Further structure-activity relationship studies on 8-substituted-3-[2- (diarylmethoxyethylidenyl)]-8-azabicyclo[3.2.1]octane derivatives at monoamine transporters

Article abstract of DOI:10.1016/j.bmc.2011.10.028

The synthesis and structure-activity relationships of 8-substituted-3-[2- (diarylmethoxyethylidenyl)]-8-azabicyclo[3.2.1]octane derivatives were investigated at the dopamine transporter (DAT), the serotonin transporter (SERT) and norepinephrine transporte

Full text of DOI:10.1016/j.bmc.2011.10.028

Bioorganic & Medicinal Chemistry 19 (2011) 7551–7558
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Bioorganic & Medicinal Chemistry
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Further structure–activity relationship studies on 8-substituted-3-[2-(diaryl-
methoxyethylidenyl)]-8-azabicyclo[3.2.1]octane derivatives at monoamine
transporters
Shaine A. Cararas a, Sari Izenwasser b, Dean Wade b, Amy Housman b, Abha Verma a, Stacey A. Lomenzo a,
Mark L. Trudell a,
⇑
a Department of Chemistry, University of New Orleans, New Orleans, 2000 Lakeshore Drive, LA 70148, USA
b Department of Psychiatry and Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and structure–activity relationships of 8-substituted-3-[2-(diarylmethoxyethylidenyl)]-
8-azabicyclo[3.2.1]octane derivatives were investigated at the dopamine transporter (DAT), the serotonin
transporter (SERT) and norepinephrine transporter (NET). The rigid ethylidenyl-8-azabicyclic[3.2.1]octane
skeleton imparted modestly stereoselective binding and uptake inhibition at the DAT. Additional struc-
ture–activity studies provided a transporter affinity profile that was reminiscent of the structure–activity
of GBR 12909. From these studies, the 8-cyclopropylmethyl group has been identified as a unique moiety
that imparts high SERT/DAT selectivity. In this study the 8-cyclopropylmethyl derivative 22e (DAT Ki of
4.0 nM) was among the most potent compounds of the series at the DAT and was the most DAT selective
ligand of the series (SERT/DAT: 1060). Similarly, the 8-chlorobenzyl derivative 22g (DAT Ki of 3.9 nM)
was found to be highly selective for the DAT over the NET (NET/DAT: 1358).
Received 26 August 2011
Revised 5 October 2011
Accepted 10 October 2011
Available online 18 October 2011
Keywords:
GBR 12909
Dopamine transporter
Serotonin transporter
Norepinephrine transporter
Dopamine uptake
Cocaine
Ó 2011 Elsevier Ltd. All rights reserved.
Tropane
1. Introduction
cocaine addiction.4–7 However, there remains overwhelming
evidence that indicates cocaine occupation of binding sites on
Despite recent trends showing a slight downward trend in illicit
drug use since 1990s, cocaine abuse in any form, either as the hydro-
chloride salt or as the free base Crack, remains a significant problem
among adolescents and adults in the United States. Reports indicate
that as recently as 2009, over 1.6 million Americans aged 12 and
over were current cocaine users.1 Although this is a small portion
of the total population the societal costs to American citizens are
estimated to be in the billions of dollars based on costs related to de-
creased productivity, crime, and health care.2 Unfortunately, to date
there is no effective medication available for the treatment of co-
caine-addicted individuals.
During the past decade tremendous progress has been made to-
wards understanding the role of the dopamine transporter (DAT) in
the pharmacological mechanism of cocaine addiction. However,
despite these advances, a clinically approved medication for
cocaine addiction targeting the DAT has yet to be identified.3 Some
recent studies have suggested that the DAT may not be the sole
contributor to the addiction pathology of cocaine and that the sero-
tonin transporter (SERT) is also involved in the brain circuitry of
the DAT contributes significantly to the stimulant, reinforcing ef-
fects and abuse liability associated with cocaine use.8–16 As such,
the DAT remains an important target for medication development
and selective DAT inhibitors may yet prove to be viable
medications for cocaine abuse.17–19
Although a plethora of structure–activity studies on a variety of
compound classes that exhibit high affinity for the DAT have been
completed,20–26 a highly selective DAT inhibitor that would mirror
the selective serotonin reuptake inhibitor (SSRI) antidepressants
(e.g., Citalopram and Paroxetine) has yet to be discovered.27 There
are several DAT ligands that exhibit 100-fold selectivity over SERT
and norepinephrine transporters (NET) in binding affinity, and
there are very few ligands that are DAT selective by three orders
of magnitude over SERT.28–30 However, there exists no DAT ligands
that inhibit dopamine, serotonin and norepinephrine uptake with
comparable selectivity.3 This disconnection between DAT binding
affinity and dopamine uptake inhibition has been suggested to be
one of the problems that may be contributing to the lack of success
in advancing a DAT selective agent for clinical development as a
cocaine medication.3
The aryl-1,4-dialkylpiperazines GBR 12909 (1) and GBR 12935
(2) have served as useful pharmacological probes for the
⇑
Corresponding author. Tel.: +1 504 280 7337; fax: +1 504 280 6860.
E-mail addresses: MLTCM@jazz.ucc.uno.edu, mtrudell@uno.edu (M.L. Trudell).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.028

7552
S. A. Cararas et al. / Bioorg. Med. Chem. 19 (2011) 7551–7558
development of selective dopamine uptake inhibitors.31 Early
studies with GBR 12909 explored the potent and somewhat selec-
tive affinity of these ligands for the DAT.32 This selectivity
prompted further investigations with 1 that led to the identifica-
tion of GBR 12909 as a potential agonist-based substitution ther-
apy for cocaine addiction.33–35 It exhibited slower onset and
longer duration of action than cocaine while possessing much
higher affinity for the DAT with a slower dissociation rate. GBR
12909 was also shown to antagonize cocaine-induced elevation
of extracellular dopamine levels.35 The favorable pharmacological
profile of 1 led to in vivo studies that demonstrated the ability of
GBR 12909 to suppress cocaine self-administration behavior in
rhesus monkeys,36 and exhibit non-stimulant properties in hu-
mans.37 Although the development of GBR 12909 as a cocaine
medication was halted after Phase 1 human safety studies identi-
fied problematic cardiovascular effects, it has served as an impor-
tant template for structure activity studies directed toward
cocaine medication development.26
While studying the structure–activity relationships (SAR) of GBR
12909, a variety of ring systems have been shown to replace the
piperazine scaffold and provide compounds with high affinity for
the DAT. These include various piperidine (e.g., 3 and 4) and tropane
(e.g., 5 and 6) scaffolds.25,26,38,39 These studies have led to the devel-
opment of a well-defined pharmacophore for high affinity and
selectivity.26 Previous efforts in our laboratories have identified a
novel class of GBR-related 8-alkylaryl-3-[2-(diarylmethoxyethy-
lidenyl)]-8-azabicyclo[3.2.1]octane derivatives that exhibit potent
and selective affinity for the DAT.39,40 These preliminary in vitro
studies demonstrated GBR-analogues 7–12 exhibited GBR-like
affinity at the DAT but were up to 300-fold more selective for the
DAT over the SERT.38,39 The potent and highly selective pharmaco-
logical profile of these 8-alkylaryl-3-[2-(diarylmethoxyethylide-
nyl)]-8-azabicyclo[3.2.1]octane derivatives has prompted further
investigation of the SAR of this novel class of DAT ligands in search
of potent highly selective ligands. It is well documented that N-sub-
stitution on 3-phenyltropane-based and benztropine-based DAT
inhibitors has little effect on affinity and selectivity, while N-substi-
tution on piperidine analogues of GBR 12909 has been shown to im-
prove DAT selectivity over SERT.25,41 In lieu of the structural
similarities of our lead compound 12 with 1 and the piperidine
derivative 3, our attention has focused on the substituents attached
to the nitrogen atom of the tropane ring system to optimize ligand-
transporter selectivity. Herein we report the synthesis and biologi-
cal evaluation at monoamine transporters of a series of novel highly
DAT selective 8-substituted-3-[2-(diarylmethoxyethylidenyl)]-8-
azabicyclo[3.2.1]octane derivatives (Fig. 1).
145 °C in neat chlorodiphenylmethane or bis(4-fluorophenyl)meth-
ylchloride, respectively. This procedure required no workup and
after column chromatography of the reaction mixture the ethers
17 (59%) and 18 (66%) were obtained in good yields. The ethers 17
and 18 were converted into the corresponding N-secondary amine
19 and 20, by decarbonylation with hydrazine monohydrate. Con-
comitant N-alkylation with the appropriate alkylbromide furnished
the 8-substituted-3-diarylmethoxyethylidenyl-8-azabicyclo[3.2.1]
octane 21 and 22 in overall yields of 30–60% for the two-step
process.
2.2. Biology
All compounds were tested as the oxalate salts. Binding affinities
for the dopamine, serotonin and norepinephrine transporters were
determined by the ability of the drug to displace the radiolabeled li-
gands [3H]WIN 35,428, [3H]citalopram, and [3H]nisoxetine, respec-
tively, from the monoamine transporters obtained from rat brain
tissue using previously reported assays.40,43,44 The binding affinities
of all compounds listed in Table 1 were initially determined for the
DAT. The compounds that exhibited DAT binding affinities with Ki
values <100 nM were evaluated at the serotonin and norepinephrine
transporters to determine transporter selectivity. Those compounds
that exhibited low binding affinity (Ki >100 nM) at the DAT were
typically not evaluated further.
2.3. Structure–activity relationships
As expected the 8-alkyl desfluorobenzhydryl analogues 21a,
21b, 21c, 21d and 21e were found to exhibit modest affinity for
the DAT, similar to the 8-methyl analogue 7 (Ki value of 130 nM)
previously reported by Zhang and coworkers.38 The ethyl analogue
21a was equipotent with the propyl analogue 21b. However, the
allyl analogue 21c was more potent than the propyl analogue
21b while the propargyl analogue 21d was less potent than either
the propyl analogue 21b or the allyl analogue 21c. The 8-cyclopro-
pymethyl analogue 21e (DAT Ki of 169 nM) exhibited weak affinity
at the DAT similar to that of the allyl analogue 21c. Alternatively,
the substituted 8-benzyl analogues 21f–h all exhibited high affin-
ity and were four to seven-fold more potent than the unsubstituted
congener 11. It is noteworthy, that among the desfluorobenzhydryl
derivatives 21, the 8-(4-fluorobenzyl)-congener 21f exhibited very
high selectivity for DAT over both SERT (DAT/SERT = 585) and NET
(DAT/NET = 477).
The 4,40-difluorobenzhydryl analogues 22a–g proved to be gen-
erally more potent than the corresponding desfluoro analogues
21a–g. This was consistent with the SAR of GBR 12909. The 8-alkyl
congeners 22a,b also exhibited similar DAT affinity to the methyl
analogue 8 (Ki of 19 nM).38 Further, the allyl analogue 22c (DAT Ki
of 21 nM) was twice as potent as the ethyl (22a) and the propyl ana-
logues (22b). However, it was quite surprising to discover that the
8-cyclopropylmethyl analogue 22e (DAT Ki of 4.0 nM), was nearly
threefold more potent than the other 8-alkyl derivatives and was
slightly more potent than GBR 12909. The 8-cyclopropylmethyl
analogue 22e exhibited low nanomolar affinity at the DAT similar
to the benzyl derivatives 22f–h (Ki values of 3–6 nM). However,
22e was significantly more selective for the DAT over the SERT than
22f–h. In fact the greater than 1000-fold SERT/DAT selectivity ob-
served for 22e is among the highest reported for any DAT li-
gand.28–30,44 In addition, 22e was also selective for DAT over NET
(NET/DAT: 625) albeit less than the SERT.
2. Results and discussion
2.1. Chemistry
The synthesis of the 8-substituted-3-diarylmethoxyethylidenyl-
8-azabicyclo[3.2.1]octane analogues commenced with the protec-
tion of the nitrogen atom of 3-tropinone (13). Previous studies had
revealed that reduction of the basicity of the nitrogen atom signifi-
cantly enhanced the yield of subsequent reactions on the tropane
scaffold, presumably by facilitating the chromatography.39 This
enhancement of the overall yield was worthy of the additional steps
involved in the protection/deprotection chemistry. To this end, the
carbamate 14 was prepared in 92% on a multigram scale (Scheme
1). The carbamate 14 was subjected to the Masamune–Roush
modification of the Horner–Wadsworth–Emmons olefination reac-
tion and afforded the ethylidenyl ester 15 in 87% yield.42 Reduction
of the ester with lithium aluminium hydride furnished the allylic
alcohol 16 in 89% yield. The allylic alcohol 16 was then converted
into the corresponding benzhydryl ethers 17 or 18 by heating at
In general, the 8-benzyl analogues 22f–h exhibited high affinity
at DAT and were equipotent with GBR 12909. However, these
analogues were consistently more selective for DAT over SERT
and NET relative to 1. It is noteworthy that within the series, SERT
affinity was always greater than NET affinity for the 8-benzyl

S. A. Cararas et al. / Bioorg. Med. Chem. 19 (2011) 7551–7558
7553
X
X
O
O
N
N
R
N
3 R = CH2Ph
1 X = F, GBR 12909
2 X = H, GBR 12935
4 R = (CH2)3Ph
X
X
R
N
R
N
X
X
F
O
5 R = CH3
7 R = CH3, X = H
O
6 R = (CH2)3Ph
8 R = CH3, X = F
9 R = (CH2)3Ph, X = H
10 R = (CH2)3Ph, X = F
11 R = CH2Ph, X = H
12 R = CH2Ph, X = F
F
Figure 1. GBR 12909-related dopamine transporter ligands.
O
EtO
O
EtO
N
N
N
i
iii
ii
87%
92%
87%
O
13
14
15
CO2CH3
O
O
EtO
O
R
R
N
N
N
X
X
X
iv
vi
70-91%
16
O
OH
X
21a X = H, R = Et
21b X = H, R = Pr
17 R = CO2Et, X = H (59%)
19 R = H, X = H (60%)
v
v
21c X = H, R = CH2CH=CH2
21d X = H, R = CH2C≡ CH
21e X = H, R = CH2Cp
21f X = H, R = 4-F-Bn
21g X = H, R = 4-Cl-Bn
21h X = H, R = 4-CH3-Bn
18 R = CO2Et, X = F (66%)
20 R = H, X = F (66%)
22a X = H, R = Et
Reagents and conditions:
22b X = H, R = Pr
i) EtOCOCl, K2CO3, toluene, Δ.
ii) LiCl, (CH3O)2POCH2CO2CH3, DBU, CH3CN.
iii) LiAlH4, THF, 0 °C.
iv) (Ph)2CHCl or (4-F-Ph)2CHCl, neat, 145 °C.
v) N2H4•H2O, HOCH2CH2OH, Δ.
vi) RBr, K2CO3, DMF, 80 °C.
22c X = H, R = CH2CH=CH2
22d X = H, R = CH2C≡ CH
22e X = H, R = CH2Cp
22f X = H, R = 4-F-Bn
22g X = H, R = 4-Cl-Bn
22h X = H, R = 4-CH3-Bn
Scheme 1. Synthesis of 8-Substituted-3-[2-(diarylmethoxyethylidenyl)]-8-azabicyclo[3.2.1]octanes.
derivatives, while the NET affinity was greater for the 8-alkyl cong-
eners. Among the benzyl derivatives the 4-chlorobenzyl analogue
22g exhibited the greatest NET/DAT selectivity, with DAT affinity
1358-times more potent than NET. The SERT/DAT selectivity of
168 was relatively high compared to GBR 12909, but significantly
less than the 8-cyclopropylmethyl derivative 22e.
for GBR 12909 derivatives than the 3-phenyltropanes or benztro-
pines. This assessment is based upon the significantly improved
DAT affinity and selectivity imparted by substitution of the 8-benzyl
substituent. These results are consistent with the SAR of the piperi-
dine derivatives of GBR 12909 and the related pharmaco-
phore.26,45,46 However, the high affinity of the 8-cyclopropyl
methyl analogue 22e seems to be an anomaly in this system. It is
unclear at this time what specific effect the 8-cyclopropylmethyl
Despite the presence of a tropane scaffold, these analogues 21
and 22 exhibited structure–activity more similar to that described

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S. A. Cararas et al. / Bioorg. Med. Chem. 19 (2011) 7551–7558
Table 1
Inhibition of [3H] WIN 35,428, [3H] Citalopram and [3H] Nisoxetine
F
R
R
N
N
O
O
F
21
22
Compda
Code
R
clogPb
DAT (Ki, nM)c
SERT (Ki, nM)c
NET (Ki, nM)c
SERT/DAT
NET/DAT
1
11
12
GBR 12909
SAC-I-37
6.24
6.49
6.77
5.12
5.41
5.64
5.93
5.49
5.78
4.91
5.19
5.54
5.83
6.63
6.92
7.09
7.38
7.00
7.29
10.6 1.9d
48 5.0
7.9 0.5
286 85
40 3.0
245 20
41 7.7
143 15
132 0d
5420 74
2220 740
496 22
1393 147
NT
13
113
281
47
29
Bn
Bn
Et
Et
Pr
Pr
ALB-IV-41
SAC3-129
SAC3-151
SAC3-111
SAC3-155
SAC3-133
SAC3-149
SAC3-157
SAC3-171
SAC3-159
SAC3-169
SAC5-165
SAC5-75
21a
22a
21b
22b
21c
22c
21d
22d
21e
22e
21f
22f
21g
22g
21h
22h
5958 786
5463 318
3461 314
3577 117
2847 349
3120 669
149
133
167
89
69
CH2CH@CH2
CH2CH@CH2
CH2C„CH
CH2C„CH
CH2Cp
CH2Cp
4-F-Bn
4-F-Bn
4-Cl-Bn
21
2
149
536 49
NTe
169 44
4.0 2.7
7.0 1.9
4.9 1.3
7.6 1.0
3.9 2.0
13 2.0
6.1 1.1
4239 225
4094 1528
1145 144
544 89
656 193
746 192
405 98
2502 157
3336 1879
3151 650
2714 216
5297 618
1772 441
2750 1267
1060
585
234
72
168
57
625
477
643
554
1358
136
450
SAC5-161
SAC5-77
SAC5-167
SAC5-137
4-Cl-Bn
4-CH3-Bn
4-CH3-Bn
68
a
b
c
All compounds were tested as the oxalate salts. NT:Not tested.
See Ref. 47.
All values are the mean SEM of three experiments preformed in triplicate.
IC50 values taken from ref. 46.
d
e
The compound decomposed in solution and a reliable Ki value could not be obtained.
moiety has on molecular recognition at the monoamine transport-
ers. Relative to the benzyl analogues, 22e has a diminished capacity
for p–p-interactions at the binding site. In addition, with ClogP va-
4. Experimental section
4.1. General
lue of 5.83 (Table 1), 22e is significantly less lipophilic than the 8-
benzyl derivatives (e.g., 12, ClogP 6.77).47 Presumably, the structural
variations present around the nitrogen atom in 22e do not contrib-
ute to enhanced DAT affinity but lead to diminished SERT and NET
affinity relative to the benzyl analogues 22f–h.
All chemicals were purchased from Aldrich Chemical Company
and used as received unless otherwise noted. Anhydrous acetoni-
trile (CH3CN), chloroform (CHCl3), dichloromethane (CH2Cl2),
methanol (MeOH), tetrahydrofuran (THF) and toluene, were pur-
chased from Mallinckrodt Baker, Inc. Thin layer chromatography
(TLC) 20 ꢀ 20 cm glass plates precoated with 250
lm silica gel
3. Conclusion
were purchased from Sorbent Technologies and used to monitor
reactions via visualization with short-wave UV light, iodine, potas-
sium permanganate, 2,4-dinitrophenyl hydrazine or Dragondorff’s
reagent. Chromatography was performed over Sorbent Technolo-
gies Silica Gel 60 Å (230–400 mesh). High-pressure hydrogenations
were carried out on a Parr apparatus. Proton and carbon NMR were
recorded on a Varian-400 MHz nuclear magnetic resonance spec-
trometer at ambient temperature in deuterated chloroform, ace-
tone or water from Cambridge Isotope Laboratories, Inc. 1H NMR
chemical shifts are reported as d values (ppm) relative to tetra-
methylsilane. Splitting patterns are designated as: s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet. 13C NMR chemi-
cal shifts are reported as d values (ppm) relative to chloroform-d
(77.0 ppm). Optical rotations were measured on Autopol III auto-
polarimeter at the sodium D line (2 mL sample cell).
In summary, a series of 8-substituted-3-diarylmethoxyethylide-
nyl-8-azabicyclo-[3.2.1]octane analogues were synthesized and
evaluated for their affinities at dopamine, serotonin and norepi-
nephrine transporters. The rigid ethylidinyl-8-azabicyclic[3.2.1]
octane skeleton imparted modestly stereoselective binding and up-
take inhibition. Additional structure–activity studies provided a
transporter affinity profile that was reminiscent of the SAR of GBR
12909. From these studies, the 8-cyclopropylmethyl group has been
identified as a unique moiety that imparts high SERT/DAT selectiv-
ity. In this study the 8-cyclopropylmethyl derivative 22e was among
the most potent compounds of the series and is one of the most DAT
selective ligands reported to date. Similarly, the 8-chlorobenzyl
derivative 22g was found to be highly selective for the DAT over
the NET. Because of the high selectivity of these compounds for
the DAT over the other monoamine transporters, 22e and 22g offer
a unique opportunity to explore the feasibility of the development
of a DAT selective cocaine pharmacotherapy. In addition, these
highly selective DAT ligands may find additional application toward
medicationdevelopment for other disease statesthat affect dopmin-
ergic pathways (ADHD and Parkinson’s disease).48,49 The pharmaco-
logical evaluation of these analogues is currently under
investigation and will be reported in due course.
4.2. 8-Ethoxycarbonyl-8-azabicyclo[3.2.1]octan-3-one (14)
In a dry 500 mL round-bottom flask was added a stir-bar,
3-tropinone (10.0 g, 0.072 mol) and K2CO3 (0.050 g). Anhydrous
toluene (90 mL) was added followed by 3 equiv of ethylchlorofor-
mate (21 mL, 0.22 mol) via syringe, drop wise. The flask was fitted
with a condenser, nitrogen bubbler and heated to reflux overnight

S. A. Cararas et al. / Bioorg. Med. Chem. 19 (2011) 7551–7558
7555
with stirring. The mixture was concentrated under vacuum and the
4.5. 3-(2-Diphenylmethoxyethylidenyl)-8-ethoxycarbonyl-8-
brown oil was dissolved in CH2Cl2 (100 mL) and washed with H2O
(100 mL). The layers were separated in a separatory funnel and the
aqueous layer extracted with CH2Cl2 (3 ꢀ 50 mL). The organics
were combined and dried over Na2SO4, filtered and concentrated
under vacuum. The residue was purified by column chromatogra-
phy (SiO2, EtOAc/hexane, 1:3) to afford 14 (13.1 g, 92%) as a light
yellow oil. 1H NMR: d 4.51 (br s, 2H), 4.16 (q, J = 7.2 Hz, 2H), 2.63
(br s, 2H), 2.31 (dd, J = 17.4, 1.6 Hz, 2H), 2.07–2.03 (m, 2H), 1.65
(d, J = 7.6 Hz, 2H), 1.25 (t, J = 7.2 Hz, 3H). 13C NMRd 208.3, 154.1,
61.6, 53.2, 49.1, 29.5(2), 28.8(2), 14.9.
azabicyclo[3.2.1]-octane (17)
In a dry 10 mL round-bottom flask was added the alcohol 16
(1.1 g, 0.005 mol), diphenylchloromethane (1.5 equiv, 1.3 mL,
0.007 mol) and a stir bar. This was heated neat to 145 °C for 2 h un-
der N2. The mixture was cooled to room temperature, dissolved in
pure ethyl acetate and purified immediately by column chroma-
tography (60% w/w SiO2, EtOAc/hexane, 1:4) to afford 17 (1.3 g,
66%) as a clear light yellow oil. 1H NMR: d 7.3–7.22 (m, 10H),
5.63–5.61 (m, 1H), 5.39 (s, 1H), 4.36–4.20 (m, 2H), 4.18–4.10 (m,
2H), 4.04–3.98 (m, 2H), 2.55–2.45 (m, 1H), 2.27–2.07 (m, 1H),
1.95–1.86 (m, 2H), 1.64–1.59 (m, 2H), 1.30–1.21 (m, 3H). 13C
NMR: d 153.6, 142.0 (2), 138.2(4), 128.1(4), 126.7(2), 123.0, 82.3,
64.2, 62.2, 55.1, 54.8, 43.8, 36.9, 29.1, 28.8(2), 14.2. Anal. Calcd
for: C25H29NO3: C, 76.70; H, 7.47; N, 3.58. Found: C, 76.54; H,
7.26; N, 4.45.
4.3. 8-Ethoxycarbonyl-3-methoxycarboethylidenyl-8-azabi-
cyclo[3.2.1]octane (15)
To a dry 500 mL round-bottom flask was added 1.2 equiv
(3.4 g, 0.080 mol) LiCl and a stir bar and sealed under a nitrogen
balloon. Acetonitrile (180 mL) was added followed by 1.2 equiv of
trimethylphosphonoacetate (6.04 mL, 0.037 mol). The mixture
was allowed to stir at room temperature for 30 min to dissolve
the LiCl. DBU (4.60 mL, 0.031 mol) was added via syringe, drop
wise, over 10 min. The protected tropinone 14 (13 g, 0.066 mol)
was dissolved in CH3CN (60 mL) and syringed into the flask. The
mixture was all allowed to stir at room temperature overnight
under N2. The mixture was concentrated under reduced pressure
and the light brown oil was dissolved in CH2Cl2 (100 mL) and
washed with H2O (50 mL). The layers were separated in a separ-
atory funnel and the aqueous layer was extracted with CH2Cl2
(3 ꢀ 50 mL). The organics were combined and dried over Na2SO4,
filtered and concentrated under vacuum. The residue was purified
by column chromatography (SiO2, EtOAc/hexane, 1:4) to afford 15
(14.5 g, 87%) as a light yellow oil. 1H NMR: d 5.79 (s, 1H), 4.39 (br
s, 2H), 4.19 (t, J = 6.8 Hz, 2H), 3.69 (s, 3H), 2.69 (br s, 1H), 2.36 (br
s, 1H), 2.12 (d, J = 14.4 Hz, 1H), 1.94 (s, 2H), 1.59 (d, J = 7.2 Hz,
2H), 1.28 (t, J = 6.8 Hz, 4H). 13C NMR: d 165.6, 155.2, 153.2,
118.2, 60.4, 53.6, 53.4, 50.3, 42.1, 35.5, 27.9(2), 14.2. Anal. Calcd
for: C13H19NO4: C, 61.64; H, 7.56; N, 5.53. Found: C, 61.46; H,
7.66; N, 5.48.
4.6. 3-[2-Bis(4-fluorophenyl)methoxyethylidenyl]-8-
ethoxycarbonyl-8-azabicyclo-[3.2.1]octane (18)
In a dry 10 mL round-bottom flask was added the alcohol 16
(1.4 g, 0.006 mol), bis-(4-fluorophenyl)methylchloride (1.5 equiv,
1.7 mL, 0.009 mol) and a stir bar. This was heated neat to 145 °C
for 2 h under N2. The mixture was cooled to room temperature, dis-
solved in EtOAc and purified immediately by column chromatogra-
phy (60% w/w SiO2, EtOAc/hexane, 1:4) to afford 18 (2.0 g, 75%) as
a clear light yellow oil. 1H NMR: d 7.3–7.26 (m, 4H), 7.03–6.98 (m,
4H), 5.63–5.61 (m, 1H), 5.39 (s, 1H), 4.36–4.20 (m, 2H), 4.18–4.10
(m, 2H), 4.04–3.98 (m, 2H), 2.55–2.45 (m, 1H), 2.27–2.07 (m,
1H), 1.95–1.86 (m, 2H), 1.64–1.59 (m, 2H), 1.30–1.21 (m, 3H). 13C
NMR: d 158.1(2), 153.5, 138.3, 138.1, 134.4(4), 128.5(2), 128.3(2),
123.1, 115.0, 81.2, 69.4, 64.1, 62.0, 55.2, 54.8, 43.8, 36.9, 29.2,
14.3. Anal. Calcd for C25H27F2NO3: C, 70.24; H, 6.37; N, 3.28. Found:
C, 70.55; H, 6.50; N, 3.09.
4.7. 3-(2-Diarylmethoxyethylidene)-8-azabicyclo[3.2.1]octane
(19 or 20)
4.4. 8-Ethoxycarbonyl-3-hydroxymethylethylidenyl-8-azabi-
cyclo[3.2.1]octane (16)
In a 250 mL round-bottom flask was added ethylene glycol
(88 mL), KOH (4.4 g, 0.079 mol), NH2NH2ꢁH2O (0.75 mL,
0.015 mol) and the corresponding protected tropane (17 or 18)
(0.003 mol). The mixture was heated to reflux for 3 h. The mixture
was then cooled to room temperature and poured into water
(100 mL) and extracted with Et2O (6 ꢀ 75 mL). The combined or-
ganic layers were dried over Na2SO4, filtered and concentrated un-
der reduced pressure. The resulting oil was purified by column
chromatography (SiO2, CHCl3/MeOH/NH4OH 90:9:1) to afford the
nortropane 19 or 20 as a light yellow oil. Compound 19: Yield
(0.62 g, 60%). 1H NMR: d 7.36–7.22 (m, 10H), 5.51–5.48 (m, 1H),
5.40 (s, 1H), 4.05–3.97 (m, 2H), 3.57–3.54 (m, 2H), 2.42 (d,
J = 13.6 Hz, 1H), 2.26 (d, J = 14.0 Hz, 1H) 2.05 (t, J = 14.0 Hz, 2H)
1.84 (br s, 2H), 1.73–1.68 (m, 2H) 1.64–1.59 (m, 1H), 1.51–1.47
(m, 1H). 13C NMR: d 142.1(2), 138.0(4), 128.1(4), 126.8(2), 122.9,
82.3, 64.1, 55.1, 54.7, 43.8, 36.9, 29.1, 28.8(2). Anal. Calcd for
Lithium aluminum hydride (1.2 equiv, 0.87 g, 0.023 mol) and a
stir bar were added to a clean, dry 250 mL round-bottom flask
and sealed under a nitrogen balloon. THF (35 mL) was added
via syringe and the solution brought to 0 °C in an ice-water bath.
The ester 15 (3.9 g, 0.015 mol) was dissolved in THF (15 mL) and
added drop wise over 20 min via syringe. The mixture was stirred
at 0 °C for 1.5 h and quenched by the slow addition of 10% KOH
(11 mL). Stirring was continued for an additional hour and the
reaction mixture was decanted into a separatory funnel. The
white solid was washed with diethyl ether (4 ꢀ 30 mL) and the
organics added to the seperatory funnel. The organics were
washed with phosphate buffer (75 mL) and the buffer solution
was extracted with diethyl ether (4 ꢀ 30 mL). The organics were
dried over Na2SO4, filtered and concentrated under reduced pres-
sure. The resulting alcohol was purified by column chromatogra-
phy (SiO2, EtOAc/hexane, 1:1) to yield 16 (3.0 g, 89%) as a light
yellow oil. 1H NMR: d 5.58 (m, 1H), 4.32 (s, 1H), 4.16 (m, 1H),
2.53 (d, J = 13.6 Hz, 2H), 2.41(d, J = 14 Hz, 2H), 2.23 (d,
J = 14.4 Hz, 2H), 2.03 (d, J = 13.6 Hz, 2H), 1.89 (m, 3H), 1.61 (t,
J = 7.6 Hz, 2H), 1.52 (t, J = 7.6 Hz, 3H). 13C NMR: d 153.7, 134.4,
127.7, 60.7, 57.7, 53.6, 41.6, 34.5(2), 27.8(2), 14.3. Anal. Calcd
for: C12H19NO3: C, 63.98; H, 8.50; N, 6.22. Found: C, 63.76; H,
8.61; N, 6.43.
C
22H25NOꢁ0.25 H2O: C, 81.75; H, 7.93; N, 4.33. Found: C, 81.57;
H, 7.86; N, 4.35. Compound 20: Yield (577 mg, 66%). 1H NMR: d
7.36–7.22 (m, 4H), 7.02–6.97 (m, 4H), 5.51–5.48 (m, 1H), 5.40 (s,
1H), 4.05–3.97 (m, 2H), 3.57–3.54 (m, 2H), 2.42 (d, J = 13.6 Hz,
1H), 2.27 (d, J = 14.0 Hz, 1H), 2.05(t, J = 14.0 Hz, 2H), 1.84 (br s,
2H), 1.73–1.68 (m, 2H), 1.64–1.59 (m, 1H), 1.51–1.47 (m, 1H). 13C
NMR: d 158.14(2), 138.3, 138.1, 134.4(4), 128.5(2), 128.3(2),
123.1, 115.0, 81.2, 69.4, 64.1, 55.2, 54.8, 43.8, 36.9, 29.2. Anal. Calcd
for C22H23F2NOꢁ0.50 H2O: C, 72.50; H, 6.63; N, 3.84. Found: C,
72.55; H, 6.50; N, 3.84.

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S. A. Cararas et al. / Bioorg. Med. Chem. 19 (2011) 7551–7558
4.8. General procedure of the synthesis of 21 and 22
4.14. 8-Allyl-3-[2-bis(4-fluorophenyl)methoxyethylidenyl]-8-
azabicyclo-[3.2.1]octane (22c)
In a clean, dry 25 mL round-bottom flask was added 600 mg
K2CO3, the nortropane (19 or 20) (2.0 mmol), DMF (15 mL) and
the alkyl bromide (2.2 mmol) and a stir bar. The mixture was
heated to 80 °C (oil bath) overnight under N2. The mixture was
cooled to room temperature and added to a separatory funnel
followed by addition of H2O (50 mL). The mixture was extracted
with diethyl ether (4 ꢀ 50 mL) and the combined organic layers
were dried over Na2SO4, filtered and concentrated under reduced
pressure. The oil was purified by column chromatography (SiO2,
EtOAc) and the pure free base was converted into the oxalate
salt.
Yellow oil (158 mg, 66%). 1H NMR: d 7.29–7.26 (m, 4H), 7.03–
6.98 (m, 4H), 5.94 (m, 1H), 5.48 (br s, 1H), 5.34 (s, 1H), 5.16 (m,
2H), 4.01–3.90 (m, 2H), 3.28 (br s, 2H), 3.08 (br s, 2H), 2.56 (br s,
1H), 2.12 (m, 2H), 1.97–1.86 (m, 2H), 1.55–1.43(m, 3H). Anal. Calcd
for C25H27F2NOꢁC2H2O4ꢁH2O: C, 64.40; H, 6.21; N, 2.78. Found: C,
64.32; H, 6.02; N, 2.70.
4.15. 3-(2-Diphenylmethoxy-ethylidene)-8-(3-prop-2-ynyl)-8-
azabicyclo[3.2.1]octane (21d)
Yellow oil (169 mg, 61%). 1H NMR d 7.35–7.23 (m, 10H), 5.51 (t,
J = 6.4 Hz, 1H), 5.39 (s, 1H), 4.05–3.93 (m, 2H), 3.40 (d, J = 12 Hz,
2H), 3.22 (s, 2H), 2.57 (d, J = 16 Hz, 1H), 2.20–2.13 (m, 2H), 1.98
(d, J = 12 Hz, 1H), 1.85 (br s, 2H) 1.59–1.56 (m, 2H) 1.44 (t,
4.9. 3-(2-Diphenylmethoxyethylidenyl)-8-ethyl-8-
azabicyclo[3.2.1]octane (21a)
Yellow oil (207 mg, 50%). 1H NMR: d 7.35–7.22 (m, 10H), 5.20 (t,
J = 6.6 Hz, 1H), 5.39 (s, 1H), 4.05–3.94 (m, 2H), 3.34 (d, J = 16 Hz,
2H), 2.64 (d, J = 16 Hz, 1H), 2.55 (q, J = 7 Hz, 2H), 2.28–2.25 (d,
J = 13.2 Hz, 1H), 2.13–2.10 (d, J = 14 Hz, 1H), 1.95 (d, J = 14.4 Hz,
1H), 1.87 (s, 2H), 1.56 (t, J = 9.2 Hz, 1H), 1.44 (t, J = 8.8 Hz, 1H),
1.15 (t, J = 7.2 Hz, 3H). Anal. Calcd for C24H29NOꢁC2H2O4: C, 71.37;
H, 7.14; N, 3.20. Found: C, 71.56; H, 7.39; N, 3.10.
J = 4.4 Hz, 1H). Anal. Calcd for
68.98; H 6.60; N 2.98. Found: C 68.67; H 6.36; N 2.93.
C
25H27NOꢁC2H2O4ꢁ1.25H2O:
C
4.16. 3-[2-Bis(4-fluorophenyl)methoxyethylidenyl]-8-(3-prop-
2-ynyl)-8-azabicyclo-[3.2.1]octane (22d)
Yellow oil (130 mg, 63%). 1H NMR: d 7.29–7.26 (m, 4H), 7.03–
6.98 (m, 4H), 5.50 (t, J = 8 Hz, 1H), 5.34 (s, 1H), 4.01–3.90 (m,
2H), 3.41 (br s, 2H), 3.20 (s, 2H), 2.57 (d, J = 14 Hz, 1H), 2.21–2.12
(m, 2H), 1.88–1.86 (m, 1H), 1.56 (s, 2H), 1.53–1.39 (m, 3H). Anal.
Calcd for C25H25F2NOꢁC2H2O4ꢁH2O: C, 64.66; H, 5.83; N, 2.79.
Found: C, 64.80; H, 5.50; N, 2.59.
4.10. 3-[2-Bis(4-fluorophenyl)methoxyethylidenyl]-8-ethyl-8-
azabicyclo[3.2.1]-octane (22a)
Yellow oil (140 mg, 66%). 1H NMR: d 7.29–7.26 (m, 4H), 7.03–
6.98 (m, 4H), 5.49 (s, 1H), 5.33 (s, 1H), 4.01–3.91 (m, 2H), 3.30
(d, J = 12 Hz, 2H), 2.58–2.47 (m, 3H), 2.19 (d, J = 16 Hz, 1H), 2.07
(d, J = 16 Hz, 1H), 1.91–1.85 (m, 3H), 1.49 (t, J = 7.6 Hz, 1H), 1.38
(t, J = 7.7 Hz, 1H), 1.11 (t, J = 7.6 Hz, 3H). Anal. Calcd for
4.17. 3-(2-Diphenylmethoxyethylidenyl)-8-cyclopropylmethyl-
8-azabicyclo-[3.2.1]octane (21e)
Yellow oil (227 mg, 45%). 1H NMR: d 7.36–7.23 (m, 10H), 5.48 (t,
J = 6.8 Hz, 1H), 5.39 (s, 1H), 4.05–3.94 (m, 2H), 3.43 (m, 2H), 2.55 (d,
J = 12 Hz, 1H), 2.33 (d, J = 4 Hz, 2H), 2.20–2.07 (m, 3H), 1.93–1.80
(m, 3H), 1.60–1.37 (m, 1H), 0.94 (m, 1H), 0.52 (m, 2H), 0.12 (m,
2H). Anal. Calcd for C26H31NOꢁC2H2O4ꢁ1.25H2O: C, 69.12; H, 7.36;
N, 2.88. Found: C, 69.00; H, 6.99; N, 2.83.
C
24H27F2NOꢁC2H2O4: C, 65.95; H, 6.17; N, 2.96. Found: C, 65.10;
H, 5.88; N, 2.65.
4.11. 3-(2-Diphenylmethoxyethylidenyl)-8-propyl-8-
azabicyclo[3.2.1]octane (21b)
Yellow oil (72 mg, 42%). 1H NMR d 7.35–7.22 (m, 10H), 5.54 (s,
1H), 5.39 (s, 1H), 4.05–3.94 (m, 2H), 3.38 (br s, 2H), 2.49 (br s, 2H),
2.16 (d, J = 13.2 Hz, 2H), 1.98–1.91 (m, 3H), 1.61 (m, 5H), 0.95 (t,
J = 7.4 Hz, 3H). Anal. Calcd for C25H31NOꢁC2H2O4ꢁH2O: C, 69.06; H,
7.51; N, 2.98. Found: C, 69.67; H, 7.30; N, 3.01.
4.18. 3-[2-Bis(4-fluorophenyl)methoxyethylidenyl]-8-
cyclopropylmethyl-8-azabicyclo[3.2.1]octane (22e)
Yellow oil (140 mg, 65%). 1H NMR: d 7.29–7.26 (m, 4H), 7.03–
6.98 (m, 4H), 5.46 (br s, 1H), 5.35 (s, 1H), 4.01–3.90 (m, 2H), 3.42
(m, 2H), 2.56 (br s, 1H), 2.31 (m 2H), 2.17 (m, 1H), 2.08 (d,
J = 14.8 Hz, 1H), 1.93 (d, J = 14.4 Hz, 1H), 1.83 (m, 2H), 1.58–143
(m, 2H), 0.51 (d, J = 7.2 Hz, 2H), 0.11 (d, J = 4 Hz, 2H). Anal. Calcd
for C26H29F2NOꢁC2H2O4ꢁH2O: C, 64.98; H, 6.43; N, 2.71. Found: C,
64.62; H, 6.17; N, 2.50.
4.12. 3-[2-Bis(4-fluorophenyl)methoxyethylidenyl]-8-propyl-8-
azabicyclo[3.2.1]-octane (22b)
Yellow oil (64 mg, 68%). 1H NMR: d7.29–7.26 (m, 4H), 7.03–6.98
(m, 4H), 5.45 (br s, 1H), 5.35 (s, 1H), 3.98–3.93 (m, 2H), 3.26 (br s,
2H), 2.56 (d, J = 12 Hz, 1H), 2.38 (t, J = 8.0 Hz, 2H), 2.16–2.04 (m,
2H), 1.92–1.84 (m, 3H), 1.56–1.50 (m, 3H), 1.36 (s, 1H), 0.92 (t,
J = 7.2 Hz, 3H). Anal. Calcd for C25H29F2NOꢁC2H2O4ꢁ1.5 H2O: C,
63.02; H, 6.66; N, 2.72. Found: C, 62.91; H, 6.89; N, 2.72.
4.19. 3-(2-Diphenylmethoxyethylidenyl)-8-(4-fluorobenzyl)-8-
azabicyclo[3.2.1]-octane (21f)
Yellow oil (77 mg, 67%). 1H NMR: d 7.38–7.21 (m, 12H), 7.04–
6.97 (m, 2H), 5.52 (t, J = 6.4 Hz, 1H), 5.39 (s, 1H), 4.04–3.94 (m,
2H), 3.58 (s, 2H), 3.22 (s, 2H), 2.62 (d, J = 13.2 Hz, 1H), 2.25–2.22
(m, 1H), 2.14–2.10 (m, 1H), 1.92 (m, 3H), 1.60–1.53 (m, 2H). Anal.
Calcd for C29H30FNOꢁC2H2O4ꢁH2O: C, 69.52; H, 6.40; N, 2.62. Found:
C, 69.30; H, 6.58; N, 2.44.
4.13. 8-Allyl-3-(2-diphenylmethoxyethylidenyl)-8-
azabicyclo[3.2.1]octane (21c)
Yellow oil (132 mg, 67%). 1H NMR: d 7.35–7.21 (m, 10H), 5.96
(br s, 1H), 5.52 (br s, 1H), 5.39 (s, 1H), 5.21–5.14 (m, 2H), 4.05–
3.93 (m, 2H), 3.30 (d, J = 12 Hz, 2H), 3.11 (d, J = 5.6 Hz, 2H), 2.63
(br s, 1H), 2.24 (br s, 1H), 2.16–2.12 (m, 1H), 1.98–1.95 (m, 1H),
1.86 (m, 2H), 1.60–1.43 (m, 2H). Anal. Calcd for C25H29NOꢁ-
C2H2O4ꢁ1.25H2O: C, 68.04; H, 7.19; N, 2.94. Found: C, 68.55; H,
6.88; N, 2.78.
4.20. 8-(4-Fluorobenzyl)-3-[2-bis(4-fluorophenyl)
methoxyethylidenyl]-8-azabicyclo[3.2.1]octane (22f)
Yellow oil (73 mg, 87%). 1H NMR: d7.37–7.25 (m, 6H), 7.06–6.98
(m, 6H), 5.58 (t, J = 6.8 Hz, 1H), 5.35 (s, 1H), 4.01–3.92 (m, 2H), 3.56

S. A. Cararas et al. / Bioorg. Med. Chem. 19 (2011) 7551–7558
7557
(s, 2H), 3.21 (br s, 2H), 2.59 (d, J = 13.6 Hz, 1H), 2.19–1.90 (m, 5H),
1.56 (m, 1H), 1.42 (t, J = 7.6 Hz, 1H). Anal. Calcd for C29H28F3NOꢁ-
C2H2O4: C, 67.26; H, 5.46; N, 2.53. Found: C, 67.44; H, 5.45; N, 2.56.
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
non-specific binding) using a Brandel Cell Harvester (Gaithersburg,
MD). After filtration, the filters were washed with three additional
3 mL washes and transferred to scintillation vials. Absolute ethanol
(0.5 mL) and Beckman Ready Value Scintillation Cocktail (2.75 mL)
were added to the vials which were counted the next day at an effi-
ciency of about 36%. Under these assay conditions, an average
experiment yielded approximately 6000 dpm total binding per
sample and approximately 250 dpm non-specific binding. Nonspe-
4.21. 3-(2-Diphenylmethoxyethylidenyl)-8-(4-chlorobenzyl)-8-
azabicyclo[3.2.1]-octane (21g)
Yellow oil (95 mg, 93%). 1H NMR: d 7.39–7.21 (m, 14H), 5.51 (br
s, 1H), 5.38 (s, 1H), 4.01–3.97 (m, 2H), 3.59–3.55 (m, 2H), 3.17 (br s,
2H), 2.56(m, 1H), 2.17–2.09 (m, 2H), 1.89 (m, 3H), 1.58–1.44 (m,
2H). Anal. Calcd for C32H30ClNOꢁC2H2O4ꢁH2O: C, 67.44; H, 6.21; N,
2.54. Found: C, 67.30; H, 5.98; N, 2.44.
cific binding was defined as binding in the presence of 100 lM co-
caine. Ki values were derived from 14 point competition assays
using increasing concentrations of unlabeled compounds (0.05
4.22. 8-(4-Chlorobenzyl)-3-[2-bis(4-fluorophenyl)
methoxyethylidene]-8-azabicyclo-[3.2.1]octane (22g)
nM to 100 l
M) against 1.5 nM [3H]WIN 35,428. Data were
analyzed with GraphPad Prism software (San Diego, California).
Yellow oil (56 mg, 74%). 1H NMR: d7.35–7.23 (m, 8H), 7.04–6.97
(m, 4H), 5.48 (t, J = 6.8 Hz, 1H), 5.35 (s, 1H), 4.01–3.91 (m, 2H), 3.56
(s, 2H), 3.19 (br s, 2H), 2.60–2.56 (d, J = 14.4 Hz, 1H), 2.20 (d,
J = 14 Hz, 1H), 2.10 (d, J = 14 Hz, 1H), 1.96–1.86 (m, 3H), 1.56 (m,
4.26. [3H]Citalopram binding assay
Brains from male Sprague-Dawley rats weighing 200–225 g
(Taconic Labs) were removed, midbrain dissected and rapidly fro-
zen. Membranes were prepared by homogenizing tissues in 25 vol-
umes (w/v) of 50 mM Tris containing 120 mM NaCl and 5 mM KCl,
(pH 7.4 at 25 °C), using a Brinkman Polytron (setting 6 for 20 s) and
centrifuged at 20,000g for 10 min at 4 °C. The resulting pellet was
resuspended in buffer, recentrifuged and resuspended in buffer to
a concentration of 7.5 mg/ml. Ligand binding experiments were
conducted in assay tubes containing 4.0 ml buffer for 60 min at
room temperature, essentially as previously described.39 Each tube
contained 1.4 nM [3H]citalopram (GE Healthcare) and 1.5 mg mid-
brain tissue (original wet weight). Nonspecific binding was deter-
1H), 1.42 (t, J = 8.8 Hz, 1H). Anal. Calcd for
C
29H28F2ClNOꢁ-
C2H2O4ꢁH2O: C, 63.32; H, 5.49; N, 2.38. Found: C, 63.70; H, 5.26;
N, 2.63.
4.23. 3-(2-Diphenylmethoxyethylidenyl)-8-(4-methylbenzyl)-8-
azabicyclo[3.2.1]-octane (21h)
Yellow oil (74 mg, 60%). 1H NMR: d 7.39–7.11 (m, 14H), 5.49 (t,
J = 6.4 Hz, 1H), 5.39 (s, 1H), 4.04–3.97 (m, 2H), 3.56 (s, 2H), 3.21 (br
s, 2H), 2.59 (d, J = 13.6 Hz, 1H), 2.33 (s, 3H), 2.23–2.19 (m, 1H),
2.11–2.08 (m, 1H), 1.93 (m, 3H), 1.57–1.43 (m, 2H). Anal. Calcd
for C30H33NOꢁC2H2O4ꢁH2O: C, 72.29; H, 7.01; N, 2.63. Found: C,
72.30; H 6.98; N 2.44.
mined using 1 lM citalopram. 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, Maryland). The filters were washed
twice with 5 ml cold buffer and transferred to scintillation vials.
Beckman 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, California). Data were
analyzed by using GraphPad Prism software (San Diego, California).
4.24. 3-[2-Bis(4-fluorophenyl)methoxyethylidenyl-8-(4-
methylbenzyl)-8-azabicyclo-[3.2.1]octane (22h)
Yellow oil (153 mg, 85%). 1H NMR: d 7.21–7.16 (m, 4H), 7.04 (d,
J = 8.0 Hz, 4H), 6.94–6.90 (m, 4H), 5.39 (t, J = 6.8 Hz, 1H), 5.27 (s,
1H), 3.90–3.86 (m, 2H), 3.49 (s, 2H) 3.16–3.13 (m, 2H), 2.54–2.50
(d, J = 16 Hz, 1H), 2.26 (s, 3H), 2.14 (d, J = 14 Hz, 1H), 2.01 (d,
J = 14 Hz, 1H), 1.86–1.83 (m, 3H), 1.46 (t, J = 8 Hz, 1H), 1.33 (t,
J = 8 Hz, 1H). Anal. Calcd for C30H31F2NOꢁC2H2O4ꢁH2O: C, 66.65; H,
6.29; N, 2.42. Found: C, 66.30; H 5.98; N 2.44.
4.27. [3H]Nisoxetine binding assay
Frontal cortex of male Sprague-Dawley rats was removed and
frozen. Membranes were prepared by homogenizing tissues in
50 mM Tris (120 mM NaCl, 5 mM KCl; pH 7.4 at 25 °C) and centri-
fuging (50,000g for 10 min at 4 °C. The resulting pellet was then
washed and centrifuged two more times. The final pellet was
resuspended to a concentration of 80 mg/ml (original wet weight).
Assays were conducted in the above Tris buffer, essentially as pre-
viously described.40 Volume totaled 0.5 mL with tissue concentra-
tion of 8 mg/tube. [3H]Nisoxetine (specific activity 80 Ci/mmol;
final concn 0.5 nM, New England Nuclear, Boston, MA) was added
and the incubation continued for 1 h on ice. Incubations were ter-
minated by rapid filtration through Whatman GF/B filters, presoa-
ked in 0.05% polyethylenimine (PEI). Nonspecific binding was
4.25. [3H]WIN 35,428 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 homog-
enized 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 glucose, pH adjusted to 7.4) using
a Teflon/glass homogenizer and centrifuged at 20,000g for 10 min
at 4 °C. The resulting pellet was then washed two more times by
resuspension in ice-cold buffer and centrifugation at 20,000g for
10 min at 4 °C. Fresh homogenates were used in all experiments.
Binding assays were conducted in modified Krebs-HEPES buffer
on ice, essentially as previously described.40 The total volume in
each tube was 0.5 mL and the final concentration of membrane
after all additions was approximately 0.3% (w/v) corresponding
defined using 1
was conducted to assess displacement of nisoxetine at a concentra-
tion of 1 M of the unknown compound. If there was greater than
lM desipramine. For these assays, an initial screen
l
50% displacement of nisoxetine, a Ki value was determined in sub-
sequent studies.
to 150–300
lg of protein/sample. Increasing concentrations of
Acknowledgments
the drug being tested were added to triplicate samples of mem-
brane suspension. Five minutes later, [3H]WIN 35,428 (final con-
centration 1.5 nM) was added and the incubation was continued
for 1 h on ice. The incubation was terminated by the addition of
This research was supported by the National Institute on Drug
Abuse, (MLT, DA11528 and DA023916) and by a Ruth L. Kirschstein
National Research Service Award (SAC, DA14155).

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S. A. Cararas et al. / Bioorg. Med. Chem. 19 (2011) 7551–7558
26. Prisinzano, T.; Rice, K. C. In Dopamine Transporters, Chemistry, Biology and
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