Structure-activity relationship studies on a novel series of (S)-2β-substituted 3α-[bis(4-fluoro- or 4-chlorophenyl)methoxy] tropane analogues for in vivo investigation

Article abstract of DOI:10.1021/jm060762q

In general, 3α-(diphenylmethoxy)tropane (benztropine)-based dopamine uptake inhibitors do not demonstrate cocaine-like pharmacological activity in models of psychostimulant abuse and have been proposed as potential medications for the treatment of cocaine addiction. However, several (S)-2-carboalkoxy- substituted-3α-[bis(4-fluorophenyl)methoxy]tropane analogues were discovered to stimulate locomotor activity and substitute in subjects trained to discriminate cocaine, suggesting a role of the 2-position substituent in mediating these cocaine-like actions. Herein, we describe the synthesis of a series of novel N- and 2-substituted-3α-[bis-(4-fluoro- or 4-chlorophenyl)methoxy]tropane analogues. Most of these analogues demonstrated high affinity binding to the dopamine transporter (DAT; K_i = 1.8-40 nM), and selectivity over the other monoamine transporters and muscarinic M ₁ receptors. When the (S)-2-carboalkoxy substituent was replaced with (S)-2-ethenyl, the resulting analogue 11 demonstrated the highest DAT binding affinity in the series (K_i = 1.81 nM) with DAT selectivity over serotonin transporters (SERT; 989-fold), norepinephrine transporters (NET; 261-fold) and muscarinic receptors (90-fold). When the 4′-F groups of compounds 5 (K_i = 2.94 nM) and 8 (K_i = 6.87 nM) were replaced with 4′-Cl in the (S)-2-carboalkoxy series, DAT binding affinities were slightly reduced (K_i = 12.6 and 14.6 nM for 6 and 7, respectively), yet inhibition of dopamine uptake potency remained comparably high (IC₅₀ range = 1.5-2.5 nM). Interestingly, the 4′-Cl analogue (±)-6 substituted less in rats trained to discriminate cocaine than the 4′-F analogue (±)-5. These studies demonstrate that manipulation of the 2-, N-, and 3-position substituents in the 3α-(diphenylmethoxy)tropane class of dopamine uptake inhibitors can result in ligands with high affinity and selectivity for the DAT, and distinctive in vivo pharmacological profiles that cannot be predicted by their effects in vitro.

Full text of DOI:10.1021/jm060762q

J. Med. Chem. 2006, 49, 6391-6399
6391
Structure-Activity Relationship Studies on a Novel Series of (S)-2â-Substituted
3r-[Bis(4-fluoro- or 4-chlorophenyl)methoxy]tropane Analogues for in Vivo Investigation
Mu-Fa Zou,† Jianjing Cao,† Theresa Kopajtic,‡ Rajeev I. Desai,‡,§ Jonathan L. Katz,‡ and Amy Hauck Newman†,*
Medicinal Chemistry and Psychobiology Sections, National Institute on Drug Abuse-Intramural Research Program,
National Institutes of Health, DHHS, Baltimore, Maryland 21224
ReceiVed June 27, 2006
In general, 3R-(diphenylmethoxy)tropane (benztropine)-based dopamine uptake inhibitors do not demonstrate
cocaine-like pharmacological activity in models of psychostimulant abuse and have been proposed as potential
medications for the treatment of cocaine addiction. However, several (S)-2-carboalkoxy-substituted-3R-
[bis(4-fluorophenyl)methoxy]tropane analogues were discovered to stimulate locomotor activity and substitute
in subjects trained to discriminate cocaine, suggesting a role of the 2-position substituent in mediating these
cocaine-like actions. Herein, we describe the synthesis of a series of novel N- and 2-substituted-3R-[bis-
(4-fluoro- or 4-chlorophenyl)methoxy]tropane analogues. Most of these analogues demonstrated high affinity
binding to the dopamine transporter (DAT; Ki ) 1.8-40 nM), and selectivity over the other monoamine
transporters and muscarinic M1 receptors. When the (S)-2-carboalkoxy substituent was replaced with (S)-
2-ethenyl, the resulting analogue 11 demonstrated the highest DAT binding affinity in the series (Ki ) 1.81
nM) with DAT selectivity over serotonin transporters (SERT; 989-fold), norepinephrine transporters (NET;
261-fold) and muscarinic receptors (90-fold). When the 4′-F groups of compounds 5 (Ki ) 2.94 nM) and
8 (Ki ) 6.87 nM) were replaced with 4′-Cl in the (S)-2-carboalkoxy series, DAT binding affinities were
slightly reduced (Ki ) 12.6 and 14.6 nM for 6 and 7, respectively), yet inhibition of dopamine uptake
potency remained comparably high (IC50 range ) 1.5-2.5 nM). Interestingly, the 4′-Cl analogue (()-6
substituted less in rats trained to discriminate cocaine than the 4′-F analogue (()-5. These studies demonstrate
that manipulation of the 2-, N-, and 3-position substituents in the 3R-(diphenylmethoxy)tropane class of
dopamine uptake inhibitors can result in ligands with high affinity and selectivity for the DAT, and distinctive
in vivo pharmacological profiles that cannot be predicted by their effects in vitro.
Introduction
parisons of the SAR of compounds from these two classes of
dopamine uptake inhibitors further support different binding
modes or domains on the DAT protein.14,20 Further studies have
revealed that the benztropine-based dopamine uptake inhibitors
not only have distinct structural requirements from cocaine
and its 3-aryl analogues for binding to the DAT, but also have
different behavioral profiles in animal models of cocaine
abuse.21-24
Meltzer et al. synthesized all eight stereoisomers of 2-carbo-
methoxy-3-[bis(4-fluorophenyl)methoxy]tropane and discovered
that the S-(+)-enantiomer (S-(+)-difluoropine) was the most
Inhibition of dopamine uptake via the dopamine transporter
(DAT) has been characterized as being the primary mechanism
underlying the psychomotor stimulant and reinforcing actions
of cocaine and has been targeted in the discovery of potential
medications for the treatment of cocaine abuse (for recent
reviews, see refs 1-3). Many studies have been focused on
the elucidation of molecular structure and function of the
DAT, as well as characterizing the binding sites of cocaine
and other dopamine uptake inhibitors. Cocaine and its 3-aryl
analogues have been used as molecular probes to explore
the topology of the cocaine binding site(s) on the DAT.4-12
Structure-activity relationship (SAR) studies have provided
insight into the possible mode of cocaine binding to the DAT
and how this may differ from other structurally diverse dopa-
mine uptake inhibitors.1,13,14 Exploiting these differences and
further examining SAR in vivo has been a primary focus of
our drug discovery efforts toward a medication to treat cocaine
abuse and addiction.
Investigation of SAR within the 3R-(diphenylmethoxy)tropane
class of dopamine uptake inhibitors has led to the discovery
that unlike 3-aryl cocaine analogues, benztropine derivatives
do not require a substituent at the 2-position of the tropane ring
for binding with high affinity to the DAT.15-19 Recent com-
* Corresponding author. anewman@intra.nida.nih.gov; phone: (410) 550-
6568 Ext. 114; Fax: (410) 550-6855.
† Medicinal Chemistry.
‡ Psychobiology.
§ Current address: McLean Hospital/Harvard Medical School, 115 Mill
St., Belmont, MA 02478.
Figure 1. Chemical structures of cocaine (1) and 3R-[bis(4-fluorophe-
nyl)methoxy]tropane analogues (2-8).
10.1021/jm060762q This article not subject to U.S. Copyright. Published 2006 by the American Chemical Society
Published on Web 09/20/2006

6392 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Zou et al.
Scheme 1a
a Reagents and conditions: (a) LiAlH4, anhyd EtO2, 0 °C to r.t, 3 h; (b) (COCl)2, DMSO, Et3N, CH2Cl2, -78 °C; (c) Ph3P+CH3 Br-, BuLi, THF; (d)
i
(CH3O)2P(O)CH2CO2Me, Pr2NEt, CH3CN; (e) H2, Raney Ni, MeOH.
Scheme 2a
Scheme 3a
a Reagents and conditions: (a) (i) ACE-Cl, Na2CO3, 1,2-dichloroethane,
reflux, (ii) MeOH, r.t; (b) allyl bromide or butyl bromide, K2CO3, DMF,
50 °C.
To further investigate the SAR at the 2-position, a novel
series of (S)-2â-substituted 3R-[bis(4-fluoro- or 4-chlorophenyl)-
methoxy]tropanes was prepared and evaluated for binding at
the DAT, SERT, NET, and muscarinic M1 receptors. In addition,
we have recently discovered that the N-substituted-3R-[bis(4-
fluorophenyl)methoxy]tropanes can antagonize the effects of
cocaine in rodents.27,28 Therefore, several N-substituted 2â-
carboethoxy analogues were prepared and evaluated for their
potential use as tools for in vivo investigation.
a Reagents and conditions: (a) 4-(4-nitropheyl)butyryl chloride, Et3N,
CH2Cl2 0 °C to r.t; (b) H2, Raney Ni, MeOH; (C) LiAlH4, Et2O.
active19 (Figure 1, 5). Subsequently we reported an enantio-
selective synthesis of a series of S-(+)-2â-carboalkoxy-3R-[bis-
(4-fluorophenyl)methoxy]tropanes and showed that these com-
pounds were also potent dopamine uptake inhibitors.25,26 An
SAR comparison of these DAT ligands with their structurally
identical R-(-)-2â-carboalkoxy-3â-(3,4-dichlorophenyl)tropanes
demonstrated that although the S-(+)-2â-carboalkoxybenz-
tropines showed consistently lower affinities than the identically
2-substituted R-(-)-3-aryltropanes in binding to the DAT, they
inhibited dopamine uptake with high and equivalent potencies.26
Chemistry
S-2â-Carbomethoxy- and 2â-carboethoxy-3R-[bis(4-chloro-
phenyl)methoxy] tropanes (6 and 7) and their racemates were
prepared according to our previously reported procedure.25,26
In Scheme 1, S-(+)-2â-carboalkoxy-3R-[bis(4-fluorophenyl)-
methoxy]tropane (5 or 826) was reduced with LiAlH4 to give
the corresponding alcohol 9. Swern oxidation of the alcohol
provided aldehyde 10 in nearly quantitative yield. Wittig reaction

Dopamine Transporter Binding of Tropane Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6393
Table 1. Binding Data for (S) and (()-2-Substituted 3R-[Bis(4-fluoro- and chlorophenyl)methoxy]tropanes
compd
DAT binding Ki a,b (nM)
DA uptake IC50a,b (nM)
SERT Kia,b (nM)
NET Kia,b (nM)
M1 Kia,b (nM)
S-5
2.94 ( 0.36
21.3 ( 3.6f
12.6 ( 0.40
23.4 ( 1.5
14.6 ( 0.39
22.0 ( 0.84
6.87 ( 0.33
26.2 ( 3.4f
17.5 ( 0.88f
71.8 ( 4.6
1.50 ( 0.2c
2.94 ( 0.4
2.46 ( 0.2
NT
690 ( 58c
1750 ( 240
528 ( 39
NT
269 ( 39c
474 ( 65
2150 ( 325
NT
133 ( 4.2c
302 ( 43
382 ( 37
NT
(()-5c
S-6
(()-6
S-7
1.52 ( 0.2
1560 ( 91
3350 ( 154
3060 ( 150
(()-7
S-8
NT
NT
NT
NT
1.8 ( 0.2c
3.6 ( 0.4
23.4 ( 3.0d
236 ( 21g
1850 ( 270c
3740 ( 490
1640 ( 236d
286 ( 38g
629 ( 31c
1020 ( 120
2980 ( 182d
3300 ( 170g
1890 ( 130c
1860 ( 190
40.6 ( 8.0d
61400 ( 11000g
(()-8c
4,4-diCle
cocaine
a Each Ki value represents data from at least three independent experiments, each performed in triplicate. Ki values were analyzed by PRISM. b Binding
methods were conducted as previously reported29,30 except that the DAT assay was run in sucrose buffer. c Data from ref 25 and included for reference.
d Data from ref 29 and included for reference. e 3R-(bis-Cl-phenylmethoxy)tropane. f The Ki values for these compounds at DAT were assessed using different
methods29,30 than those used for the other compounds. In our experience the values obtained using those methods give Ki values that are approximately
3-fold higher than those obtained with the methods used for the other compounds. g Data from ref 30 and included for reference. NT ) not tested.
of 10 with methyltriphenylphosphonium bromide gave alkene
11, while Horner-Wadsworth-Emmons reaction of the alde-
hyde 10 with trimethyl phosphonoacetate afforded the R,â-
unsaturated ester 12, as a single crystalline isomer, which was
reduced by catalytic hydrogenation to give compound 13.
In Scheme 2, compound 9 was converted to the ester 14 by
reaction with 4-(p-nitrophenyl)butyric acid chloride, which was
prepared from the carboxylic acid in refluxing SOCl2. Catalytic
reduction of the nitro group of 14 with 10% Pd/C was
unsuccessful. However, reduction with Raney Ni successfully
produced the aniline 15 in good yield. The optical rotation
cocaine from saline injections. The racemates were used for
behavioral testing because multigram quantities needed for
behavioral evaluation were more conveniently synthesized as
racemates. Further, these compounds (Table 1) and previously
prepared racemates25 demonstrate high DAT affinity (though
approximately one-half as potent as the S-enantiomers) and
selectivity for the DAT over other monoamine transporters.
Compound (()-5 fully substituted for the cocaine discriminative
stimulus (Figure 2, top left panel), whereas its parent compound
(2), which lacks a substitution in the 2 position, as previously
reported,21 produced approximately 65% cocaine-appropriate
responding. Extending the pretreatment time for administration
of 2 was previously reported to result in full substitution for
cocaine.21 In contrast to (()-5 and despite having high binding
affinity at the DAT, the 4′4′′-diCl analogue, (()-6, did not
produce cocaine-like behavioral effects (Figure 2 top right). The
lack of substitution for cocaine occurred despite the study of a
range of doses from those having no effects to those that
virtually eliminated responding (Figure 2, bottom right panel).
A study of the time course of effects of (()-6 (Desai et al.,
submitted) indicated that time only diminished its cocaine-like
behavioral activity.
D
for 14 was [R]25 +3.5° (c ) 1.0, CHCl3); however, when
it was converted to 15, the rotation changed from “(+)” to
D
“(-)” (15: [R]25 -5.4° (c ) 1.0, CHCl3). To ensure that the
S-stereochemistry remained unchanged, compound 15 was
reduced to the alcohol 9 with LiAlH4. This product was
analytically identical to the original 9, and measurement of the
optical rotation showed it changed back to (+) from (-). This
experiment confirmed that no enantiomeric conversion of 15
had taken place during hydrogenation and that the optical
rotation conversion was independent of the stereochemistry at
the asymmetric C-2.
In Scheme 3, N-demethylation of the ethyl ester 825,26 using
the Oloffson procedure with ACE-Cl, resulted in the formation
of the N-nor product 16, which was easily converted to 17 and
18 by treatment with the corresponding alkyl bromides in DMF
under mildly basic conditions.
To further explore SAR at the N-position of the F-analogues,
a series of N-substituted (S)-2-substituted-3R-[bis(4-fluorophe-
nyl)methoxy]tropane analogues were synthesized and evaluated
for binding affinities at the DAT, NET, SERT, and muscarinic
M1 receptors (Table 2). Most of the compounds in this series
demonstrated high affinity for the DAT. Increasing the carbon
chain length at the 2-position of the tropane ring did not
significantly affect that affinity (e.g. 12, DAT Ki ) 4.69 nM,
and 13, DAT Ki ) 3.74 nM, compared to 5, DAT Ki ) 2.94
nM). However, increasing the steric bulk of this substituent did
result in an approximate 10-fold reduction in DAT binding
affinity (e.g. 14 and 15). DAT binding affinities were retained
by conversion of the ester to alcohol or alkene (9 and 11),
indicating that the ester function is not required for this class
of ligands to bind to the DAT with high affinity. In fact,
compound 11 (Ki ) 1.81 nM) showed the highest DAT binding
affinity in this series. This observation is consistent with
previously described results with the 3-aryltropanes, wherein
at the 2-position H-bonding interactions with the DAT protein
are not required for high affinity binding and may indicate that
lipophilicity is more important than H-bonding interactions.10,32
N-Demethylation (16) and substitution of the N-methyl of
compound 8 with other alkyl groups (17, 18) resulted in
comparable DAT binding affinities. These compounds also had
affinities at the DAT that were comparable to the analogues 3
and 4 that had no substitution in the 2-position (Table 2).
However, the 2-substituted compounds demonstrated much
Results and Discussion
Binding affinities of all novel compounds were evaluated in
radiolabeled ligand displacement assays for DAT, SERT, NET,
and muscarinic M1 receptors in rat brain. The methods for these
in vitro assays are briefly described in the Experimental Methods
Section, and results are shown in Table 1. The methods for the
SERT, NET, and M1 binding assays have been previously
described.29,30 Pirenzepine, though not exclusively labeling M1
receptors, has been shown to be a useful ligand for M1 receptor
identification.31 Inhibition of [3H]-dopamine uptake in rat syn-
aptosomes was also evaluated using a previously reported
procedure29,30 for compounds (S)-6 and (S)-7 for comparison
with the 4-F analogues (S)-5 and (S)-8, and their racemates.
We found that replacement of the 4-F groups in compounds
(S)-5 and (S)-8 with a Cl group, (S)-6 and (S)-7, modestly
reduced DAT binding affinities by 4- and 2-fold, respectively.
However, the IC50 values for inhibiting dopamine uptake
changed minimally, and remained relatively high (IC50 ) 2.5
and 1.5 nM for (S)-6 and (S)-7, respectively).
Compounds (()-5 and (()-6 were selected to compare their
cocaine-like subjective effects in rats trained to discriminate

6394 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Zou et al.
Table 2. Binding Affinities of Novel S-2-Substituted 3R-[Bis(4-fluorophenyl)methoxy]tropanes at the DAT, SERT, NET, and Muscaranic M1 Receptors
compound
R1
R2
DAT Ki ( SEM (nM)a SERT Ki ( SEM (nM)a NET Ki ( SEM (nM)a M1 Ki ( SEM (nM)a
2, (AHN 1-055) Me
H
H
H
4.11 ( 0.50b
9.70 ( 0.92c
8.79 ( 0.52c
3.40 ( 0.32
1.81 ( 0.21
4.69 ( 0.60
3.74 ( 0.07
39.9 ( 4.3
27.6 ( 0.85
8.87 ( 1.0
11.0 ( 0.94
8.18 ( 0.17
3260 ( 110b
1350 ( 150c
2850 ( 63c
910 ( 64.5
1790 ( 110
572 ( 51
1070 ( 120
3780 ( 390
1390 ( 150
2150 ( 220
2280 ( 130
2500 ( 330
844 ( 57
1490 ( 190c
1570 ( 160c
983 ( 97
11.6 ( 0.93b
3 (JHW 007)
butyl
399 ( 27c
4, (AHN 2-005) allyl
177 ( 21c
109 ( 15
163 ( 23
1380 ( 81
3110 ( 440
708 ( 100
342 ( 34
17100 ( 2500
11400 ( 1700
3200 ( 300
9
Me
Me
Me
Me
Me
Me
H
CH2OH
CHdCH2
CHdCHCO2Me
11
12
13
14
15
16
17
18
473 ( 64
269 ( 35
454 ( 43
2130 ( 160
440 ( 18
563 ( 74
935 ( 110
580 ( 16
CH2CH2CO2Me
CH2OCO(CH2) 3C6H4NO2-p
CH2OCO(CH2) 3C6H4NH2-p
CO2Et
allyl CO2Et
butyl CO2Et
a Binding methods were conducted as previously reported29,30 except that the DAT assay was run in sucrose buffer. b Included for reference (ref 34).
c Included for reference (ref 16).
Table 3. Binding Affinity Selectivity at DAT over SERT, NET, and
Muscarinic M1 Receptors
compound
SERT/DAT
NET/DAT
M1/DAT
2
3
4
5
6
8
9
11
793
139
324
234
42
269
259
989
122
286
95
148
154
179
92
171
92
279
261
57
121
53
3
41
20
45
30
275
31
90
294
832
18
12
13
14
15
50
16
12
16
17
18
4,4-diCl
cocaine
242
207
306
94
63
85
71
170
46
1928
1036
391
2
4
853
role in binding to the DAT, but clearly and adversely affects
binding at muscarinic receptors.
In general, none of these ligands demonstrated high binding
affinities to the norepinephrine (NET) and serotonin (SERT)
transporters. Therefore, with the exception of 14 and 15, which
showed relatively reduced DAT/M1 selectivity, all new com-
pounds showed high binding selectivity to the DAT over SERT,
NET, and muscarinic M1 receptors. Notably, compound 16 is the
most DAT/M1 selective benztropine reported to date (Table 3).
In summary, a series of novel S-2â-substituted-3R-[bis(4-
fluorophenyl)methoxy]tropanes and several N- or bis(4-chloro-
phenyl)methoxy-substituted-(S)-2â-carboethoxy analogues were
synthesized. Binding evaluation revealed that all of the com-
pounds showed high affinity for the DAT and demonstrated
high DAT selectivity over the NET, SERT, and muscarinic
M1 receptor. Several structurally variant substitutions at the
2-position of the tropane ring were well tolerated, although
groups with steric bulk caused a slight decrease in DAT binding
affinity, suggesting some limitations in tolerance to steric bulk
Figure 2. Effects of cocaine (1), 2, (()-5, (()-6, and 4′′,4′′-diCl-
BZT (3R-(bis-Cl-phenylmethoxy)tropane) in rats trained to discriminate
injections of cocaine (10 mg/kg; 29.4 µmol/kg) from saline. Ordi-
nates: percentage of responses on the cocaine-appropriate lever (top
panels) or rates at which responses were emitted as a percentage of
responses after saline administration (bottom panels). Abscissae: drug
dose in µmol/kg (log scale). Each point represents the effect in six
rats. The percentage of responses emitted on the cocaine-appropriate
lever was considered unreliable and not plotted, if fewer than half of
the subjects responded at that dose. Note that 2 failed to substitute for
cocaine with a pretreatment time of 5 min, whereas (()-5 substituted
for the discriminative stimulus effects of cocaine. In contrast, neither
(()-6 nor 4′′,4′′-diCl-BZT fully substituted for cocaine. Effects of 2
and 4′′,4′′-diCl-BZT were previously published.21
higher selectivity for the DAT over muscarinic receptors than
did the 2-unsubstituted compounds (Table 3). These data suggest
that the 2-carboalkoxy group may not be playing a significant

Dopamine Transporter Binding of Tropane Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6395
D
above procedure. Mp: 88-89.5 °C. [R]24 +17.0° (c ) 1.0,
at this position. Substitution at the tropane nitrogen, in the
2-carboethoxy series, resulted in retention of high DAT binding
affinity and selectivity. Indeed, these analogues (16, 17, and
18) demonstrated the highest DAT/ M1 selectivity reported to
date for the benztropine class of dopamine uptake inhibitors.
Behavioral studies demonstrated that the F-analogue (()-5
produced cocaine-like discriminative stimulus effects in rats.
However, when the 4-Fs on the 3R-diphenylmethoxy substituent
were replaced with Cl groups, the resulting (()-6 was unlike
cocaine. A comparison of binding profiles across the monoamine
transporters and muscarinic receptors between 5 and 8 and all
the other benztropine analogues reported to date,1,33 including
6 and 7 herein, does not explain the different behavioral profiles
of these compounds. Hence, further investigation into the role
of the 2-substituent and its affect on the rate of DAT occupancy
is underway. In addition, compounds 7, 11, 13, 17, and 18 are
also currently being evaluated in animal models of cocaine abuse
in order to further characterize SAR in vivo and define the role
of various substituents on the tropane ring, in this intriguing
class of dopamine uptake inhibitors.
CHCl3); IR: 1733, 1222, cm-1; H NMR (CDCl3) δ 1.22 (t, J )
1
7.0 Hz, 3H), 2.16-1.65 (m, 6H), 2.18 (s, 3H), 2.68 (m, 1H), 3.08
(m, 1H), 3.58 (m, 1H), 3.97 (d, J ) 4.8 Hz, 1H), 4.22-4.00 (m,
2H), 5.30 (s, 1H), 7.30-7.16 (m, 8H), ppm; GC-MS (m/z) 447
(M+); anal. (C24H27NCl2O3) for C, H, N.
(()-7 was prepared in 74% yield by the same procedure. Mp:
87.5-89.5 °C. The IR, H NMR, and GC-MS spectra for (()-7
1
were identical to S-(+)-7. Anal. (C24H27NCl2O3) for C, H, N.
S-(+)-2â-Hydroxymethyl-3R-[bis(4-fluorophenyl)methoxy]-
tropane (9). A solution of 5 or 826 (3.03 mmol) in 8 mL of
anhydrous diethyl ether was added dropwise to a suspension of
LiAlH4 (115 mg, 3.03 mmol) in the same solvent (8 mL) at 0 °C.
After the addition, the ice-H2O bath was removed and the reaction
mixture was allowed to warm to room temperature for 3 h. H2O
(0.3 mL) was added carefully at 0 °C, followed by the addition of
0.5 mL of aqueous NaOH (2 N). The resulting mixture was filtered,
the filtrate was dried (K2CO3) and filtered, and the ether was
removed in vacuo. The residue was purified by flash column
chromatography (eluting with 5-10% (CHCl3/MeOH/NH4OH;
D
CMA) to give the product (1.02 g, 90%) as a colorless oil. [R]25
+37.5° (c ) 1.0, CHCl3); 1H NMR (CDCl3) δ 2.14-1.79 (m, 7H),
2.22 (s, 3H), 3.12 (m, 1H), 3.24 (m, 1H), 3.46 (dd, J ) 2.8, 10.4
Hz, 1H), 3.56 (d, J ) 6.0 Hz, 1H), 3.93 (dd, J ) 2.4, 10.0 Hz,
1H), 5.38 (s, 1H), 6.98 (m, 4H), 7.22 (m, 4H), ppm; Anal. (C22H25-
NF2O2) for C, H, N.
Experimental Section
All chemicals and reagents were purchased from Aldrich
Chemical Co. or Lancaster Synthesis, Inc., and used without further
purification. All melting points were determined on a Thomas-
S-(+)-2â-Formyl-3R-[bis(4-fluorophenyl)methoxy]tropane (10).
To a solution of oxalyl chloride (1.64 mL, 2.0 M solution in CH2-
Cl2, 3.28 mmol) under argon at -78 °C was added a solution of
DMSO (469 mg, 6.02 mmol) in dry CH2Cl2 (1 mL). After 0.5 h,
a solution of the alcohol 9 (1.02 g, 2.73 mmol) in dry CH2Cl2 (3
mL) was added and the reaction mixture was stirred for 1 h at -78
°C, followed by addition of triethylamine (1.71 mL, 12.3 mmol) at
the same temperature. The reaction mixture was then allowed to
warm to room temperature and diluted with H2O (20 mL). The
organic layer was separated and the aqueous layer was extracted
with CH2Cl2 (3×20 mL). The combined organic layers were dried
(K2CO3) and concentrated to yield the aldehyde (994 mg, 98%).
1
Hoover melting point apparatus and are uncorrected. The H and
13C NMR spectra were recorded on a Bruker AC-300 or a Varian
Mercury Plus 400 instruments. Proton chemical shifts are reported
as parts per million (δ , ppm) relative to tetramethylsilane (0.00
ppm) as an internal standard. Coupling constants are measured in
hertz. Chemical shifts for 13C NMR spectra are reported as δ relative
to deuterated chloroform (CDCl3, 77.5 ppm, CD3OD 49.3). Infrared
spectra were recorded as a neat film on NaCl plates with a Perkin-
Elmer Spectrum RX I FT-IR system. Mass spectra were recorded
on a Hewlett-Packard (Palo Alto, CA) 5971A mass-selective ion
detector in the electron-impact mode with sample introduction via
an HP-1 (cross-linked methylsilicone gum) 25 m × 0.2 mm i.d.,
50 µm film thickness. 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 oven temperature was 100 °C, held for 3 min, programmed
to 295 °C at 15 °C/min, and maintained at 295 °C for 10 or 23
min. Microanalyses were performed by Atlantic Microlab, Inc.
(Norcross, GA) and agree with ( 0.4% of calculated values). All
column chromatography was performed using silica gel (Merck,
230-400 mesh, 60 Å).
IR: 1723, 1222, cm-1; H NMR (CDCl3) δ 2.21-1.58(m, 7H),
2.23(s, 3H), 3.06(m, 1H), 3.57(m, 1H), 3.99(m, 1H), 5.37(s, 1H)
6.98(m, 4H), 7.24(m, 4H), 9.59(s, 1H), ppm. The aldehyde was
used in the following steps without further purification.
1
S-(+)-2â-Ethenyl-3R-[bis(4-fluorophenyl)methoxy]tropane (11).
Butyllithium (0.22 mL, 2.5 M solution in hexane, 0.55 mmol) was
added dropwise to a suspension of methyltriphenylphosphonium
bromide (195 mg, 0.55 mmol) in dry THF (3 mL) at 0 °C, under
argon. The resulting yellow-orange solution was stirred for 30 min,
and the ice-H2O bath was then removed. The crude aldehyde 10
(169 mg, 0.46 mmol) in 2 mL of THF was added, and the reaction
mixture was stirred overnight at room temperature. The mixture
was diluted with H2O (20 mL), and the two layers were separated.
The aqueous layer was extracted with CHCl3 (3 × 20 mL). The
combined organic layers were dried (K2CO3) and concentrated.
The residue was purified by column chromatography to afford the
S-(+)-2â-Carbomethoxy-3R-[bis(4-chlorophenyl)methoxy]tro-
pane (6). S-(+)-alloecgonine methyl ester26 (309 mg, 1.55 mmol),
4,4′-dichlorobenzhydrol (786 mg, 3.10 mmol), p-toluenesulfonic
acid monohydrate (443 mg, 2.33 mmol), and benzene (15 mL) were
placed in a 50 mL round-bottom flask fitted with a Dean-Stark
trap and condenser. The reaction mixture was heated to reflux for
24 h. The solvent was then removed, and the residue was diluted
with water (20 mL), basified with NH4OH to pH 9, and extracted
with CHCl3 (3 × 20 mL). The combined organic layer was dried
(K2CO3) and concentrated. The residue was purified by column
chromatography (CHCl3/MeOH/NH4OH, 97:3:1) to afford S-6 (472
mg, 70%) as an oil, which solidified slowly to a white solid after
D
product (105 mg, 62%) as an oil. [R]25 +5.6° (c ) 1.0, CHCl3);
IR: 1222 cm-1; H NMR (CDCl3) δ 2.08-1.75 (m. 6H), 2.21
1
(s, 3H), 2.47 (m, 1H), 2.99 (m, 1H), 3.07 (m, 1H), 3.32 (d, J )
5.2 Hz, 1H), 4.88 (d, J ) 17.2 Hz, 1H), 4.96 (d, J ) 10.4 Hz,
1H), 5.37 (s, 1H), 5.96 (m, 1H), 6.98 (m, 4H), 7.30 (m, 4H), ppm;
13C NMR δ 24.9, 25.4, 36.1, 42.0, 50.1, 60.6, 65.9, 74.0, 79.8,
114.1, 115.1, 115.3, 128.3, 128.5, 138.5, 140.9, 160.8, 163.3, ppm;
GC-MS (m/z) 369 (M+), 368 (M - 1); Anal. (C23H25NF2O) for
C, H, N.
standing at room temperature. Mp: 109-110 °C (lit.33 mp: 110-
D
112 °C). [R]25 +17.3° (c ) 1.0, CHCl3); IR: 1732, 1069, cm-1
;
1H NMR (CDCl3) δ 2.17-1.68 (m, 6H), 2.18 (s, 3H), 2.71 (m,
1H), 3.09 (m, 1H), 3.59 (m, 1H), 3.68(s, 3H), 3.95 (d, J ) 4.8 Hz,
1H), 5.32 (s, 1H), 7.30-7.12 (m, 8H), ppm; GC-MS (m/z) 433
(M+); Anal. (C23H25NCl2O3) for C, H, N.
S-(+)-2â-(2′-(methoxycarbonyl)eth-1′-enyl)-3R-[bis(4-fluo-
rophenyl)methoxy] tropane (12). To a suspension of LiCl (80
mg, 1.89 mmol) in dry acetonitrile (8 mL) at room temperature
under an argon atmosphere were added trimethyl phosphonoacetate
(343 mg, 1.89 mmol), N,N-diisopropylethylamine (203 mg, 1.57
mmol), and the aldehyde (10, 583 mg, 1.57 mmol). The reaction
mixture was allowed to stir for 24 h, acetonitrile was then removed
under reduced pressure. The residue was diluted with H2O (20 mL)
(()-6 was prepared by the same procedure in 75% yield. Mp:
108-110 °C. The IR, 1H NMR, and GC-MS spectra for (()-6 were
identical to S-(+)-6. Anal. (C23H25NCl2O3) for C, H, N.
S-(+)-2â-Carboethoxy-3R-[bis(4-chlorophenyl)methoxy]tro-
pane (7). S-(+)-7 was prepared in 78% yield according to the

6396 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Zou et al.
and extracted with CHCl3 (3 × 20 mL). The combined organic
layers were dried (K2CO3) and concentrated. The crude product
was purified by column chromatography (3% CMA) to afford the
product (580 mg, 86%) as a colorless oil, which solidified slowly
S-(+)-N-Nor-2â-Carboethoxy-3R-[bis(4-fluorophenyl)meth-
oxy]tropane (16). Compound 8 (652 mg, 1.57 mmol) was dissolved
in anhydrous 1,2-dichloroethane (10 mL). To the solution were
added 1-chloroethyl chloroformate (ACE-Cl, 0.68 mL, 6.28 mmol)
and Na2CO3 (833 mg, 7.85 mmol), and the mixture was warmed
to reflux for 3 h. TLC showed complete absence of starting material.
After cooling to room temperature, the reaction mixture was filtered.
The solvent in the filtrate was removed in vacuo, and the residue
was dissolved in MeOH (20 mL). The reaction mixture was then
stirred at reflux for 1 h. MeOH was removed in vacuo. The residue
was diluted with H2O (50 mL), basified to pH 9 with NaHCO3,
and extracted with CHCl3 (3 × 50 mL). The combined organic
layer was dried (K2CO3) and concentrated. The residue was purified
by column chromatography (eluting with 5% CMA) to give the
product (610 mg, 97%) as an oil. [R]25D +33.8° (c ) 1.0, CHCl3);
IR: 3326, 1728, 1212, cm-1; 1H NMR (CDCl3) δ 1.23 (t, J ) 7.2
Hz, 3H), 1.97-1.65 (m, 5H), 2.22-2.06 (m, 2H), 2.65 (s, 1H),
3.48 (m, 1H), 3.79 (m, 1H), 3.83 (d, (J ) Hz, 1H), 4.17-4.04 (m,
2H), 5.37 (s, 1H), 7.00 (dd, J ) Hz, 4H), 7.25 (m, 4H), ppm; 13C
NMR δ 14.6, 28.9, 29.2, 36.0, 50.6, 53.5, 55.8, 51, 71.2, 80.6, 115.4,
115.6, 128.5, 128.7, 138.3, 161.0, 163.4, 173.2, ppm; GC-MS (m/z)
401 (M+); Anal. (C23H25NF2O3) for C, H, N.
D
to a colorless solid Mp 80-82 °C. [R]25 +26.5° (c ) 1.0,
1
CHCl3); IR: 1718, 1222, cm-1; H NMR (CDCl3) δ 2.16-1.78
(m, 6H), 2.20 (s, 3H), 2.58 (d, J ) 8.0 Hz, 1H), 3.12-3.00
(m, 2H), 3.34 (d, J ) 6.0 Hz, 1H), 3.70 (s, 3H), 5.35 (s, 1H), 5.68
(dd, J ) 1.2, 16 Hz, 1H), 7.08-6.97 (m, 5H), 7.24 (m, 4H), ppm;
13C NMR δ 25.4, 25.9, 36.4, 42.3, 49.4, 51.8, 60.9, 65.4, 73.6,
80.4, 115.5, 115.7, 120.8, 128.5, 128.6, 138.3, 151.0, 161.0, 163.4,
167.2, ppm; GC-MS(m/z) 427 (M+); Anal. (C25H27NF2O3) for
C, H, N.
S-(+)-2â-(2′-(Methoxycarbonyl)ethyl)-3R-[bis(4-fluorophenyl)-
methoxy] tropane (13). A large spatula tip of Raney Ni (in H2O)
was placed in a Parr bottle and washed (×3) with MeOH. The
unsaturated compound 12 (580 mg, 1.36 mmol) in MeOH (40 mL)
was added, and the mixture was shaken under an atmosphere of
H2 (40 psi) for 1.5 h. GC-MS showed the complete absence of
starting material. The reaction mixture was then filtered, and the
solvent was evaporated under reduced pressure. The residue was
purified by column chromatography (Et2O/ Et3N ) 95/5) to give
(510 mg. 88%) as an oil. [R]25D +24.8° (c ) 1.0, CHCl3); 1H NMR
(CDCl3) δ 2.06-1.48(m, 9H), 2.17(t, J ) Hz, 2H), 2.21(s, 3H),
2.94(m, 1H), 3.03 (m, 1H), 3.16 (d, J ) 5.2 Hz, 1H), 3.66 (s, 3H),
5.34 (s, 1H), 6.99 (m, 4H), 7.25 (m, 4H), ppm; 13C NMR δ 24.8,
25.9, 28.3, 32.6, 35.9, 42.4, 45.6, 51.9, 61.3, 65.3, 73.1, 79.7, 115.4,
115.6, 128.5, 128.6, 138.6, 138.7, 160.9, 163.3, 174.1, ppm; GC-
MS(m/z) 429 (M+); Anal. (C25H29NF2O3) for C, H, N.
S-(+)-N-Allyl-2â-carboethoxy-3R-[bis(4-fluorophenyl)methoxy]-
tropane (17). Compound 16 (85 mg, 0.21 mmol) and allyl bromide
(51 mg, 0.42 mmol) were combined in DMF (3 mL). To the solution
was added K2CO3 (58 mg, 0.42 mmol), and the mixture was heated
to 50 °C overnight. The reaction was quenched with H2O (20 mL),
and the mixture was extracted with CHCl3 (3 × 30 mL). The
combined organic layer was dried (K2CO3) and concentrated. The
residue was purified by column chromatography (eluting with 3%
S-(+)-2â-{[4-(4′-Nitrophenyl)butryl]oxymethyl}-3R-[bis(4-
fluorophenyl) methoxy]tropane (14). 4-(4′-Nitrophenyl)butyric
acid (414 mg, 1.98 mmol) in SOCl2 (8 mL) was stirred at reflux
for 3 h. Excess of SOCl2 was removed in vacuo, and the residue
was dissolved in CH2Cl2 (20 mL). To the solution was added
alcohol 9 (492 mg, 1.32 mmol), followed by the slow addition of
triethylamine (1.1 mL, 7.9 mmol) at 0 °C. The reaction mixture
was then allowed to stir at room temperature for 3 h. The mixture
was diluted with H2O (30 mL), the two layers were separated, and
the aqueous layer was further extracted with CH2Cl2 (3 × 20 mL).
The combined organic layers were dried (K2CO3) and concentrated.
The residue was purified by column chromatography (eluting with
Et2O/Et3N ) 97/3) to give the product (644 mg, 87%) as an oil.
D
CMA) to give the product (86 mg, 92%) as an oil. [R]25 +23.2°
(c ) 1.0, CHCl3); IR: 1730, 1221, cm-1; 1H NMR (CDCl3) δ 1.23
(t, J ) 7.2 Hz, 3H), 2.18-1.58 (m, 6H), 2.69 (m, 1H), 2.81 (dd, J
) 7.2, 13.6 Hz, 1H), 2.94 (dd, J ) 5.6, 13.6 Hz, 1H), 3.15 (m,
1H), 3.68 (m, 1H), 4.00 (d, J ) 2.0 Hz, 1H), 4.18-4.02 (m, 2H),
5.12-5.02 (m, 2H), 5.34 (s, 1H), 5.78 (m, 1H), 6.98 (m, 4H), 7.25
(m, 4H), ppm; 13C NMR δ 14.2, 24.6, 25.9, 36.3, 51.8, 56.7, 59.8,
60.2, 60.4, 70.7, 80.3, 115.2, 115.4116.3, 128.3, 128.4, 136.8, 138.3,
138.4, 160.8, 163.3, 172.5, ppm; GC-MS (m/z) 441 (M+); Anal.
(C26H29NF2O3) for C, H, N.
S-(+)-N-Butyl-2â-carboethoxy-3R-[bis(4-fluorophenyl)methoxy]-
tropane (18). Compound 18 was obtained in 87% yield by the
procedure described above for 17 using n-butyl bromide as the
alkylating agent. [R]25D +20.4° (c ) 1.0, CHCl3); IR: 1729, 1603,
1223, cm-1; 1H NMR (CDCl3) δ 0.86 (t, J ) 7.2 Hz, 3H), 1.24 (t,
J ) 7.2 Hz, 3H), 1.40-1.20 (m. 4H), 2.21-1.74 (m, 8H), 2.68
(m, 1H), 3.12 (m, 1H), 3.69 (m, 1H), 3.99 (d, J ) 4.8 Hz, 1H),
4.09 (m, 2H), 5.34 (s, 1H), 6.98 (m, 4H), 7.23 (m, 4H), ppm; 13C
NMR δ 14.3, 14.4, 20.7, 24.9, 26.2, 31.5, 36.5, 52.1, 53.5, 60.6,
60.7, 61.0, 71.1, 80.5, 110.0, 115.4, 115.6, 128.5, 128.6, 138.8,
161.0, 163.5, 172.7, ppm; GC-MS (m/z) 457 (M+); Anal. (C27H33-
NF2O3) for C, H, N.
D
1
[R]25 +3.5° (c ) 1.0, CHCl3); H NMR (CDCl3) δ 2.08-1.62
(m, 11H), 2.15 (s, 3H, N-CH3), 2.72 (t, J ) 7.6 Hz, 2H, COCH2),
3.07-2.98 (m, 2H), 3.30 (d, J ) 5.2 Hz, 1H), 4.00 (dd, J ) 9.2,
11.2 Hz, 1H, CH2O), 4.09 (dd, J ) 6.0, 11.2 Hz, 1H, CH2O), 5.34
(s, 1H, OCHAr2), 6.96 (m, 4H, Ar-H), 7.24 (m, 4H, Ar-H), 7.32
(d, J ) 8.8 Hz, 2H, Ar-H), 8.15 (d, J ) 8.8 Hz, 2H, Ar-H),
ppm; 13C NMR δ 24.2, 25.5, 25.9, 33.3, 34.9, 35.4, 45.8, 60.8,
62.8, 66.2, 70.1, 79.4, 115.1, 115.3, 123.7, 128.3, 128.4, 129.2,
138.4, 146.5, 149.2, 160.8, 163.2, 172.8, ppm; Anal. (C32H34N2F2O5)
for C, H, N.
Biological Assays. Dopamine Transporter Binding Assay.
Brains from male Sprague-Dawley rats weighing 200-225 g
(Taconic Labs) were removed, and striatum was dissected and
quickly frozen. Membranes were prepared by homogenizing tissues
in 20 volumes (w/v) of ice cold modified sucrose phosphate buffer
(0.32 M sucrose, 7.74 mM Na2HPO4, 2.26 mM NaH2PO4, pH
adjusted to 7.4) using a Brinkman Polytron (setting 6 for 20 s) and
centrifuged at 20000g for 10 min at 4 °C. The resulting pellet was
resuspended in buffer, recentrifuged, and resuspended in buffer to
a concentration of 10 mg/mL. Ligand binding experiments were
conducted in assay tubes containing 0.5 mL sucrose phosphate
S-(-)-2â-{[4-(4′-Aminophenyl)butyryl]oxymethyl}-3R-[bis(4-
fluorophenyl) methoxy]tropane (15). A spatula tip of Raney Ni
(in H2O) was placed in a Parr bottle and washed (×3) with MeOH.
A solution of the nitro compound 14 (523 mg, 0.98 mmol) and
MeOH/EtOAc (mL/mL) was shaken under an atmosphere of H2
(30 psi) overnight. TLC showed complete absence of starting
material. The mixture was then filtered, and the filtrate was
concentrated. The residue was purified by column chromatography
D
(5% CMA) to give the product as an oil. [R]25 -5.4° (c)1.0,
1
CHCl3); IR: 1727, 1603, 1221, cm-1; H NMR (CDCl3) δ 2.18-
3
1.63 (m, 11H), 2.15 (s, 3H), 2.51 (t, J ) 7.6 Hz, 2H), 3.07-2.98
(m, 2H), 3.30 (d, J ) 5.2 Hz, 1H), 3.70-3.40 (brs, 2H, NH2), 3.95
(dd, J ) 9.2, 11.0 Hz, 1H), 4.10 (dd, J ) 6.0, 11.0 Hz, 1H), 5.32
(s, 1H), 6.63 (d, J ) 8.8 Hz, 2H), 6.96 (m, 6H), 7.22 (m, 4H),
ppm; 13C NMR δ 24.6, 25.7, 27.0, 33.8, 34.5, 35.9, 42.1, 45.8,
61.1,63.0, 66.2, 70.4, 79.6, 115.3, 115.5, 128.6, 128.7, 129.5, 131.6,
138.6, 144.7, 161.0, 163.4, 173.7, ppm; Anal. (C32H36N2F2O3) for
C, H, N.
buffer for 120 min on ice. Each tube contained 0.5 nM H WIN
35428 (specific activity 84 Ci/mmol) and 1.0 mg striatal tissue
(original wet weight). Nonspecific binding was determined using
0.1 mM cocaine HCl. Incubations were terminated by rapid filtration
through Whatman GF/B filters, presoaked in 0.05% PEI (polyethyl-
eneimine), using a Brandel R48 filtering manifold (Brandel
Instruments Gaithersburg, MD). The filters were washed twice with
5 mL cold buffer and transferred to scintillation vials. Beckman

Dopamine Transporter Binding of Tropane Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6397
Ready Safe (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). Data were analyzed
by using GraphPad Prism software (San Diego, CA).
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 benzilate). 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 centrifugation at 1000g
for 10 min at 4 °C. The supernatant was saved and recentrifuged
at 10000g for 20 min at 4 °C. The supernatant was then discarded,
and the pellet was gently resuspended 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 con-
centration, 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, Gaith-
ersburg, 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).
Drug Discrimination Procedure. Cocaine Discrimination.
Male Sprague-Dawley rats (Charles River, Wilmington, MA)
weighing approximately 350 g were individually housed with free
access to water under a 12 h light/dark cycle (lights on 07.00 h).
All testing was between 09:00 and 15:00 h. Rats were fed approx-
imately 15 g standard lab chow daily at least 30 min after testing.
Rats were tested in two-lever operant-conditioning chambers
(modified Med Associates, model ENV 007, St. Albans, VT; inside
29.2 × 24.2 × 21 cm)) housed within light- and sound-attenuating
enclosures with white noise present throughout testing. Ambient
illumination of the chamber was by a lamp in the top center of the
front panel. Levers were set 17 cm apart, with a pair of green light
emitting diodes (LEDs) above one lever and a pair of yellow LEDs
above the other. A force on the lever of 0.4 N through 1 mm was
required to register a response and produced a click; reinforced
responses dispensed one 45 mg pellet (BioServe, Frenchtown, NJ)
into the centrally located food tray.
Serotonin Transporter Binding Assay. Brains from male
Sprague-Dawley rats weighing 200-225 g (Taconic Labs, Ger-
mantown, NY) were removed, and midbrain 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 15 mg/mL. Ligand binding experiments were
conducted in assay tubes containing 0.5 mL of 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 mM fluoxetine. Incubations were terminated by rapid
filtration through Whatman GF/B filters, presoaked in 0.3%
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 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 mM
for competition against binding of [3H]Citalopram, in at least three
independent experiments, each performed 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, Ger-
mantown, NY) were removed, and 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 mM desipramine. Incubations were terminated by rapid filtration
through Whatman GF/B filters, presoaked in 0.05% polyethyl-
enimine, 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 using
a Beckman 6000 liquid scintillation counter (Beckman Coulter
Instruments, Fullerton, CA). Each compound was tested with con-
centrations ranging from 0.01 nM to 100 mM for competition
against binding of [3H]nisoxetine, in at least three independent
experiments, each performed in triplicate. Data were analyzed by
using GraphPad Prism software (San Diego, CA).
Muscarinic M1 Binding Assay. Whole frozen rat brains exclud-
ing 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 Sciences, Boston,
MA) 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% polyethylenimine in water) using a Brandel
Rats were initially trained to press both levers under a fixed-
ratio 20-response schedule of food reinforcement. Following i.p.
cocaine injection, responses on only one lever were reinforced;
following saline, responses on the other lever were reinforced. The
assignment of cocaine- and saline-appropriate levers was counter-
balanced across rats. Immediately after injection, rats were placed
inside the experimental chambers and a 5 min time-out period was
initiated, during which all lamps were extinguished and responding
had no scheduled consequences. All lamps were then illuminated
and responses on the appropriate lever were reinforced. Responses
on the inappropriate lever reset the FR 20 response requirement
on the appropriate lever. Each food presentation was followed by
a 20 s time-out period. Sessions ended after 20 food presentations
or 15 min, whichever occurred first.

6398 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Zou et al.
(11) Kotian, P.; Muscarella, S. W.; Abraham, P.; Lewin, A. H.; Boja,
J. W.; Kuhar, M. J.; Carroll, F. I. Synthesis, Ligand Binding and
Quantitative Structure-Activity Relationship Study of 3â-(4′-
substituted phenyl)-2â-heterocyclic Tropanes: Evidance for an
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Affinity, Dopamine Uptake Inhibition, and Locomotor Stimulant
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J. A. The Uptake Inhibitors Cocaine and Benztropine Differentially
Alter the Conformation of the Human Dopamine Transporter. J. Biol.
Chem. 2001, 276, 29012-29018.
(14) Ukairo, O. T.; Bondi, C. D.; Newman, A. H.; Kulkarni, S. S.;
Kozikowski, A. P., Pan, S.; Surratt, C. K. Recognition of benztropine
by the dopamine transporter (DAT) differs from that of the classical
dopamine uptake inhibitors cocaine, methylphenidate and mazindol
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Training sessions for which cocaine (C) and saline (S) injections
were administered were ordered in an SCCS sequence, with test
sessions conducted after consecutive SC or CS training sessions.
Test sessions were conducted if the subject attained criterion
performance on both of the immediately preceding saline and
cocaine training sessions. The criteria were at least 85% appropriate
responding overall and during the first FR of the session. Test
sessions were identical with training sessions, with the exception
that 20 consecutive responses on either lever were reinforced. Before
test sessions, different doses of cocaine or doses of test compounds
were administered.
For each rat, the overall response rate and the percentage of
responses occurring on the cocaine-appropriate lever were calcu-
lated. The mean values were calculated for each measure at each
drug dose tested. Data from any rat which failed to respond at a
rate exceeding 0.02 responses per second was not included in the
calculation of mean cocaine-appropriate responding at that dose.
If less than three rats met the response rate requirement, no mean
value was calculated for percentage of cocaine-appropriate respond-
ing at that dose.
(15) Newman, A. H.; Allen, A. C.; Izenwasser, S.; Katz, J. L. Novel 3R-
Diphenylmethoxytropane Analogues are Potent Dopamine Uptake
Inhibitors without Cocaine-like Behavioral Profiles. J. Med. Chem.
1994, 37, 2258-2261.
(16) Newman, A. H.; Kline, R. H.; Allen, A. C.; George, C.; Izenwasser,
S.; Katz, J. L. Novel 4′- and 4′,4′′-Substituted-3R-(diphenylmethoxy)-
tropane Analogues are Potent and Selective Dopamine Uptake
Inhibitors. J. Med. Chem. 1995, 38, 3933-3940.
Acknowledgment. This work was supported by funds from
the Intramural Research Program, National Institute on Drug
Abuse, National Institutes of Health. Dr. Desai was supported
by a National Institutes of Health Visiting Fellowship.
(17) Kline, R. H.; Izenwasser, S.; Katz, J. L.; Newman, A. H. 3′-Chloro-
3R-(diphenylmethoxy)tropane But Not 4′-Chloro-3R-(diphenyl-
methoxy)tropane Produces a Cocaine-like Behavioral Profile. J. Med.
Chem. 1997, 40, 851-857.
(18) Agoston, G. E.; Wu, J. H.; Izenwasser, S.; George, C.; Katz, J. L.;
Kline, R. H.; Newman, A. H. Novel N-Substituted 4′,4”-Difluoro-
3R-(diphenylmethoxy)-tropane Analogues: Selective Ligands for the
Dopamine Transporter. J. Med. Chem. 1997, 40, 4329-4339.
(19) Meltzer, P. C.; Liang, A. Y.; Madras, B. K. The Discovery of an
Unusually Selective and Novel Cocaine Analog: Difluoropine.
Synthesis and Inhibition of Binding at Cocaine Recognition Sites. J.
Med. Chem. 1994, 37, 2001-2010.
(20) Loland, C. J.; Desai, R. I.; Gerstbrein, K.; Sitte, H. H.; Newman, A.
H.; Katz, J. L.; Gether, U. Relationship Between Conformational
Changes in the Dopamine Transporter and Cocaine-like Subjective
Effects of Uptake Inhibitors, J. Biol. Chem. 2006, submitted.
(21) Katz, J. K.; Izenwasser, S., Kline, R. H., Allen, A. C.; Newman, A.
H. Novel 3R-Diphenylmethoxytropane Analogs: Selective Dopamine
Uptake Inhibitors with Behavioral Effects Distinct from Those of
Cocaine. J. Pharmacol. Exp. Ther. 1999, 288, 302-315.
(22) Katz, J. L.; Kopajtic, T.; Agoston, G. E.; Newman, A. H. Effects of
N-Substituted Analogues of Benztropine: Diminished Cocaine-like
Effects in Dopamine Transporter Ligands. J. Pharmacol. Exp. Ther.
2004, 288, 302-315.
(23) Woolverton, W. L.; Hecht, G. S.; Agoston, G. E.; Katz, J. L.;
Newman, A. H. Further Studies of the Reinforcing Effects of
Benztropine Analogs in Rhesus Monkeys. Psychopharmacology
2001, 154, 375-382.
(24) Li, S.-M.; Newman, A. H.; Katz, J. L. Place Conditioning and
Locomotor Effects of N-Substituted, 4′, 4′′-Difluorobenztropine
Analogues in Rats. J. Pharmacol. Exp. Ther. 2005, 313, 1223-1230.
(25) Zou, M.-F.; Agoston, G. E.; Belov, Y.; Kopajtic, T.; Katz, J. L.;
Newman, A. H. Enantioselective Synthesis of S-(+)-2â-Carboalkoxy-
3R-[bis(4-fluorophenyl)-methoxy]tropanes as Novel Probes for the
Dopamine Transporter. Bioorg. Med. Chem. Lett. 2002, 12, 1249-
1252.
(26) Zou, M.-F.; Kopajtic, T.; Katz, J. L.; Newman, A. H. Structure-
Activity Relationship Comparison of (S)-2â-Substituted 3R-[bis(4-
fluorophenyl)-methoxy]tropanes and (R)-2â-Substituted 3â-(3,4-
Dichlorophenyl)tropanes at the Dopamine Transporter. J. Med. Chem.
2003, 46, 2908-2916.
(27) Desai, R. I.; Kopajtic, T.; Koffarnus, M.; Newman, A. H.; Katz,
J. L. Identification of a Dopamine Transporter Ligand that Blocks
the Stimulant Effects of Cocaine. J. Neurosci. 2005, 25(8), 1889-
1893.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
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