CoMFA study of novel phenyl ring-substituted 3α- (diphenylmethoxy)tropane analogues at the dopamine transporter

Article abstract of DOI:10.1021/jm980701v

A series of phenyl ring-substituted analogues of 3α- (diphenylmethoxy)tropane (benztropine) has been prepared as novel probes for the dopamine transporter. Cross-validated comparative molecular field analysis (CoMFA) models of the binding domain on the do

Full text of DOI:10.1021/jm980701v

3502
J . Med. Chem. 1999, 42, 3502-3509
CoMF A Stu d y of Novel P h en yl Rin g-Su bstitu ted 3r-(Dip h en ylm eth oxy)tr op a n e
An a logu es a t th e Dop a m in e Tr a n sp or ter
Amy Hauck Newman,* Sari Izenwasser,† Michael J . Robarge, and Richard H. Kline‡
Psychobiology Section, National Institute on Drug Abuse - Intramural Research Program, National Institutes of Health,
5500 Nathan Shock Drive, Baltimore, Maryland 21224
Received December 14, 1998
A series of phenyl ring-substituted analogues of 3R-(diphenylmethoxy)tropane (benztropine)
has been prepared as novel probes for the dopamine transporter. Cross-validated comparative
molecular field analysis (CoMFA) models of the binding domain on the dopamine transporter
were constructed using 37 geometry-optimized structures of these compounds and their
corresponding binding affinities (Ki values) for the displacement of [3H]WIN 35,428 or potency
of [3H]dopamine uptake inhibition (IC50 values) in rat caudate putamen tissue. The most
predictive model (q2 ) 0.78) correlated the steric component of CoMFA to the dependent variable
of [3H]WIN 35,428 binding affinities. A novel series of seven phenyl ring-substituted analogues
of 3R-(diphenylmethoxy)tropane was prepared, and our best molecular model was used to
accurately predict their binding affinities. This study is the first to provide a CoMFA model
for this class of dopamine uptake inhibitors. This model represents an advancement in the
design of novel dopamine transporter ligands, based on 3R-(diphenylmethoxy)tropane, and
further substantiates structure-activity relationships that have previously been proposed for
this class of compounds. This CoMFA model can now be used to predict the binding affinities
of novel 3R-(diphenylmethoxy)tropane analogues at the dopamine transporter and will be useful
in the design of molecular probes within this class of dopamine uptake inhibitors.
In tr od u ction
laboratories as a complement. Specifically, the use of
comparative molecular field analysis (CoMFA) has been
described for the cocaine series of compounds by
Carroll9-11 and by MacKerell12,13 and their colleagues.
These studies primarily focused on the 3â-position of
the tropane ring of cocaine and identified structural
features with optimal electrostatic and steric factors as
determinants of high-affinity binding at the dopamine
transporter. These resulting cross-validated models
have provided a high predictive capacity to assess
relative steric and electrostatic contributions of substi-
tution at the 3-position on the tropane ring. These
models, in turn, allowed the inference of the binding site
environment in terms of these factors and subsequently
provided guidance in the design of optimally active
3-substituted cocaine analogues. These reports demon-
strate the value of 3D-QSAR techniques such as CoMFA
in the design of third-generation high-affinity ligands
at the dopamine transporter. To date, however, the
molecular models of the dopamine transporter have
been based on cocaine and its analogues9-15 providing
limited descriptor sampling for ligands that bind to this
protein. Nevertheless, other structural classes of dopam-
ine uptake inhibitors may access binding domains on
the dopamine transporter that are distinctive from
cocaine.16,17 Thus, molecular models derived from struc-
tural classes of dopamine transporter ligands, other
than cocaine and its analogues, will undoubtedly provide
further insight into structure-activity relationships and
binding domain structure and function and will also
provide new leads toward the development of highly
selective and potent ligands for this molecular target.
We have been investigating a series of dopamine
uptake inhibitors based on the tropane-based drug 3R-
Over the past decade, a significant research effort has
been directed toward the elucidation of neurochemical
mechanisms underlying the reinforcing and addictive
properties of cocaine. Cocaine is known to bind with
moderate and equipotent affinity to all three of the
monoamine neurotransmitter transporters and subse-
quently inhibits the reuptake of dopamine, serotonin,
and norepinephrine into their respective presynaptic
neurons. However, it is the inhibition of dopamine
uptake that appears to play a pivotal role in cocaine’s
reinforcing actions.1,2 Although recent studies in dopam-
ine transporter knockout mice have provided evidence
that this may not be the exclusive neurochemical target
for these actions,3-5 there is significant support for the
further elucidation of its role in the abuse liability of
cocaine and other psychostimulants. Moreover, the
development of a mechanistically based cocaine-abuse
pharmacotherapeutic continues to focus on drugs that
selectively target the dopamine transporter.6-8
The development of novel dopamine uptake inhibitors,
as molecular probes for the dopamine transporter as
well as potential leads for the treatment of cocaine
abuse, has been the focus of numerous laboratories, i.e.,
for review see refs 6 and 7. Although classical medicinal
chemistry has primarily been implemented to design
these compounds, both quantitative structure-activity
relationship (QSAR) and other computer-aided drug
design approaches have been developed by several
† Current address: Department of Neurology, University of Miami
School of Medicine, 1501 NW 9th Ave, Miami, FL 33136.
‡ Current address: Cocaine Treatment Discovery Program, Chem-
istry & Pharmaceutics Branch, MDD, NIDA/NIH, 5600 Fishers Ln,
Rockville, MD 20857.
10.1021/jm980701v This article not subject to U.S. Copyright. Published 1999 by the American Chemical Society
Published on Web 08/24/1999

CoMFA of Phenyl-Substituted Tropanes at the DAT
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3503
(diphenylmethoxy)tropane (benztropine). In the course
of these studies, we have undertaken chemical modifi-
cation of both the 3-position18-20 and the tropane
nitrogen.21 Structure-activity relationships derived
from these series of compounds demonstrated a signifi-
cant divergence from the cocaine series of compounds,
i.e., for review see ref 22. In addition, several studies
describing the 2-carbomethoxy-substituted-3R-(diphe-
nylmethoxy)tropanes also suggest a divergence in struc-
ture-activity relationships between these two classes
of tropane-based dopamine uptake inhibitors.23,24 These
differences, coupled with a distinctive behavioral profile
in animal models of cocaine abuse, have suggested that
this class of dopamine uptake inhibitors may be access-
ing a different binding domain at the dopamine trans-
porter as compared to cocaine.25,26
To identify the location and structure of the binding
domain on the dopamine transporter to which this class
of dopamine uptake inhibitors binds, several approaches
may be taken. First, the development of structure-
activity relationships provides insight into the physical
and electronic environment in which the drug molecules
may interact. Further, molecular tools such as irrevers-
ible ligands and radiolabeled ligands are extremely
useful in identifying specific peptide residues that are
covalently attached to the drug molecule and allow
subsequent characterization of those sites. Since the
development of these types of compounds requires the
incorporation of electrophiles (i.e. NCS, N3) or radionu-
clides (i.e. 125I) into the pharmacophore, steric and
electronic requirements for optimal binding must be
determined so as to precisely position these chemical
moieties to achieve both high-affinity binding and, in
the case of irreversible ligands, covalent attachment. To
optimize the probability of designing these molecular
probes, we have initiated molecular modeling studies
using the active analogue approach.27-29 In the present
study, we focused our attention on the 3-position diphe-
nyl ether of the benztropine series of molecules to (1)
provide direction in design of optimal structures for
high-affinity binding at the dopamine transporter and
(2) compare these models to those described for this
position in the cocaine series of compounds.
We have previously described a large series of aryl
ring-substituted 3R-(diphenylmethoxy)tropane ana-
logues and described structure-activity relationships
at this position.18-20 Furthermore, we have discovered
that replacing the diphenyl ether moiety with a number
of aryl and arylalkyl substituents uniformly resulted in
significant reduction in binding affinity at the dopamine
transporter.22 Therefore, the focus of the present study
was to develop a molecular model in the 3R-diphenyl
ether series that could be used to accurately predict the
binding affinities of novel compounds that may prove
useful for future studies of the dopamine transporter.
Our current study utilized the molecular fields from the
geometry-optimized structures of a series of 37 3R-
(diphenylmethoxy)tropane analogues for correlation to
either inhibition of [3H]WIN 35,428 binding to dopamine
transporters in rat caudate putamen or inhibition of [3H]-
dopamine reuptake in the same tissue. The results of
these studies were envisioned to provide new leads
toward the development of benztropine-based dopamine
uptake inhibitors. In addition, a comparison of the
analyses of the binding pocket could be made that has
been described by others to be accessed by the cocaine
series of compounds.9-13 To this end, we describe herein
the derivation of a valid CoMFA model based on a series
of phenyl-ring substituted 3R-(diphenylmethoxy)tropane
analogues. Subsequently, a series of seven novel com-
pounds were synthesized and their binding affinities at
the dopamine transporter were predicted. Experimental
data were obtained for both binding affinity and dopam-
ine uptake potency for these compounds and compared
to the Ki values derived from the best predictive CoMFA
model.
Ta ble 1. CoMFA Training Set Compounds
[3H]WIN 35,428 binding
[3H]DAUI
[3H]WIN 35,428 binding
[3H]DAUI
IC50, nMa
compd
R′
4-F
4-Cl
4-Br
4-Me
4-OMe
4-F
4-Cl
4-Br
4-Me
4-OMe
4-CN
4-NO2
4-Et
R′′
4-F
4-Cl
4-Br
4-Me
4-OMe
H
H
H
H
H
Ki, nM (% error)a
IC50, nMa
compd
R′
R′′
Ki, nM (% error)a
8
9
11.8 (11)
20.0 (14)
91.6 (13)
420 (7)
2000 (7)
32.2 (10)
30.0 (12)
37.9 (7)
187 (5)
78.4 (8)
196 (9)
197 (8)
520 (8)
1920 (7)
635 (10)
71.0
75.0
34.0
2540
2880
48.0
115
29.0
512
468
222
219
984
4460
2160
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
4-OH
H
3-Cl
3-CF3
3,4-diCl
3,4-diCl
2-NH2
2-F
2-Me
4-Br
2-Cl
3-F
3-F
H
H
H
H
4-F
H
H
H
H
4-F
H
3-F
H
H
4-F
297 (13)
118 (9)
22.0 (7)
187 (5)
18.9 (14)
21.1 (19)
1840 (8)
50.0 (12)
309 (6)
15.2 (19)
228 (9)
47.4 (11)
68.5 (12)
27.9 (11)
23.3 (8)
677
403
228
457
24.0
47.0
373
140
1200
27.2
977
407
250
181
139
10
11
12
13
14
15
16
17
18
19
20
21
22
H
H
H
H
4-t-Bu
4-CF3
3,4-diF
3-F
H
a
Each value represents data from at least three independent experiments, each performed in triplicate (data from refs 18-20 and 26).

3504 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Newman et al.
F igu r e 1. Stereoview of CoMFA training set. Compounds were aligned based on non-hydrogen atoms in the tropane ring.
Ta ble 2. CoMFA Analysis Results
cross-validated
analysis
no.
dependent data
type (descriptors)
CoMFA fields
(cutoff values)
probe atom
(grid size)
predictive q2 values
(no. components)
PRESS
values
1
2
3
4
5
6
7
8
[3H]WIN binding
electronic/steric
(30/30)
electronic/steric
(30/30)
steric
(30)
steric
(30)
electronic/steric
(30/30)
electronic/steric
(30/30)
C (sp3)
(2 Å)
0.37 (3)
0.65 (4)
0.67 (3)
0.78 (4)
0.25 (2)
0.37 (4)
0.59 (5)
0.61 (6)
0.57
0.43
0.40
0.34
0.56
0.52
0.43
0.42
[3H]WIN binding
(cLogP)
C (sp3)
(2 Å)
[3H]WIN binding
C (sp3)
(1 Å)
[3H]WIN binding
(cLogP)
C (sp3)
(1 Å)
[3H]DAUI
C (sp3)
(2 Å)
[3H]DAUI
(cLogP)
C (sp3)
(2 Å)
[3H]DAUI
steric
(30)
steric
(30)
C (sp3)
(1 Å)
[3H]DAUI
(cLogP)
C (sp3)
(1 Å)
Molecu la r Mod elin g - CoMF A
alignment resulted. In addition, two regioisomers ex-
isted for each enantiomer in the 2- and 3-substituted
analogues. We tested all four possible sets of alignments
and determined that using equally weighted enanti-
omers with the substituent oriented in the space
between the aryl rings gave the best model. Logarithms
of 1/inhibition of [3H]WIN 35,428 binding (Ki) and 1/[3H]-
dopamine uptake inhibition (IC50) were used as the
dependent variables to more evenly distribute the data.
Partial least-squares (PLS) analyses were performed
using steric (Lennard-J ones, cutoff range 5-30 kcal)
and electrostatic (Coulombic, 30 kcal cutoff) fields either
in combination or individually. Additionally, molar
refractivity (CMR) and partition coefficients (cLogP)
were calculated using CMR and CLOGP modules within
SYBYL, respectively. Since the use of several descriptors
with even moderately sized training sets may lead to
artificially significant correlation coefficients, correla-
tions were determined with standard CoMFA weighting
and only one additional descriptor.
Molecular modeling studies were performed using the
SYBYL software package (Tripos version 6.3, R4000)
installed on a Silicon Graphics IRIS Indigo XZ worksta-
tion running IRIX 5.3. Use of the algorithm CoMFA
allowed the development of a 3D-QSAR model that
describes the structure-activity relationships for a
series of analogues.27 Once derived, a valid CoMFA
model was utilized to design more active compounds by
predicting the pharmacological activities of proposed
analogues. Additionally, the results from these models
can be used to predict the characteristics of ligand-
receptor interactions.
The structures for members of our data set were
derived from the known X-ray crystal structure of 4′-
chloro-3R-(diphenylmethoxy)tropane and molecular frag-
ments and standard bond lengths and angles from the
SYBYL structural library.19 Optimized geometries and
partial charges of the original X-ray structure as well
as all derived structures were obtained using the AM130
model Hamiltonian as implemented in the MOPAC
program (version 6.0) using the PRECISE convergence
criteria. The compounds that were used as a training
set in the CoMFA analyses are listed in Table 1. For
the CoMFA studies all compounds were aligned by
fitting together the non-hydrogen atoms of the tropane
ring (Figure 1). As some of the compounds in the
training set have a chiral benzylic center resulting from
asymmetric aryl substitution, two obvious choices for
The results of the CoMFA analyses can be seen in
Table 2. The most predictive model, as indicated by the
highest cross-validated (leave-one-out method used)
correlation (q2 ) 0.78), column filtering < 0.5 kcal/mol,
resulted from the PLS analysis of the compounds in the
training set with respect to inhibition of [3H]WIN 35,-
428 binding. In the final model, only the standard (30
kcal/mol cutoff) steric fields derived from the use of an
sp3-hybridized probe atom (the use of a H probe was

CoMFA of Phenyl-Substituted Tropanes at the DAT
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3505
Sch em e 1a
a
(a) NaBH4; (b) SOCl2; (c) 160 °C.
Ta ble 3. Physical Properties of Compounds 4a -4g
also analyzed and found to give no improvement to the
model) in a matrix of grid points spaced at 1 Å and
cLogP descriptors were used as the independent vari-
ables. The seven test compounds were derived from the
same AM1 geometry-optimized X-ray crystal structure
as was used for the training set compounds. The
optimized geometries and partial charges for these
compounds were also obtained using an AM1 Hamilto-
nian, and the non-hydrogen atoms of their respective
tropane rings were fit with the same atoms of the
compounds in the training set prior to predicting bind-
ing affinities.
recryst
solvent
MS
%
yield
compd
mp, °C (M+)
formula
4a
4b
4c
4d
4e
4f
EtOAc/acetone 152-154 405
C
C
C
C
C
C
C
21H24NOBr2F
21H24NOClF
21H25NOBrCl
22H27NOFCl
22H28NOCl
65
40
45
46
61
35
25
EtOAc
EtOAc
EtOAc
acetone
EtOAc
EtOAc
155-157 359
175-177 387
136-138 339
134-136 321
165-167 393
129-131 352
22H24NOClF4
21H25N2O3Cl
4g
binding with affinities of 23.4-187 nM (Ki). Inhibition
of [3H]dopamine uptake was determined for each of the
compounds (IC50 ) 104-795 nM). Since the most
predictive model was based on the binding affinities,
these data were then compared to the predicted values.
Ch em istr y
The synthesis of compounds 4a -4g is depicted in
Scheme 1, and they were prepared as previously de-
scribed for the first series of ortho- and meta-substituted
3R-(diphenylmethoxy)tropanes.20 In brief, commercially
available substituted benzophenones 1a -1g were re-
duced to their corresponding benzhydrols 2a -2g using
NaBH4 in absolute ethanol or methanol. Conversion to
the benzhydryl chlorides 3a -3g was achieved in reflux-
ing thionyl chloride. The excess thionyl chloride was
removed in vacuo, and the benzhydryl chloride was
added neat or in a minimal volume of anhydrous ether
to an equimolar portion of tropane, at 160 °C. The
reaction mixture was allowed to stir for 20-60 min, at
this temperature. The hydrochloride salts were isolated
via an ion-pairing extraction technique as previously
described19 and recrystallized to give 25-65% yield of
the desired products 4a -4g. Selected physical proper-
ties of these compounds are depicted in Table 3.
Resu lts a n d Discu ssion
Molecular modeling studies, using CoMFA, were
undertaken to aid in the design of novel dopamine
transporter ligands as well as to provide insight into
the steric and electronic nature of the binding pocket
in which this series of 3R-(diphenylmethoxy)tropane
analogues interacts. The CoMFA training set in which
the compounds were energy-minimized and superim-
posed based on the non-hydrogen atoms on the tropane
ring can be seen in Figure 1. Several predictive models
were derived using a series of 37 phenyl ring-substituted
3R-(diphenylmethoxy)tropane analogues and either [3H]-
WIN 35,428 binding (Ki values) or [3H]dopamine uptake
inhibition (IC50 values) constants as the dependent
variables (Table 2). CoMFA fields were generated that
included either electronic and steric components or
steric components alone. A 1 or 2 Å sp3 carbon grid size
was evaluated for each model. This optimal model
included the additional descriptor cLogP, a calculated
measure of the partition coefficient. The inclusion of
additional descriptors in QSAR model development is
a common tactic, since expanded sampling of descriptor
space is more likely to yield a predictive model. With
P h a r m a cology
The compounds were evaluated for displacement of
[3H]WIN 35,428 binding to the dopamine transporter
and for inhibition of [3H]dopamine uptake in rat caudate
putamen tissue, as described previously.19,20 All of the
compounds monophasically displaced [3H]WIN 35,428

3506 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Newman et al.
Ta ble 4. Predicted versus Actual Results of Radiolabeled
Binding and Uptake Experiments
confirmed the structure-activity relationship derived
previously18-20 which showed that small halogens (F,
Cl) in the meta or para positions of one or both phenyl
rings are well-tolerated at the dopamine transporter.
Compounds that have sterically bulkier groups or
strongly electronic groups in these positions bind with
considerably lower affinity. Furthermore, compounds
with substituents in the ortho positions likewise have
lower binding affinities at the dopamine transporter,
possibly due to unfavorable steric or electronic interac-
tions with the protein. In addition, the effect these
substituents may have on the torsional angle in which
the phenyl rings reside may also provide a less than
optimal interaction at the binding site. On the basis of
these results, we have chosen to retain the optimal
substituents of small halogens in the para and/or meta
positions in our subsequent structure-activity studies
in which the N-CH3 group has been replaced with
various aryl and alkylaryl substituents.21 Further, it
was deemed unwise to synthesize potential irreversible
ligands with either the radiolabel or the electrophile at
the 3-position diphenyl ether since both steric bulk and
electronic properties of these substituents were pre-
dicted to contribute negatively to binding affinity at the
dopamine transporter. Subsequently, we have developed
a photoaffinity label that binds irreversibly and radio-
labels dopamine transporters, with these substituents
attached to the N-butylphenyl moiety.31 Interestingly,
peptide mapping studies using this photoaffinity label
along with proteolytic and immunological precipitation
techniques have provided further evidence to support
the concept that distinctive binding domains are ac-
cessed by the 3R-(diphenylmethoxy)tropanes versus the
tropane-based cocaine analogues.32
inhibition of [3H]WIN
35,428 binding Ki, nM
[3H]DA uptake
exptla
inhibitiona
compd substitution
(% error)
predb
IC50 ( SEM, nM
4a
4b
4c
4d
4e
4f
3′-Br, 4′′-F
3′-Cl, 4′′-F
3′-Br
3′-CH3, 4′′-F
3′-CH3
38.2 (14)
23.4 (18)
27.0 (12)
30.9 (9)
78.0 (7)
88.1 (10)
187 (10)
31.4
25.3
47.9
39.8
61.0
63.7
64.7
179 ( 51
123 ( 23
104 ( 27
108 ( 23
226 ( 70
182 ( 36
795 ( 199
3′-CF3, 4′′-F
3′-NO2
4g
a
Each experimental value represents data from at least three
independent experiments, each performed in triplicate. b The most
predictive model (analysis 4 in Table 3), without cross-validation,
was used to predict these Ki values.
the additional descriptor, the corresponding QSAR
equation becomes:
activityn ) constantn + An(stericxyz) +
Bn(electrostaticxyz) + ... + A′n(stericxyz) +
B′n(stericxyz) + ... + C(cLogP)
in which the activity of compound n is proportional to
the summation of the three-dimensional steric and
electrostatic terms and the cLogP value for compound
n. In our own case, the inclusion of cLogP as an
independent variable led to an improvement in our
model from 0.67 to 0.78 (cf. Table 2). While steric and
electrostatic considerations are likely strong determi-
nants of the calculated partition coefficient, the im-
provement of our model by inclusion of this additional
term indicates that it encodes structural information not
exhaustively sampled by the steric and electrostatic
terms calculated by CoMFA. The most predictive model
in this study provided a q2 value of 0.78 (analysis 4 in
Table 2) and demonstrated that the steric component
of this series is a significant determinant of binding
affinity at the dopamine transporter. The best CoMFA
model was then used to predict the binding affinities of
a new series of phenyl ring-substituted 3R-(diphenyl-
methoxy)tropane analogues. Compounds 4a -4g were
prepared and evaluated for their binding affinity at the
dopamine transporter, and these experimental values
were then compared to the values predicted by the best
model. As can be seen in Table 4, the final PLS analysis
(non-cross-validated) closely predicted the Ki values for
the test set compounds 4a -4f.
The one exception in this series was the 3′-NO2-
substituted analogue whose predictive value was lower
than its actual value (Ki ) 64.7 nM vs 187 nM). Based
on our observations19,22 and these data, the disparity of
the predicted versus the actual activity for this analogue
may be due to the fact that the NO2 substituent has
significant electron-withdrawing character that may
contribute negatively to its binding at the dopamine
transporter. Since the most predictive model chosen
considered only the steric component, electronic contri-
bution of the NO2 group is not considered, and this may
account for the less accurate prediction of the Ki value
for compound 4g. Nevertheless, this study demonstrates
that the CoMFA model described herein will be useful
in predicting the binding affinities of phenyl ring-
substituted 3R-(diphenylmethoxy)tropane analogues and
Currently we are employing the same methods de-
scribed herein to derive molecular models for optimal
N-substituents33 and modeling the binding pocket that
accommodates these substituents. Our earlier studies
demonstrated that binding selectivity for the dopamine
transporter versus the m1 muscarinic receptor could not
be achieved by modification in the 3-position of the
benztropine molecule; substituents that improved bind-
ing affinity at the dopamine transporter generally
retained high binding affinity at muscarinic receptors.
Conversely, substituents that decreased binding affinity
at muscarinic receptors had essentially the same effect
on dopamine transporter binding. However, in the
N-substituted 4′,4”-difluoro-substituted series of 3R-
(diphenylmethoxy)tropane analogues, a separation in
the binding affinities for the dopamine transporter and
muscarinic receptors was achieved.21 Therefore we
envision that the further development of predictive
models for the dopamine transporter, making use of this
new series of N-substituted compounds, will enhance
our capability of designing more selective ligands for the
dopamine transporter. Further, the determination of
whether a predictive model can be derived for these
compounds at the m1 muscarinic receptor will also prove
useful in the design of third-generation molecules with
improved selectivity and potency at the dopamine
transporter.
Finally, previously reported CoMFA studies on the
3-position of the tropane ring of cocaine also suggest
that the steric component is a predominant factor in the

CoMFA of Phenyl-Substituted Tropanes at the DAT
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3507
removal of all SOCl2). Resultant oils were determined spec-
troscopically to be the desired benzhydryl chlorides 3a -3g.
These benzhydryl chlorides were individually added to a
tropine (5-10 mmol) melt (160 °C) over 5 min. The reactions
were allowed to proceed for 10-45 min resulting in brown oils
which solidified upon cooling. The crude products were dis-
solved in CHCl3 (30 mL) and transferred to separatory funnels.
Ion pairing of the desired products was carried out via
extraction with 2.8N HCl (30 mL) as previously described.19
The phases were mixed and separated and the aqueous phase
was further extracted with CHCl3 (2 × 25 mL). The organic
fractions were combined, washed with 2.8 N HCl (2 × 25 mL),
dried over anhydrous Na2SO4, filtered, and concentrated in
vacuo resulting in light brown foams which were recrystallized
to give the pure products 4a -4g as HCl salts (25-65% yield).
P h a r m a cology. 1. Dop a m in e Tr a n sp or t er Bin d in g
Assa y. A detailed description of the binding methods can be
found in ref 34. Briefly, 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
of 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 centrifugation at 20000g
for 10 min at 4 °C. Fresh homogenates were used in all
experiments.
binding affinities of these analogues with electrostatics
playing a smaller yet significant role.9-13 QSAR in the
cocaine series of compounds reflects opposite optimal
stereochemistry at the 3-position as well as a different
rank order of substituents that give optimal binding at
the dopamine transporter as compared to the benz-
tropine analogues. A comparison of these QSAR inves-
tigations with our CoMFA model, along with classical
structure-activity studies, further substantiates the
hypothesis that the cocaine and benztropine classes of
compounds interact with different binding domains.
Therefore the present study demonstrates that a CoM-
FA model can be derived for the 3R-(diphenylmethoxy)-
tropane class of dopamine uptake inhibitors and can be
useful in designing novel ligands with which to further
elucidate the role of the dopamine transporter in the
reinforcing effects and abuse liability of psychomotor
stimulants such as cocaine. Furthermore, the binding
site model derived from these studies confirms differ-
ences in binding domains that are likely interacting
with these tropane-based classes of molecules and
underscores the possibility of developing dopamine
uptake inhibitors that are structurally and pharmaco-
logically distinctive from cocaine and hence may have
therapeutic utility in the treatment of its abuse.
Binding assays were conducted in modified Krebs-HEPES
buffer on ice. The total volume in each tube was 0.5 mL and
the final concentration of membrane after all additions was
0.5% (w/v) corresponding to 200-300 µg of protein/sample.
Triplicate samples of membrane suspension were preincubated
for 5 min in the presence or absence of the compound being
tested. [3H]WIN 35,428 (2-â-carbomethoxy-3-â-(4-fluorophe-
nyl)tropane 1,5-naphthalenedisulfonate, specific activity 82.4
Ci/mmol; New England Nuclear, Boston, MA, final concentra-
tion 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
to reduce nonspecific binding) using a Brandel cell harvester
(Gaithersburg, MD). The filters were washed with three
additional 3 mL washes and transferred to scintillation vials.
Absolute ethanol (0.5 mL) and Beckman Ready Value scintil-
lation cocktail (2.75 mL) were added to the vials which were
counted the next day at an efficiency of about 36%. Under
these assay conditions, an average experiment yielded ap-
proximately 6000 dpm total binding per sample and ap-
proximately 250 dpm nonspecific binding, 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, each performed in triplicate.
2. [3H]Dop a m in e Up ta k e Assa y. Rats were sacrificed by
decapitation and their brains removed to an ice-cooled dish
for dissection of the caudate putamen. [3H]Dopamine uptake
was measured in a chopped tissue preparation as described
previously.35 Briefly, the tissue was chopped into 225 µm slices
on a McIllwain tissue slicer with two successive cuts at an
angle of 90°. The strips of tissue were suspended in oxygenated
modified Krebs-HEPES buffer (see above), which was pre-
gassed with 95% O2/5% CO2 and warmed to 37 °C. After
rinsing, aliquots of tissue slice suspensions were incubated in
buffer in glass test tubes at 37 °C to which either the drug
being tested or no drug was added, as appropriate. After a 5
min incubation period in the presence of drug, [3H]dopamine
(final concentration 15 nM, specific activity 50 Ci/mmol;
Amersham Corp., Arlington Heights, IL) was added to each
tube. After 5 min the incubation was terminated by the
addition of 2 mL of ice-cold buffer to each tube and filtration
under reduced pressure over glass fiber filters (presoaked in
0.1% poly(ethylenimine) in water). The filters were rinsed and
Exp er im en ta l Meth od s
Ch em istr y. Melting points were determined on a Thomas-
Hoover melting point apparatus and are uncorrected. H and
1
13C NMR spectra were recorded using a Bruker (Billerica, MA)
AC-300 spectrometer. Samples were dissolved in the indicated
deuterated solvents, and chemical shifts are reported as parts
per million (δ) relative to tetramethylsilane (Me4Si, 0.00 ppm)
as internal standard. Chemical shifts for 13C spectra were
reported as relative to deuterated chloroform (CDCl3, 77.0
ppm). Mass spectra were obtained using a Hewlett-Packard
(Palo Alto, CA) 5971A mass selective ion detector (MSD) in
the electron-impact mode. Samples were introduced into the
MSD via an HP-5890 series II gas chromatograph (GC) fitted
with an HP-1 (cross-linked methyl silicone gum, 25 m × 0.2
mm i.d., 50 µm film thickness) column. 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. The initial GC oven temperature was 100
°C, held for 3.0 min, increased to 295 °C at a rate of 15 °C/
min, and held at this final temperature for 10 min. Infrared
spectra were recorded with a Perkin-Elmer 1600 series FTIR
spectrophotometer with KBr disks. Elemental analysis per-
formed by Atlantic Microlab, Inc. (Norcross, GA) agreed to
within 0.4% of the calculated values. The TLC solvent used
was CHCl3/CH3OH/NH4OH (89:10:1) unless otherwise indi-
cated. All chemicals and reagents were purchased from Lan-
caster Synthesis, Inc. or Aldrich Chemical Co.
Gen er a l Syn th etic Meth od for Su bstitu ted 3R-(Dip h e-
n ylm eth oxy)tr op a n es (4a -4g). Commercially available ben-
zophenones 1a -1g (10 mmol) were dissolved in absolute
ethanol or methanol (75-100 mL) and NaBH4 (10 mmol) was
added. Reactions were typically allowed to proceed for 1-2 h
after which time the excess alcohol was removed in vacuo. The
dry white residue was taken up in H2O (50 mL) and ether (50
mL), and after the two phases were mixed and separated, the
aqueous phase was further extracted with ether (2 × 25 mL).
The ether fractions were combined, washed with H2O (2 × 20
mL), dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. Reaction products were obtained in yields of 93-
98%. Resultant benzhydrols 2a -2g (5-10 mmol) were dis-
solved in SOCl2 (10 mL) in an atmosphere of argon. The
reaction was allowed to proceed overnight at ambient tem-
perature. Excess SOCl2 was removed in vacuo (addition of 2
small portions of dry toluene and removal in vacuo ensured

3508 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
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3R-(diphenylmethoxy)tropane Analogues: Selective Ligands for
the Dopamine Transporter. J . Med. Chem. 1997, 40, 4329-4339.
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placed in scintillation vials to which 1 mL of methanol and 2
mL of 0.2 M HCl were added to extract the accumulated [3H]-
dopamine. Radioactivity was determined by liquid scintillation
spectrometry at an efficiency of approximately 30%. The
reported values represent specific uptake from which nonspe-
cific binding to filters was subtracted.
3. An a lysis of Da ta . Saturation and displacement data
were analyzed by the use of the nonlinear least-squares curve-
fitting computer program LIGAND.36 Data from replicate
experiments were modeled together to produce a set of
parameter estimates and the associated standard errors of
these estimates. In each case, the model reported fit signifi-
cantly better than all others according to the F test at p <
0.05. The Ki values reported are the dissociation constants
derived for the unlabeled ligands. Uptake data were analyzed
using standard analysis of variance and linear regression
techniques.37 IC50 values were calculated using the linear
portion of the concentration-response curve (linear regression
p < 0.05).
Ack n ow led gm en t . R.H.K. was supported by a
NIDA-sponsored National Research Council Fellowship.
M.J .R. was funded by a National Institutes of Health
Intramural Research Training Award Fellowship. We
thank Richard Loeloff and Phyllis Terry for expert
technical assistance. We also thank Dr. Brian Hoffman
for valued insight and editorial assistance on the final
version of this manuscript. Animals used in this study
were maintained in facilities fully accredited by the
American Association for the Accreditation of Labora-
tory Animal Care (AAALAC), and all experimentation
was conducted according to the guidelines of the Insti-
tutional Care and Use Committee of the Intramural
Research Program, National Institute on Drug Abuse,
NIH, and the Guide for Care and Use of Laboratory
Animals of the Institute of Laboratory Animal Resources,
National Research Council, Department of Health,
Education and Welfare, Publication (NIH) 85-23, revised
1985.
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