mL of absolute
ethanol was refluxed for 2.5 h. After this time, the ice-cold
reaction mixture was filtered and solvent was removed under
reduced pressure. The residue was taken up in CHCl3 (10 mL)
and washed with 40% (w/v) KOH solution. The organic phase
was dried with Na2SO4 and concentrated in vacuo to give
compound 5b. The free base was converted to the oxalate salt
(0.75 g, 41%, from 1a): mp 202 °C; 1H NMR (400 MHz, CDCl3)
δ 1.41-1.46 (m, 4H), 1.77-1.91 (m, 6H), 2.05 (d, 2H), 2.32 (t,
2H), 2.68 (t, 2H), 3.16 (s, 2H), 3.52 (t, 1H), 5.34 (s, 1H), 6.95
(m, 4H), 7.24 (m, 4H); 13C NMR (101 MHz, CDCl3): δ 27.08,
27.22, 32.61, 36.59, 42.94, 52.74, 58.60, 70.27, 79.69, 115.25,
115.46, 128.37, 128.45, 138.71, 138.74, 160.56, 162.99; IR 3369
cm-1. Anal. (C24H30F2N2O‚C2H2O4‚1.5H2O) C, H, N.
N-(2-Benzamidoethyl)-3r-[(bis(4′-fluorophenyl)meth-
oxy]tropane Oxalate (6a). Compound 5a (0.54 g, 1.45 mmol)
was dissolved in CHCl3 (10 mL) and stirred with 5 mL (0.73
g, 8.70 mmol) of aqueous NaHCO3 solution. To this biphasic
solution, benzoyl chloride (0.17 mL, 1.45 mmol) in CHCl3 (2
mL) was added slowly at room temperature. The mixture was
stirred vigorously for 30 min. The aqueous layer was sepa-
rated, and the organic layer was washed with aqueous Na2CO3
solution (25 mL × 2), dried over anhydrous MgSO4, and
evaporated to give an oily product. Purification by column
chromatography gave the free base as a foam, which was
converted to the oxalate salt (yield, 0.20 g, 29%): mp 206-
N-(2-Ethoxyethan-2-ol)-3r-[(bis(4′-fluorophenyl)meth-
oxy]tropane Hydrobromide (4b). Compound 1a (1.20 g,
3.65 mmol) was heated to 100 °C for 10 h with 2-chloro-
ethoxyethanol (0.55 g, 4.40 mmol) and anhydrous K2CO3 (0.76
g, 5.50 mmol) in DMF (10 mL). The reaction was worked up
by extraction with ethyl acetate (50 mL × 2) from water (25
mL). The organic layer was washed with brine (25 mL × 2)
and dried over anhydrous MgSO4, and the solvent was
evaporated under reduced pressure. The crude product was
purified by column chromatography. The free base was con-
verted to the HBr salt (yield, 0.50 g, 32%): mp 115-119 °C;
1H NMR (300 MHz, CDCl3) δ 1.83-2.18 (m, 10H), 2.59 (m,
2H), 3.32 (m, 2H), 3.59-3.69 (m, 6H), 5.36 (s, 1H), 6.96-7.02
(m, 4H), 7.23-7.28 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 26.14,
38.99, 60.23, 73.05, 80.02, 115.60, 115.86, 128.38, 128.73,
128.83, 138.66, 160.84, 162.02; IR 3369 cm-1. Anal. (C24H29F2-
NO3‚1HBr‚1H2O) C, H, N.
N-[2(2-Chloroethoxy)ethyl]-3r-[(bis(4′-fluorophenyl)-
methoxy]tropane (4c). Compound 4c was obtained from 1a
(3.00 g, 9.10 mmol), 2-(bis-chloroethyl)ether (3.91 g, 27.30
mmol), and anhydrous K2CO3 (3.75 g, 27.10 mmol) in DMF
(20 mL) as described for compound 4b. The crude product was
purified by column chromatography, eluting with n-hexane/
ethyl acetate (4:1) to give a clear oil (yield, 1.00 g, 25%): 1H
NMR (300 MHz, CDCl3) δ 1.84-1.95 (m, 6H), 2.15-2.19 (m,
2H), 3.59-3.65 (m, 3H), 3.69-3.76 (m, 4H), 4.23-4.26 (m, 4H),
5.38 (s, 1H), 6.96-7.04 (m, 4H), 7.24-7.29 (m, 4H); 13C NMR
(75 MHz, CDCl3) δ 28.21, 31.31, 43.13, 53.22, 64.14, 70.11,
70.27, 71.49, 80.23, 115.58, 115.86, 128.65, 128.76, 138.68,
138.72, 153.77, 160.84, 164.10.
N-[2(2-Morpholin-4-ylethoxy)ethyl]-3r-[(bis(4′fluoro-
phenyl)methoxy]tropane Oxalate (4d). Compound 4c (1.00
g, 2.29 mmol) was stirred and heated with morpholine (0.20
g, 2.29 mmol) and anhydrous K2CO3 (0.63 g, 4.56 mmol) in

Structure-Activity Relationships of Tropanes
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3395
208 °C; 1H NMR (300 MHz, CDCl3) δ 1.91-2.01 (m, 4H), 2.27-
2.30 (m, 4H), 2.83-2.86 (t, 2H), 3.48 (m, 2H), 3.62-3.70 (m,
3H), 5.36 (s, 1H), 6.96-7.04 (m, 4H), 7.23-7.27 (m, 4H), 7.32-
7.49 (m, 3H), 7.85-7.88 (d, 2H), 8.10 (broad s, 1H); 13C NMR
(75 MHz, CDCl3) δ 26.10, 35.74, 37.36, 51.57, 60.19, 68.88,
80.18, 115.66, 115.94, 127.54, 128.13, 128.64, 128.75, 128.90,
129.83, 131.82, 134.51, 138.43, 138.47, 160.88, 164.14, 167.92.
Anal. (C29H30F2N2O2‚C2H2O4) C, H, N.
N-(2-Benzylaminoethyl)-3r-[(bis(4′-fluorophenyl)meth-
oxy]tropane Hydrobromide (6b). To a suspension of LiAlH4
(0.32 g, 8.40 mmol) in THF (30 mL) a solution of compound
6a (0.50 g, 1.05 mmol) in THF (10 mL) was added slowly at 0
°C under inert atmosphere. The reaction mixture was heated
to reflux for 4 h. The product was carefully worked up by
adding 4 mL of water to a well-cooled (0 °C) reaction mixture
followed by 2.00 mL of 15% (w/v) aqueous NaOH solution. The
white suspension was stirred with ethyl acetate (50 mL) and
dried (MgSO4). The organic layer was filtered and evaporated
to obtain an oily product, which was purified by column
chromatography, and the free base was converted to the HBr
salt (yield, 0.19 g, 39%): mp 243-247 °C (dec); 1H NMR (300
MHz, CDCl3) δ 1.79-2.12 (m, 9H), 2.51-2.55 (m, 2H), 2.69-
2.73 (t, 2H), 3.20 (m, 2H), 3.53-3.56 (m, 1H), 3.81 (s, 2H), 5.35
(s, 1H), 6.95-7.02 (m, 4H), 7.24-7.32 (m, 9H); 13C NMR (75
MHz, CDCl3) δ 26.44, 36.44, 47.51, 52.24, 54.25, 59.17, 69.76,
79.80, 115.50, 115.78, 127.15, 127.33, 128.51, 128.68, 128.79,
129.10, 138.96, 140.63, 160.78, 164.04; IR 3307 cm-1. Anal.
(C29H32F2N2O‚2HBr) C, H, N.
N-(2-Nicotinamidoethyl)-3r-[(bis(4′-fluorophenyl)meth-
oxy]tropane Oxalate (6c). This compound was synthesized
as compound 6a using 1.00 g (2.69 mmol) of compound 5a,
8.00 mL of aqueous NaHCO3 (1.35 g, 16.10 mmol) and 0.48 g
(2.69 mmol) of nicotinoyl chloride hydrochloride in 15 mL of
CHCl3 by stirring at room temperature for 2 h. The free base
was converted to the oxalate salt (yield, 0.28 g, 21%): mp 153-
155 °C; 1H NMR (300 MHz, CDCl3) δ 1.87-1.95 (m, 6H), 2.18-
2.21 (m, 2H), 2.63-2.67 (t, 2H), 3.26 (m, 2H), 3.49-3.56 (m,
2H), 3.59 (m, 1H), 5.37 (s, 1H), 6.96-7.04 (m, 4H), 7.25-7.29
(m, 4H), 7.36-7.40 (q, 1H), 7.52 (broad s, 1H), 8.15-8.18 (dt,
1H), 8.70-8.72 (dd, 1H), 8.98 (d, 1H); 13C NMR (75 MHz,
CDCl3) δ 26.50, 36.74, 51.28, 54.18, 59.06, 69.45, 79.95, 115.27,
115.54, 115.98, 116.27, 128.63, 128.96, 130.60, 135.57, 138.79,
148.16, 160.83, 164.09, 165.69. Anal. (C28H29F2N3O2‚C2H2O4‚
0.5H2O) C, H, N.
3-{3r-[(Bis(4′-fluorophenyl)methoxy]tropan-8-yl}pro-
pionic Acid (7a). Compound 1a (3.00 g, 9.10 mmol) was
stirred with anhydrous K2CO3 (1.88 g, 13.60 mmol) in DMF
(15 mL) under argon, and ethyl 3-bromopropionate (1.98 g,
10.94 mmol) was added. The reaction mixture was stirred and
heated to 80 °C for 2 h. The product was isolated by extraction
with ethyl acetate (50 mL × 2) after diluting the reaction
mixture with 30 mL of water. The organic layer was washed
with brine (50 mL × 2), dried over anhydrous MgSO4, and
evaporated under reduced pressure to give 2.90 g (6.75 mmol)
of oily product: 1H NMR (400 MHz, CDCl3) δ 1.22-1.26 (t,
3H), 1.77-1.81 (m, 2H), 1.87-1.90 (m, 4H), 2.05-2.08 (m, 2H),
2.44-2.48 (t, 2H), 2.62-2.66 (t, 2H), 3.14 (m, 2H), 3.49-3.52
(m, 1H), 4.08-4.13 (q, 2H), 5.33 (s, 1H), 6.94-6.99 (m, 4H),
7.22-7.25 (m, 4H); 13C NMR (101 MHz, CDCl3) δ 15.19, 27.10,
35.39, 36.72, 48.58, 59.01, 60.89, 70.14, 79.81, 115.30, 115.51,
128.39, 128.47, 138.65, 138.69, 160.63, 163.05, 172.54.
pound 7a (2.80 g, 6.98 mmol) in DMF (25 mL), 1.72 g (8.34
mmol) of dicyclohexylcarbodiimide and 1.13 g (8.36 mmol) of
1-hydroxybenzotriazole hydrate were added, and the mixture
was stirred under inert atmosphere. Aniline (0.70 mL, 7.68
mmol) was added to the reaction mixture slowly. The reaction
mixture was cooled to 0 °C, and 2.13 mL (15.3 mmol) of
triethylamine was added. The mixture was then stirred at
room temperature for 72 h. The mixture was diluted with
water (50 mL) and extracted with CHCl3 (50 mL × 2). The
organic layer was washed with brine (50 mL × 2) and dried
over anhydrous MgSO4, and the solvent was evaporated under
reduced pressure. The residue was purified by column chro-
matography, and the free base was converted to the oxalate
salt (yield, 1.10 g, 33%): mp 188-190 °C; 1H NMR (300 MHz,
CDCl3) δ 1.94-2.00 (m, 6H), 2.18-2.24 (q, 2H), 2.44-2.48 (t,
2H), 2.69-2.73 (t, 2H), 3.35 (m, 2H), 3.66-3.68 (m, 1H), 5.41
(s, 1H), 6.99-7.06 (m, 4H), 7.24-7.33 (m, 7H), 7.45-7.48 (m,
2H), 11.51 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 26.45, 34.37,
36.92, 48.67, 58.27, 69.61, 80.00, 115.47, 115.61, 115.75,
115.89, 119.80, 123.90, 128.67, 128.78, 129.37, 138.73, 138.77,
139.22, 160.86, 164.12, 171.51. Anal. (C29H30F2N2O2‚C2H2O4‚
0.25H2O) C, H, N.
3-{3r-[(Bis(4′-chlorophenyl)methoxy]tropan-8-yl}pro-
pionic Acid (7b). This compound was prepared from the free
base of compound 1e (5.1 g, 14.07 mmol) as described for
compound 7a (yield, 6.00 g, 92% crude): 1H NMR (400 MHz,
CDCl3) δ 1.22-1.25 (t, 3H), 1.76-1.89 (m, 6H), 2.04-2.06 (m,
2H), 2.43-2.46 (t, 2H), 2.61-2.64 (t, 2H), 3.13 (m, 2H), 3.50
(m, 1H), 4.08-4.13 (q, 2H), 5.31 (s, 1H), 7.20-7.26 (m, 8H);
13C NMR (101 MHz, CDCl3) δ 15.19, 27.11, 35.43, 36.76, 48.62,
58.98, 60.89, 70.39, 79.84, 128.19, 128.69, 133.22, 141.14,
172.55. The saponification of the ester, similar as for 7a, gave
the acid 7b (yield, 5.00 g, 88% crude): 1H NMR (400 MHz,
CDCl3) δ 1.80-1.89 (m, 4H), 2.01-2.11 (m, 4H), 2.34 (m, 2H),
2.71 (m, 2H), 3.36 (m, 2H), 3.49 (m, 1H), 5.28 (s, 1H), 7.15-
7.24 (m, 8H), 8.39 (s, 1H); 13C NMR (101 MHz, CDCl3) δ 26.36,
34.60, 35.59, 49.05, 59.20, 69.21, 80.09, 128.13, 128.84, 133.47,
140.54, 171.55.
N-[3-(N-Phenyl)propionamido]-3r-[(bis(4′-chlorophen-
yl)methoxy]tropane (8b). This compound was prepared from
compound 7b (5.00 g, 11.52 mmol) as described for compound
8a (yield, 2.00 g, 34%): mp 64-68 °C; oxalate salt, mp 190-
192 °C; 1H NMR (400 MHz, CDCl3) δ 1.94-1.99 (m, 6H), 2.16-
2.21 (m, 2H), 2.45-2.48 (t, 2H), 2.69-2.72 (t, 2H), 3.34 (m,
2H), 3.64-3.66 (m, 1H), 5.36 (s, 1H), 7.19-7.29 (m, 11H),
7.42-7.45 (m, 2H), 11.42 (s, 1H); 13C NMR (101 MHz, CDCl3)
δ 26.89, 34.75, 37.28, 48.99, 58.52, 69.89, 80.04, 119.55, 123.66,
128.15, 128.82, 129.07, 133.43, 138.81, 140.79, 170.84. Anal.
(C29H30Cl2N2O2‚1/3H2O) C, H, N.
N-(4-Dimethylaminobutyl)-3r-[(bis(4′-fluorophenyl)-
methoxy]tropane Oxalate (10). A solution of 4-(dimethyl-
amino)butyric acid hydrochloride (0.42 g, 2.50 mmol) in 3 mL
of anhydrous pyridine was treated with 1,1′-carbonyldiimid-
azole (0.41 g, 2.50 mmol), and the mixture was stirred for 1 h.
After this time, a solution of compound 1a (0.33 g, 1.00 mmol)
in 5 mL of absolute CHCl3 was added and the mixture was
stirred overnight. After hydrolysis by addition of water (10
mL), the organic phase was dried with Na2SO4 and all volatiles
were removed in vacuo: 1H NMR (400 MHz, CDCl3) δ 1.77-
1.97 (m, 8H), 2.14-2.39 (m, 11H), 3.64 (m, 1H), 4.10 (m, 1H),
4.16 (m, 1H), 4.64 (m, 1H), 5.38 (s, 1H), 6.94-6.99 (m, 4H),
7.23-7.27 (m, 4H); 13C NMR (101 MHz, CDCl3) δ 24.07, 28.02,
29.73, 32.13, 36.03, 37.00, 38.00, 46.09, 51.16, 54.39, 59.70,
70.33, 115.31, 115.33, 115.52, 115.54, 128.17, 128.25, 128.28,
128.36, 138.10, 160.51, 160.55, 162.94, 162.97, 168.07; IR 1632.
The above product was dissolved in ethanol (20 mL). A 4 N
NaOH solution (5 mL) was added, and the reaction mixture
was stirred for 2 h at room temperature. Evaporation of the
reaction mixture under reduced pressure gave the product 7a,
which was used for the next step without further purification
(yield, 2.80 g, 100% crude): 1H NMR (300 MHz, CDCl3) δ 2.03-
2.52 (m, 8H), 2.92-2.96 (m, 2H), 3.23 (m, 2H), 3.79-3.86 (m,
2H), 4.02 (m, 1H), 5.20 (s, 1H), 6.98-7.04 (m, 4H), 7.22-7.38
(m, 4H), 9.38 (broad s, 1H); 13C NMR (75 MHz, CDCl3) δ 25.10,
34.75, 57.28, 57.39, 78.40, 115.53, 115.88, 128.64, 128.75,
To a solution of the crude amide in absolute THF (5 mL)
was added LiAlH4 (0.052 g, 1.40 mmol) at 0 °C, and the
reaction mixture was strirred at room temperature for 1 h.
Hydrolysis by the addition of Glauber’s salt (Na2SO4‚10H2O)
and filtration, followed by recrystallization of its oxalate salt
from acetone/diethyl ether, yielded 10 (yield, 0.37 g, 63%): mp
138.02, 138.07, 160.94, 164.20, 171.44; IR 1737, 3351 cm-1
.
1
124-126 °C; H NMR (400 MHz, CDCl3) δ 1.49 (m, 2H), 1.56
N-[3-(N-Phenyl)propionamido]-3r-[(bis(4′-fluorophen-
yl)methoxy]tropane Oxalate (8a). To a solution of com-
(m, 2H), 1.83 (d, 2H), 1.94 (m, 2H), 2.16-2.25 (m, 4H), 2.24
(s, 6H), 2.32 (t, 2H), 2.53 (t, 2H), 3.39 (s, 2H), 3.59 (t, 1H),

3396 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Kulkarni et al.
5.33 (s, 1H), 6.93-6.98 (m, 4H), 7.20-7.26 (m, 4H); 13C NMR
(101 MHz, CDCl3) δ 25.94, 35.87, 36.54, 45.85, 52.25, 59.22,
59.79, 69.59, 79.95, 115.38, 115.56, 128.35, 128.44, 138.37,
138.40, 160.63, 163.06. Anal. (C26H34F2N2O‚1.5C2H2O4‚1.25H2O)
C, H, N.
for the unlabeled ligands. Data were analyzed with either
LIGAND or GraphPad Prism software (San Diego, CA).
Serotonin Transporter Binding Assay. Brains from
male Sprague-Dawley rats weighing 200-225 g (Taconic Labs,
Germantown, NY) were removed; the midbrain was dissected
and rapidly frozen. Membranes were prepared by homogeniz-
ing tissues in 20 volumes (w/v) of 50 mM Tris containing 120
mM NaCl and 5 mM KCl (pH 7.4 at 25 °C) using a Brinkman
Polytron and centrifuged at 50000g for 10 min at 4 °C. The
resulting pellet was resuspended in buffer, recentrifuged, and
resuspended in buffer to a concentration of 15 mg/mL. Ligand
binding experiments were conducted in assay tubes containing
0.5 mL buffer for 60 min at room temperature. Each tube
contained 1.4 nM [3H]citalopram (Amersham Biosciences,
Piscataway, NJ) and 1.5 mg of midbrain tissue (original wet
weight). Nonspecific binding was determined using 10 µM
fluoxetine. Incubations were terminated by rapid filtration
through Whatman GF/B filters, presoaked in 0.3% polyethylen-
imine, using a Brandel R48 filtering manifold (Brandel Instru-
ments Gaithersburg, MD). The filters were washed twice with
3 mL of cold buffer and transferred to scintillation vials.
Beckman Ready Value (3.0 mL) was added, and the vials were
counted the next day using a Beckman 6000 liquid scintillation
counter (Beckman Coulter Instruments, Fullerton, CA). Each
compound was tested with concentrations ranging from 0.01
nM to 100 µM for competition against binding of [3H]citalo-
pram, in at least three independent experiments, each per-
formed in triplicate. Data were analyzed with GraphPad Prism
software (San Diego, CA).
Norepinephrine Transporter Binding Assay. Brains
from male Sprague-Dawley rats weighing 200-225 g (Taconic
Labs, Germantown, NY) were removed; the frontal cortex was
dissected and rapidly frozen. Membranes were prepared by
homogenizing tissues in 20 volumes (w/v) of 50 mM Tris
containing 120 mM NaCl and 5 mM KCl (pH 7.4 at 25 °C)
using a Brinkman Polytron and centrifuged at 50000g for 10
min at 4 °C. The resulting pellet was resuspended in buffer,
recentrifuged, and resuspended in buffer to a concentration
of 80 mg/mL. Ligand binding experiments were conducted in
assay tubes containing 0.5 mL of buffer for 60 min at 0-4 °C.
Each tube contained 0.5 nM [3H]nisoxetine (PerkinElmer Life
Sciences, Boston, MA) and 8 mg of frontal cortex tissue
(original wet weight). Nonspecific binding was determined
using 1 µM desipramine. Incubations were terminated by rapid
filtration through Whatman GF/B filters, presoaked in 0.05%
polyethylenimine, using a Brandel R48 filtering manifold
(Brandel Instruments Gaithersburg, MD). The filters were
washed twice with 3 mL of cold buffer and transferred to
scintillation vials. Beckman Ready Value (3.0 mL) was added,
and the vials were counted using a Beckman 6000 liquid
scintillation counter (Beckman Coulter Instruments, Fullerton,
CA). Each compound was tested with concentrations ranging
from 0.01 nM to 100 µM for competition against binding of
[3H]nisoxetine, in at least three independent experiments, each
performed in triplicate. Data were analyzed by using Graph-
Pad Prism software (San Diego, CA).
Molecular Modeling. Molecular modeling and CoMFA
studies were performed using the SYBYL 6.7 (Tripos Inc.)
package running on a Silicon Graphics Octane workstation.
Standard Tripos force field and MOPAC charges (AM1 Hamil-
tonian) were used for all energy calculations. The X-ray crystal
structures of compounds 1c, 17, and 42 were used to build
the rest of the compounds. The geometries were optimized by
conjugate gradient minimization until a convergence criteria
of 0.001 kcal/(mol Å) was achieved. Our data set contained a
total of 76 benztropine analogues synthesized previously in
the laboratory.16-21 The compounds with an asymmetric center
were modeled by following the weighting protocol described
earlier.15,20 These compounds had a range of substitutions at
the 3-diarylmethoxy group as well as on the N-8. The predic-
tive power of the molecular models was assessed by setting
aside a set of compounds as a test set. The mean/standard
deviation of the activity for the training and test sets were
1.12/0.57 and 1.04/0.35, respectively (see Supporting Informa-
tion for structures and biological activity). These compounds
were aligned using the non-hydrogen atoms of the tropane ring
(alignment I, AFI) and the centroids of the diphenyl substitu-
ent with the N-8 atom (alignment II, AFII). These alignments
were also used as inputs for rigid-body field fit analysis
(alignment FFI and FFII). The CoMFA interaction energies
were calculated using the default settings. The statistical
analyses were performed using the partial least squares (PLS)
method, leave one out (LOO) procedure. The optimum number
of components was chosen based on the highest r2cv value and
the lowest standard error of prediction. To estimate the
statistical confidence limits on the analysis, PLS analysis using
100 bootstrap groups with an optimum number of components
was performed. The CoMFA contour maps were derived from
a product of the standard deviation associated with the CoMFA
column and coefficient (SD X coeff) at each lattice point.
Biological Assays. Dopamine Transporter Binding
Assay. Male Sprague-Dawley rats (200-250 g, Taconic,
Germantown, NY) were decapitated, and their brains removed
to an ice-cooled dish for dissection of the caudate putamen.
The tissue was homogenized in 30 volumes ice-cold modified
Krebs-HEPES buffer (15 mM HEPES, 127 mM NaCl, 5 mM
KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 1.3 mM NaH2PO4, 10 mM
D-glucose, pH adjusted to 7.4) using a Brinkman Polytron and
centrifuged at 20000g for 10 min at 4 °C. The resulting pellet
was then washed two more times by resuspension in ice-cold
buffer and centrifuged at 20000g for 10 min at 4 °C. Fresh
homogenates were used in all experiments. Binding assays
were conducted in modified Krebs-HEPES buffer on ice. The
total volume in each tube was 0.5 mL, and the final concentra-
tion of membrane after all additions was 1.5 mg (original wet
weight). Triplicate samples of membrane suspension were
preincubated for 5 min in the presence or absence of the com-
pound being tested. [3H]WIN 35,428 (2-â-carbomethoxy-3-â-
(4-fluorophenyl)tropane 1,5-naphthalene disulfonate (Perkin-
Elmer Life Sciences, Boston, MA) at a final concentration 1.5
nM) was added, and the incubation was continued for 1 h on
ice. The incubation was terminated by the addition of 3 mL of
ice-cold buffer and rapid filtration through Whatman GF/B
glass fiber filter paper (presoaked in 0.1% BSA in water) using
a Brandel cell harvester (Brandel Instruments, Gaithersburg,
MD). The filters were washed with three additional 3 mL
washes and transferred to scintillation vials. Beckman Ready
Value scintillation cocktail (3 mL) was added to the vials,
which were counted using a Beckman 6000 liquid scintillation
counter (Beckman Coulter Instruments, Fullerton, CA). Non-
specific binding was defined as binding in the presence of 100
µM cocaine. Each compound was tested with concentrations
ranging from 0.01 nM to 100 µM for competition against
binding of [3H]WIN 35,428, in three independent experiments.
The Ki values reported are the dissociation constants derived
Muscarinic M1 Receptor Binding Assay. Whole frozen
rat brains excluding cerebellum (Taconic, Germantown, NY)
were thawed in ice-cold buffer (10 mM Tris-HCl, 320 mM
sucrose, pH 7.4) and homogenized with a Brinkman Polytron
in a volume of 10 mL/g of tissue. The homogenate was
centrifuged at 1000g for 10 min at 4 °C. The resulting
supernatant was then centrifuged at 10000g for 20 min at 4
°C. The resulting pellet was resuspended in a volume of 1 g/5
mL in 10 mM Tris buffer (pH 7.4). Assays were conducted in
binding buffer (10 mM Tris-HCl, 5 mM MgCl2). The total
volume in each tube was 0.5 mL, and the final concentration
of membrane after all additions was approximately 20 mg
(original wet weight). [3H]pirenzepine (PerkinElmer Life Sci-
ences, Boston, MA) at a final concentration 3 nM was added,
and the incubation was continued for 1 h at 37 °C. The
incubation was terminated by the addition of 3 mL of ice-cold
buffer (10 mM Tris-HCl, pH 7.4) and rapid filtration through
Whatman GF/B glass fiber filter paper (presoaked in 0.5%

Structure-Activity Relationships of Tropanes
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polyethylenimine in water) using a Brandel cell harvester
(Brandel Instruments, Gaithersburg, MD). The filters were
washed with two additional 5 mL washes and transferred to
scintillation vials. Beckman Ready Value scintillation cocktail
(3 mL) was added to the vials, which were counted the next
day using a Beckman liquid scintillation counter (Beckman
Coulter, Fullerton, CA). Nonspecific binding was defined as
binding in the presence of 10 µM QNB (quinuclidinyl benzi-
late). Each compound was tested with concentrations ranging
from 0.01 nM to 100 µM for competition against binding of
[3H]pirenzepine, in at least three independent experiments,
each performed in triplicate. Displacement data were analyzed
with GraphPad Prism software (San Diego, CA).
Dopamine Uptake Assay. The tissue was homogenized
in ice-cold buffer (5 mM HEPES, 0.32 M sucrose) using 10
strokes with a Teflon glass homogenizer followed by centrifu-
gation at 1000g for 10 min at 4 °C. The supernatant was saved
and recentrifuged at 10000g for 20 min at 4 °C. The super-
natant was then discarded, and the pellet was gently resus-
pended in ice-cold incubation buffer (127 mM NaCl, 5 mM KCl,
1.3 mM NaH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 1.498 mM
HEPES acid, 10 mM D-glucose, 1.14 mM L-ascorbic acid, pH
7.4) and placed on ice for 15 min. The synaptosomal tissue
preparation was incubated in buffer in glass test tubes at 37
°C to which 10 µM pargyline and either the drug being tested
or no drug was added, as appropriate. After a 10 min
preincubation in the presence of drug, [3H]dopamine (final
concentration of 0.5 nM) (Amersham Biosciences, Piscataway,
NJ) was added to each tube and the incubation was carried
on for 5 min. The reaction was terminated by the addition of
3 mL of ice-cold buffer to each tube and rapid filtration through
Whatman GF/B glass fiber filter paper (presoaked in 0.1%
polyethylenimine in water) using a Brandel cell harvester
(Brandel Instruments, Gaithersburg, MD). After filtration, the
filters were washed with two additional 5 mL washes and
transferred into scintillation vials. Beckman Ready Value
(Beckman-Coulter Instruments, Fullerton, CA) was added, and
the vials were counted the next day using a Beckman 6000
liquid scintillation counter (Beckman Coulter Instruments,
Fullerton, CA). The reported values represent specific uptake
from which nonspecific uptake was subtracted (defined as
uptake in the presence of 100 µM (-)-cocaine HCl). Data were
analyzed using the nonlinear regression analysis of GraphPad
Prism software (San Diego CA).
Acknowledgment. S.S.K. and P.G. were supported
by National Institutes of Health Visiting Fellowships.
The authors thank J. Cao for technical assistance. This
work was supported by the National Institutes on Drug
AbusesIntramural Research Program.
Supporting Information Available: Structures and
activities of compounds used for molecular modeling and
elemental analysis results. This material is available free of
charge via the Internet at http://pubs.acs.org.
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