3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-substituted-3,6-diazabicyclo[ 3.1.1]heptanes as novel potent dopamine uptake inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.03.035

A series of analogues 2a-i related to 3-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-8-(1H-indol-2-ylmethyl)-3,8-dia zabicyclo[3.2.1]octane (1) in which the 3,8-diazabicyclo[3.2.1]octane core was replaced by 3,6-diazabicyclo[3.1.1]heptane ring system has been synthesized and evaluated for their ability to inhibit DA reuptake into striatal nerve endings (synaptosomes). Biological data showed that compound 2a, the closest analogue of lead 1, possessed an increased reuptake inhibition activity over 1 (2a, K_i = 5.5 nM). Replacement of the indole ring with bioisosteric aromatic rings-benzothiophene (2b), benzofurane (2c), or indene (2d)-resulted, with the exception of 2d, in a double digit nanomolar activity. Changing the indenyl moiety of 2d with simplified aryl groups led to compounds 2e-h which displayed a similar or slightly decreased activity with respect to the ground term. Naphthalene derivative (2i) demonstrated a weaker activity than aromatic analogues.

Full text of DOI:10.1016/j.bmc.2007.03.035

Bioorganic & Medicinal Chemistry 15 (2007) 3748–3755
3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-substituted-3,6-
diazabicyclo[3.1.1]heptanes as novel potent
dopamine uptake inhibitors
Giovanni Loriga,a Stefania Ruiu,b Ilaria Manca,a Gabriele Murineddu,a
a,
Christian Dessi,b Luca Panib,c and Gerard A. Pinna
*
´
a
`
Dipartimento Farmaco Chimico Tossicologico, Universita di Sassari, via F. Muroni 23/A, I-07100 Sassari, Italy
bNeuroscienze PharmaNess S.c.a r.l., Technological Park of Sardinia ‘Polaris’ Loc. Piscinamanna, I-09010 Pula, Cagliari, Italy
cC.N.R. Institute of Biomedical Technology, Sect Cagliari, Italy
Received 19 September 2006; revised 5 March 2007; accepted 13 March 2007
Available online 16 March 2007
Abstract—A series of analogues 2a–i related to 3-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-8-(1H-indol-2-ylmethyl)-3,8-diazabicy-
clo[3.2.1]octane (1) in which the 3,8-diazabicyclo[3.2.1]octane core was replaced by 3,6-diazabicyclo[3.1.1]heptane ring system has
been synthesized and evaluated for their ability to inhibit DA reuptake into striatal nerve endings (synaptosomes). Biological data
showed that compound 2a, the closest analogue of lead 1, possessed an increased reuptake inhibition activity over 1 (2a,
Ki = 5.5 nM). Replacement of the indole ring with bioisosteric aromatic rings—benzothiophene (2b), benzofurane (2c), or indene
(2d)—resulted, with the exception of 2d, in a double digit nanomolar activity. Changing the indenyl moiety of 2d with simplified
aryl groups led to compounds 2e–h which displayed a similar or slightly decreased activity with respect to the ground term.
Naphthalene derivative (2i) demonstrated a weaker activity than aromatic analogues.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Unlike the situation with other drugs of abuse, there are
currently no safe and effective pharmacotherapies avail-
able to treat cocaine abuse.
(ꢀ)-Cocaine is the chief component of Erythroxylum
coca leaves.1 Cocaine addiction is a chronic relapsing
brain disorder characterized by neurobiological changes
leading to the illicit use of cocaine which secondly corre-
lates with exacerbation of the spread of AIDS, hepatitis
B and C, and drug resistant tuberculosis.2
From the pharmacological perspective, two approaches
of medications to aid cessation in the cocaine use have
been pursued.
One approach is the so-called cocaine replacement ther-
apy (CRT) or maintenance pharmacotherapy which
provides a medication that would have pharmacological
similarity to cocaine with the purpose to reduce the
cocaine-withdrawal symptoms and the craving for
cocaine use. For a replacement therapy to be beneficial
to treat cocaine dependence, however, it should be less
medically deleterious than the drug of abuse that it is
replacing. This goal may be obtained using agents with
a proper pharmacokinetic profile that surrogate the
supply of low and constant levels of cocaine.
Cocaine acts as an indirect dopamine agonist by block-
ing the dopamine transporter in the pleasure/reward
center of the brain3 and this blockade results in an
excess of dopamine in the synapses. The resulting
increase in synaptic dopamine levels produce an ampli-
fication of pleasure sensation such as euphoria, psych-
ostimulation, and other rewarding experiences that can
lead to drug addiction.4
Keywords: Cocaine abuse treatment; Synthesis of 3,6-diazabicy-
clo[3.1.1]heptanes; DAT inhibitors.
The second approach is that to accomplish the pharma-
cological antagonism by means of a dopamine receptor
*
Corresponding author. Tel.: +39 079228721; fax: +39 079228720;
e-mail: pinger@uniss.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.035

G. Loriga et al. / Bioorg. Med. Chem. 15 (2007) 3748–3755
3749
antagonist or partial agonist with the purpose to reduce
rewarding effect of cocaine use.
ciently flexible to permit a variety of conformations with
a more rigid 3,6-diazabicyclo[3.1.1]heptane scaffold.
This backbone-rigidification would be expected to influ-
ence bioactivity profoundly. A second objective of this
study was to seek correlations between steric properties
of N6 pendant groups and DAT reuptake inhibitory
activity.8,9
From a preclinical point of view two major classes of
therapeutic agents have been studied such as (1)
dopaminergic agents and (2) antidepressants. Other
category of compounds such as calcium channel
blockers and immune response inducers have also
been examined as potential cocaine abuse therapeutic
agents.5,6
In this context, we chose to synthesize the 3,6-diazabicy-
clo[3.1.1]heptane analogues of 1 (2a–i) bearing different
groups on the N6 amine nitrogen with the 2-[bis-(4-fluor-
ophenyl)methoxy]ethyl group at N3 and to evaluate
their DA transporter activity (Chart 1).
For all of these reasons, immediate therapies with non-
addictive and nonabusable agents are needed for the
treatment of cocaine abuse worldwide.
In the pursuit of possible medications we have focused
our attention on DA uptake blockers having original
chemical scaffolds in the belief that novel molecular
architectures may lead to desired insights into DAT
inhibitor activity.
2. Chemistry
The starting material available for the preparation of
compounds 2a–i in Chart 1, that is the 6-tert-butyloxy-
carbonyl-3,6-diazabicyclo[3.1.1]heptane (3, Scheme 1),
was synthesized as reported by us before.10
Recently, K.C. Rice and co-workers7 have introduced
the 3,8-diazabicyclo[3.2.1]octane system as new scaffold
in DAT inhibitor field (area) embedded in the struc-
ture of 3-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-8-(1H-
indol-2-ylmethyl)-3,8-diazabicyclo[3.2.1]octane (1)7
(Chart 1) which manifests considerable DAT affinity
(IC50 = 1.4 0.1 nM) and high uptake inhibition potency
(IC50 = 40 7 nM).
The N3 atom in the diazabicyclo[3.1.1]heptane nucleus
of compound 3 was alkylated by reacting with 2-[bis-
(4-fluorophenyl)methoxy]ethyl iodide (4)7 in the pres-
ence of a base to give compound 5 in good yield (see
Scheme 1).
Deprotection of the carbamic nitrogen atom of 5 by
removal of its N6-Boc group, under mild acidic
conditions, produced the desired 3-{2-[bis-(4-fluorophe-
nyl)methoxy]ethyl}-3,6-diazabicyclo[3.1.1]heptane (6).
The first objective of the present study was to replace the
3,8-diazabicyclo[3.2.1]octane template of 1 still suffi-
F
F
O
O
N
N
N
N
R
N
H
2a-i
1
F
F
2
a
R
2
f
R
N
H
S
b
c
g
S
O
h
O
d
i
e
CH3
Chart 1. Molecular structures of the lead 1 and the designed compounds 2a–i.

3750
G. Loriga et al. / Bioorg. Med. Chem. 15 (2007) 3748–3755
I
F
O
F
F
O
4
N
NH
BocN
BocN
a
5
3
F
b
F
O
N
HN
6
F
Scheme 1. Reagents and conditions: (a) K2CO3, 4-methylpentan-2-one, 115 ꢁC, 17 h; (b) HCO2H, room temperature, 4.5 h.
The availability of key intermediate 6 allowed the syn-
thesis of the designed compounds 2a–i.
The highest activity is shown by the ligand 2a having
the indol-2-yl-methyl unit linked to N6 of the N3-bis-
(4-fluorophenyl)-methoxyethyl-3,6-diazabicyclo[3.1.1]hep-
tane scaffold, with a Ki of 5.5 nM, more potent than
compound 1. Compound 2a serves as a convenient
benchmark for all of the others in terms of presenting
structure–activity relationship. Compounds 2b–d have
bioisosteric bicyclic units such as benzothiophene
(2b), benzofurane (2c), or indene (2d) in place of the
indole nucleus. These compounds, with the exception
of 2d, displayed a reduced activity, with Ki values of
35 and 37.5 nM, respectively, suggesting that the
nature of the bicyclic unit in these ligands has interest-
ing but unpredictable effects on dopamine reuptake
activity.
In particular, the compounds 2a–c, g, h were synthesized
by a two-step process starting from 6 as shown in
Scheme 2. The reaction at room temperature between
the diamine 6 and the appropriate carboxylic acids com-
mercially available 7a–c and 9g, h, in the presence of an
drying agent as N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide (EDCI), allowed us to obtain the amides
8a–c and 10g, h, respectively. Thus, the intermediate
amides underwent metal hydride reduction to afford
the target amines 2a–c, g, h.
Finally, the remaining designed compounds 2d–f, i
were synthesized by direct alkylation of 6 with the
appropriate aldehydes 11d,11 11e,12 11f,€ and 11i,13
under reductive alkylation conditions with sodium
cyanoborohydride and a catalytic amount of acetic acid
(Scheme 2).
Compound 2e, which contains a o-methylstiryl moiety
obtained simply disconnecting the C1–C2 bond of the
indenyl unit, maintained an activity that is comparable
to that of 2a.
All compounds were converted into HCl or fumarate
salts for biological evaluation.
Elimination of the methyl group of 2e gave compound
2f which showed a slight reduction of activity with
respect to the ground term (2f vs 2a).
3. Results and discussion
Replacement of the phenyl group of 2f with a thiophene
(2g) or furane (2h) ring resulted in slightly reduced activ-
ity (2g and 2h vs 2a). Naphthalene derivative (2i) exhib-
ited only a modest activity (Ki = 45.6 nM).
The ability to inhibit reuptake of [3H]DA into striatal
nerve ending (synaptosomes) of novel ligands 2 pre-
pared in this study was determined using protocols
described previously.14 The reuptake values are listed
in Table 1. For comparison purposes, data for lead
compound 1, expressed as IC50 value, and cocaine are
also reported using those extrapolated from previous
papers.7,14
On the basis of the SAR that has emerged, we conclude
that the N3-difluoro-benzhydryloxyethyl-3,6-diazabi-
cyclo[3.1.1]heptane play an important role in biological
activity and could be viewed as a novel chemical
template in DAT inhibitor design.
As shown in Table 1, in general, the majority of com-
pounds show very high potency in inhibiting DA uptake
in comparison with lead compound 1.
In this new series, moreover, five compounds, 2a, 2d, 2e,
2f, and 2h, were the most potent derivatives with Ki val-
ues ranging from 5.5 to 9.1 nM.

G. Loriga et al. / Bioorg. Med. Chem. 15 (2007) 3748–3755
3751
Q
N
Q
N
Q
a
b
N
OH
N
+
N
HN
X
X
X
O
O
6
2a, X = N
2b, X = S
2c, X = O
7a, X = N
7b, X = S
7c, X = O
8a, X = N
8b, X = S
8c, X = O
Q
Q
N
c
N
a
OH
+
Y
N
O
6
Y
N
Y
O
9g, Y = S
9h, Y = O
10g, Y = S
10h, Y = O
2g, Y = S
2h, Y = O
Q
O
d
N
+
6
R
N
R
H
11d-f,i
2d-f,i
2, 11
R
d
e
F
O
CH3
Q =
f
i
F
Scheme 2. Reagents and conditions: (a) EDCI, CH2Cl2, room temperature, 20 h; (b) LAH, THF, room temperature, 14 h; (c) DIBALH, THF, 67 ꢁC,
2 h; (d) NaCNBH3, CH3OH, CH3COOH, room temperature, 20 h.
Finally, we believe that the knowledge gained from this
study will facilitate the design of new candidates for safe
and effective treatment of cocaine abuse.
Flash chromatography (FC) was performed using
Merck silica gel 60 (230–400 mesh ASTM).
IR spectra were recorded as thin films (for oils) or
Nujol mulls (for solids) on NaCl plates with a Jasco
FT/IR 460 plus spectrophotometer and are expressed
in m (cmꢀ1).
Encouraged by these results we will continue to focus
our efforts in further exploration of this novel class of
DAT inhibitors.
All NMR spectra were taken on a Varian XL-200 NMR
spectrometer with 1H and 13C being observed at 200 and
50 MHz, respectively. Chemical shifts for H and spec-
tra were reported in d or ppm downfield from TMS
[(CH3)4Si]. Multiplicities are recorded as s (singlet), br
s (broad singlet), d (doublet), t (triplet), dd (double dou-
blet), dt (double triplet), q (quartet), m (multiplet).
1
4. Experimental
4.1. Chemistry
General informations. Melting points were obtained on
an Electrothermal IA 9100 digital melting point appara-
tus or on a Kopfler melting point apparatus and are
uncorrected.
Elemental analyses were performed by Laboratorio di
`
Microanalisi,DipartimentodiChimica,UniversitadiSas-
sari, Italy, and are within 0.4% of the calculated values.
Thin layer chromatography (TLC) was performed with
Polygramꢂ SIL N-HR/HV254 precoated plastic sheets
(0.2 mm).
All reactions involving air- or moisture-sensitive com-
pounds were performed under argon atmosphere.

3752
G. Loriga et al. / Bioorg. Med. Chem. 15 (2007) 3748–3755
Table 1. Inhibition of reuptake at DAT, Kia (nM)
solution of fumaric acid in dry methanol to a solution
of the compound in dry methanol. The solvent was
evaporated and the resulting salt was triturated with
anhydrous ether and dried under vacuum.
F
O
N
R
N
Unless otherwise specified, all materials, solvents,
reagents, and precursors 7a–c, 9g, h, and 11f were
obtained from commercial suppliers.
2a-i
F
Compoundb
R
Ki uptake (nM)
4.2. 3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-tert-
butyloxycarbonyl-3,6-diazabicyclo[3.1.1] heptane (5)
2a
5.5 0.9
A mixture of 310 (1.30 g, 6.56 mmol), 2-[bis-(4-fluor-
ophenyl)methoxy]ethyl iodide (4)7 (3.09 g, 8.26 mmol),
and K2CO3 (1.81 g, 13.12 mmol) in 4-methylpentan-2-
one (55 mL) was stirred at reflux for 17 h. The solvent
was then evaporated, the residue was taken up in
CHCl3, and the inorganic salt was filtered off. The fil-
trate was concentrated and the residue was purified by
FC eluting with petroleum ether/EtOAc 7:3, to afford
1.96 g (67%) of 5 as yellow oil: Rf 0.29 (petroleum
ether/EtOAc 7:3); bp 172 ꢁC/0.1 mmHg; IR: 1600,
N
H
2b
2c
2d
2e
2f
35 5.7
37.5 5.9
6.5 1.2
6.25 1.1
9.1 1.2
12.6 1.9
8.8 1.6
45.6 2.3
40 7c
S
O
1
1710; H NMR (CDCl3) d 1.42 (s, 9H), 1.63 (d, 1H,
J = 7.4 Hz), 2.30–2.43 (m, 1H), 2.79–2.90 (m, 4H),
3.00–3.23 (m, 2H), 3.53 (t, 2H, J = 6.0 Hz), 4.00–4.09
(m, 2H), 5.32 (s, 1H), 6.96–7.05 (m, 4H), 7.22–7.35 (m,
4H). Anal (C25H30F2N2O3) C, H, N.
CH3
4.3. 3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-3,6-diaza-
bicyclo[3.1.1]heptane (6)
A solution of 5 (0.65 g, 1.46 mmol) in 95% formic acid
(25 mL) was stirred at room temperature for 4.5 h.
The mixture was made alkaline with a 10% aqueous
solution of NaOH and extracted with Et2O. The organic
layers were dried over Na2SO4 and concentrated to
afford 0.28 g (56%) of 6 as yellow oil: Rf 0.27 (CHCl3:
S
2g
2h
2i
O
1
MeOH 9:1); bp 198 ꢁC/0.1 mmHg; IR: 1610, 3250; H
NMR (CDCl3) d 1.85 (d, 1H, J = 7.8 Hz), 2.40–2.55
(m, 1H), 2.72 (d, 2H, J = 10.6 Hz), 2.86 (t, 2H,
J = 5.6 Hz), 3.13 (d, 2H, J = 10.8 Hz), 3.55–3.63
(m, 4H), 5.35 (s, 1H), 6.95–7.08 (m, 4H), 7.22–7.35
(m, 4H). Anal (C20H22F2N2O) C, H, N.
1
(ꢀ)-Cocaine
111
5
4.4. General procedures for the preparation of 8a–c and
10g, h
a Results are means SEM for three independent experiments.
b The inhibition of reuptake experiments of all compounds were
carried out on their hydrochloride (2a) or fumarate (2b–i) salts.
c Expressed as IC50 value.
A solution of the appropriate carboxylic acid 7a–c, 9g, h
(0.73 mmol) and N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide (EDCI) (0.17 g, 0.87 mmol) in CH2Cl2
(10 ml) was allowed to stir at room temperature for
5 min before a solution of 6 (0.20 g, 0.58 mmol) in
CH2Cl2 (9 ml) was added. The reaction mixture was stir-
red at room temperature for 20 h, then washed with sat-
urated aqueous NaHCO3, H2O, and brine. The organic
layers were dried over Na2SO4 and concentrated. The
crude product was purified by FC to afford the com-
pounds 8a–c and 10g, h.
All final compounds were converted into the HCl or
HO2CCH@CHCO2H salts.
The general procedure for conversion to an HCl salt
was the addition of excess ethereal HCl solution to a
solution of the compound in ethanol or diethyl ether.
The solvent was evaporated and the resulting salt was
triturated with anhydrous ether and dried under
vacuum.
4.4.1. (3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-3,6-diaz-
abicyclo[3.1.1]heptan-6-yl)(1H-indol-2-yl)methanone (8a).
Purified by FC (eluent: Et2O); yield 85%; Rf 0.42
The general procedure for conversion to a fumarate salt
was the addition of a stoichiometric amount of a

G. Loriga et al. / Bioorg. Med. Chem. 15 (2007) 3748–3755
3753
(Et2O); mp 136–138 ꢁC (Et2O); IR: 1610, 1640, 3300; 1H
NMR (CDCl3) d 2.09 (d, 1H, J = 8.2 Hz), 2.60–2.72 (m,
1H), 2.77–2.87 (m, 2H), 2.96–3.36 (m, 4H), 3.45–3.55
(m, 2H), 4.55–4.67 (m, 1H), 4.77–4.87 (m, 1H), 5.25 (s,
1H), 6.71 (d, 1H, J = 1.2 Hz), 6.85–7.00 (m, 4H), 7.10–
7.43 (m, 7H), 7.65 (d, 1H, J = 8.0 Hz), 9.17 (br s, 1H);
13C NMR (CDCl3) d 28.97, 52.28, 54.25, 55.15, 59.95,
63.91, 66.73, 82.53, 104.33, 111.82, 115.03, 115.45,
120.60, 121.97, 124.61, 128.01, 128.37, 128.53, 129.83,
135.35, 137.75, 137.82, 159.64, 161.72, 164.53. Anal
(C29H27F2N3O2) C, H, N.
0 ꢁC, diethyl ether (40 mL) was added, and the reaction
quenched with H2O (0.37 mL), 2 M aqueous NaOH
(0.37 mL), and H2O (1.11 mL). The mixture was filtered
and evaporated to give pure 2a–c.
4.5.1. 3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-[(1H-
indol-2-yl)methyl]-3,6-diazabicyclo [3.1.1]heptane (2a).
Yield 86%; Rf 0.51 (CHCl3/MeOH 9.6:0.4); mp 108–
1
110 ꢁC (as dihydrochloride); IR: 1600, 3320; H NMR
(CDCl3) d 1.94 (d, 1H, J = 7.8 Hz), 2.40–2.50 (m, 1H),
2.88 (d, 2H, J = 10.8 Hz), 2.97 (t, 2H, J = 5.8 Hz), 3.13
(d, 2H, J = 11.4 Hz), 3.45–3.55 (m, 2H), 3.60 (t, 2H,
J = 5.8 Hz), 3.77 (s, 2H), 5.37 (s, 1H), 6.23 (s, 1H),
6.95–7.17 (m, 6H), 7.22–7.38 (m, 5H), 7.52 (d, 1H,
J = 7.6 Hz), 8.72 (br s, 1H); 13C NMR (CDCl3) d (as
dihydrochloride) 34.99, 42.26, 51.36, 53.77, 56.72,
60.08, 66.65, 82.54, 105.23, 111.88, 115.09, 115.52,
120.04, 120.44, 123.03, 126.77, 127.00, 128.39, 128.55,
137.09, 137.36, 141.47, 159.60, 164.49. Anal
(C29H29F2N3O) C, H, N.
4.4.2. (Benzo[b]thiophen-2-yl)(3-{2-[bis-(4-fluorophenyl)
methoxy]ethyl}-3,6-diazabicyclo [3.1.1]heptan-6-yl)meth-
anone (8b). Purified by FC (eluent: Et2O); yield 51%; Rf
0.29 (Et2O); IR: 1600, 1650; 1H NMR (CDCl3) d2.06 (d,
1H, J = 8.0 Hz), 2.58–2.68 (m, 1H), 2.80–2.92 (m, 2H),
2.95–3.18 (m, 3H), 3.30–3.58 (m, 3H), 4.53–4.62 (m,
1H), 4.75–4.86 (m, 1H), 5.27 (s, 1H), 6.85–7.00 (m, 4H),
7.15–7.26 (m, 4H), 7.36–7.44 (m, 2H), 7.72 (s, 1H),
7.78–7.89 (m, 2H). Anal (C29H26F2N2O2S) C, H, N.
4.5.2. 6-[(Benzo[b]thiophen-2-yl)methyl]-3-{2-[bis-(4-flu-
orophenyl)methoxy]ethyl}-3,6-diaza bicyclo[3.1.1]heptane
(2b). Yield 81%; Rf 0.21 (Et2O); mp 116–118 ꢁC (as
difumarate); IR: 1603; 1H NMR (CDCl3) d 1.84 (d,
1H, J = 7.8 Hz), 2.32–2.48 (m, 1H), 2.75–3.08 (m, 4H),
3.04 (d, 2H, J = 11.2 Hz), 3.45–3.60 (m, 4H), 3.77 (s,
2H), 5.28 (s, 1H), 5.66 (s, 1H), 6.84–7.00 (m, 4H),
7.10–7.33 (m, 6H), 7.52–7.73 (m, 2H); 13C NMR
(CDCl3) d 28.47, 30.23, 44.88, 48.55, 55.03, 59.50,
66.95, 74.49, 82.51, 115.01, 115.43, 120.94, 122.16,
122.89, 123.61, 123.97, 126.37, 126.81, 127.95, 128.11,
128.38, 128.54, 137.88, 139.60, 143.14, 159.60, 164.49.
Anal (C29H28F2N2OS) C, H, N.
4.4.3. (Benzofuran-2-yl)(3-{2-[bis-(4-fluorophenyl)meth-
oxy]ethyl}-3,6-diazabicyclo[3.1.1]heptane-6-yl)methanone
(8c). Purified by FC (eluent: Et2O); yield 60%; Rf 0.49
1
(Et2O); IR: 1620, 1640; H NMR (CDCl3) d 2.12 (d,
1H, J = 7.8 Hz), 2.58–2.70 (m, 1H), 2.80–2.90 (m, 2H),
3.00–3.40 (m, 4H), 3.42–3.60 (m, 2H), 4.55–4.65 (m,
1H), 5.02–5.12 (m, 1H), 5.27 (s, 1H), 6.85–7.00 (m,
4H), 7.15–7.55 (m, 8H), 7.66 (d, 1H, J = 7.6 Hz). Anal
(C29H26F2N2O3) C, H, N.
4.4.4.
(E)-1-(3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-
3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-(thiophen-2-yl)prop-
2-en-1-one (10g). Purified by FC (eluent: EtOAc/Et2O
6:4); yield 54%; Rf 0.23 (EtOAc/Et2O 1:1); IR: 1600,
1650; 1H NMR (CDCl3) d2.08 (d, 1H, J = 8.0 Hz),
2.42–2.58 (m, 1H), 2.80–2.88 (m, 2H), 2.90–3.28 (m,
4H), 3.49–3.54 (m, 2H), 4.40–4.50 (m, 2H), 5.29 (s,
1H), 6.22 (d, 1H, J = 15.2 Hz), 6.90–7.06 (m, 4H),
7.15–7.37 (m, 7H), 7.75 (d, 1H, J = 15.4 Hz). Anal
(C27H26F2N2O2S) C, H, N.
4.5.3. 6-[(Benzofuran-2-yl)methyl]-3-{2-[bis-(4-fluorophe-
nyl)methoxy]ethyl}-3,6-diazabicyclo [3.1.1]heptane (2c).
Yield 62%; Rf 0.63 (CHCl31/MeOH 9:1); mp 104–106 ꢁC
(as difumarate); IR: 1610; H NMR (CDCl3) d 1.91 (d,
1H, J = 8.4 Hz), 2.45–2.60 (m, 1H), 2.85–3.02 (m, 4H),
3.19 (d, 2H, J = 10.8 Hz), 3.57–3.70 (m, 4H), 3.74 (s,
2H), 5.37 (s, 1H), 6.47 (s, 1H), 6.95–7.08 (m, 4H),
7.17–7.52 (m, 8H); 13C NMR (CDCl3) d (as difumarate)
28.54, 31.20, 41.83, 47.68, 56.13, 60.36, 65.43, 67.35,
81.27, 113.98, 114.41, 119.94, 121.91, 123.10, 123.40,
127.40, 127.56, 131.03, 133.42, 135.38, 136.85, 153.41,
158.42, 163.30. Anal (C29H28F2N2O2) C, H, N.
4.4.5.
(E)-1-(3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-
3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-(furan-2-yl)prop-2-
en-1-one (10h). Purified by FC (eluent: EtOAc/Et2O
3.5:6.5); yield 44%; Rf 0.29 (EtOAc/Et2O 3.5:6.5); IR:
1610, 1660; 1H NMR (CDCl3) d1.99 (d, 1H,
J = 8.0 Hz), 2.35–2.46 (m, 1H), 2.76 (t, 2H, J = 5.4 Hz),
2.93 (d, 2H, J = 10.8 Hz), 3.16 (d, 2H, J = 11.0 Hz),
3.44 (t, 2H, J = 5.4 Hz), 4.37 (d, 2H, J = 5.8 Hz), 5.21
(s, 1H), 6.25 (d, 1H, J = 15.4 Hz), 6.41 (dd, 1H,
J = 15.4 and 3.2 Hz), 6.80–6.98 (m, 4H), 7.08–7.20 (m,
6H), 7.36 (s, 1H). Anal (C27H26F2N2O3) C, H, N.
4.6. General procedures for the preparation of 2g, h
The appropriate amide 10g, h (0.75 mmol) in dry THF
(9 mL) was treated under argon with 1.5 M DIBALH
(2.02 mL, 3.03 mmol) in toluene at room temperature.
The mixture was stirred at reflux for 2 h, then cooled
at room temperature, quenched by dropwise addition
of H2O (60 mL), and extracted with CHCl3 (3· 30
mL). The organic layers were separated, dried over
Na2SO4, and concentrated. The crude product was puri-
fied by FC to afford the amines 2g, h.
4.5. General procedures for the preparation of 2a–c
To a suspension of lithium aluminum hydride (0.37 g,
9.84 mmol) in dry THF (40 mL) at 0 ꢁC under a nitro-
gen atmosphere was added
a solution of 8a–c
(2.46 mmol) in dry THF (40 mL). The mixture was
allowed to slowly reach room temperature and was then
stirred overnight. The reaction mixture was cooled to
4.6.1. 3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-[(E)-3-
thiophen-2-ylallyl]-3,6-diazabicyclo[3.1.1]heptane (2g). Puri-
fied by FC (eluent: CHCl3/MeOH 9:1); yield 54%; Rf 0.51

3754
G. Loriga et al. / Bioorg. Med. Chem. 15 (2007) 3748–3755
(CHCl3: MeOH 9.2:0.8); mp 109–111 ꢁC (as difumarate);
82.56, 115.09, 115.52, 123.64, 125.85, 126.16, 127.84,
128.40, 128.55, 130.24, 132.38, 135.27, 137.72, 137.77,
159.67, 164.56. Anal (C30H32F2N2O) C, H, N.
1
IR: 1605; H NMR (CDCl3) d 1.95 (d, 1H, J = 7.4 Hz),
2.45–2.63 (m, 1H), 2.90–3.00 (m, 4H), 3.06 (d, 2H,
J = 11.0 Hz), 3.10–3.22 (m, 2H), 3.53–3.68 (m, 4H), 5.36
(s, 1H), 6.00 (dt, 1H, J = 15.4 and 6.4 Hz), 6.62 (d, 1H,
J = 15.4 Hz), 6.90–7.38 (m, 11H); 13C NMR (CDCl3) d
28.43, 47.21, 48.73, 54.94, 60.02, 67.05, 82.56, 115.09,
115.52, 124.19, 125.36, 127.27, 128.43, 128.59, 137.83,
159.69, 164.58. Anal (C27H28F2N2OS) C, H, N.
4.7.3.
3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-cinn-
amyl-3,6-diazabicyclo[3.1.1]heptane (2f). Purified by FC
(eluent: CHCl3/MeOH 9.5:0.5); yield 45%; Rf 0.35
(CHCl3/MeOH 9.5:0.5); mp 123–125 ꢁC (as difumarate);
1
IR: 1610; H NMR (CDCl3) d 2.01 (d, 1H, J = 7.8 Hz),
2.50–2.62 (m, 1H), 2.90–3.00 (m, 4H), 3.15 (d, 2H,
J = 11.2 Hz), 3.30 (d, 2H, J = 5.4 Hz), 3.60 (t, 2H,
J = 5.6 Hz), 3.65–3.73 (m, 2H), 5.37 (s, 1H), 6.21 (dt,
1H, J = 16.0 and 5.4 Hz), 6.52 (d, 1H, J = 16.0 Hz),
6.95–7.08 (m, 4H), 7.20–7.38 (m, 9H); 13C NMR
(CDCl3) d 28.35, 48.66, 54.92, 59.95, 67.01, 82.50,
115.03, 115.45, 124.91, 126.22, 127.47, 128.39, 128.46,
128.55, 132.59, 136.70, 137.81, 137.87, 159.62, 164.51.
Anal (C29H30F2N2O) C, H, N.
4.6.2. 3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-[(E)-3-
furan-2-ylallyl]-3,6-diazabicyclo [3.1.1]heptane (2h). Puri-
fied by FC (eluent: CHCl3/MeOH 9.2:0.8); yield 43%; Rf
0.31 (CHCl3/MeOH 9.2:0.8); mp 110–112 ꢁC (as difum-
arate); IR: 1600; 1H NMR (CDCl3) d 1.91 (d, 1H,
J = 8.4 Hz), 2.52–2.65 (m, 1H), 2.90–3.02 (m, 4H), 3.15
(d, 2H, J = 11.4 Hz), 3.25–3.35 (m, 2H), 3.59 (t, 2H,
J = 5.8 Hz), 3.70 (d, 2H, J = 6.0 Hz), 5.36 (s, 1H), 6.17
(dt, 1H, J = 14.0 and 5.2 Hz), 6.37 (d, 1H,
J = 14.0 Hz), 6.95–7.07 (m, 4H), 7.22–7.36 (m, 7H);
13C NMR (CDCl3) d 28.62, 46.80, 48.59, 55.05, 59.64,
67.07, 82.55, 107.19, 111.15, 115.08, 115.50, 120.46,
124.40, 128.44, 128.60, 130.86, 137.87, 141.74, 159.69,
164.57. Anal (C27H28F2N2O2) C, H, N.
4.7.4. 3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-[(E)-3-
(naphthalen-2-yl)allyl]-3,6-diazabicyclo[3.1.1]heptane (2i).
Purified by FC (eluent: CHCl3/MeOH 9.6:0.4); yield
57%; Rf 0.34 (CHCl3/MeOH 9.6:0.4); mp 116–118 ꢁC
(as difumarate); IR: 1605; 1H NMR (CDCl3) d 2.01
(d, 1H, J = 7.4 Hz), 2.48–2.62 (m, 1H), 2.85–3.03 (m,
4H), 3.16 (d, 2H, J = 11.2 Hz), 3.33 (d, 2H,
J = 5.6 Hz), 3.55–3.71 (m, 4H), 5.38 (s, 1H), 6.32
(dt, 1H, J = 16.2 and 5.6 Hz), 6.67 (d, 1H,
J = 16.2 Hz), 6.95–7.07 (m, 4H), 7.22–7.36 (m, 4H),
7.38–7.45 (m, 2H), 7.57 (d, 1H, J = 8.8 Hz), 7.66 (s,
1H), 7.70–7.82 (m, 3H); 13C NMR (CDCl3) d 28.59,
47.54, 48.75, 55.08, 59.86, 67.10, 82.57, 115.09,
115.52, 123.44, 125.76, 126.09, 126.21, 127.61,
127.90, 128.15, 128.46, 128.63, 132.32, 132.90,
133.54, 134.37, 137.89, 137.95, 159.70, 164.59. Anal
(C33H32F2N2O) C, H, N.
4.7. General procedures for the preparation of 2d–f, i
A solution of 6 (0.20 g, 0.58 mmol) and the required
aldehydes 11d,11 11e,12 11f, and 11i13 (0.70 mmol) in
MeOH (9 mL) was treated with few drops of acetic acid,
followed by NaCNBH3 (51 mg, 0.81 mmol). The solu-
tion was stirred at room temperature for 20 h, and then
the solvent was evaporated. The residue was dissolved in
5 mL of 1 N NH4OH solution and extracted with
diethyl ether. The organic layer was dried over Na2SO4,
evaporated, and the residue was purified by FC to afford
the amines 2d–f, i.
4.8. Biology: materials and methods
4.7.1. 3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-[(1H-
inden-2-yl)methyl]-3,6-diazabicyclo [3.1.1]heptane (2d).
Purified by FC (eluent: CHCl3/MeOH 9.5:0.5); yield
46%; Rf 0.47 (CHCl3/MeOH 9.5:0.5); mp 99–101 ꢁC
(as difumarate); IR: 1604; 1H NMR (CDCl3) d 2.03
(d, 1H, J = 5.4 Hz), 2.55–2.70 (m, 1H), 2.90–3.08 (m,
4H), 3.12–3.28 (m, 2H), 3.41 (s, 2H), 3.55–3.68 (m,
4H), 3.70–3.77 (m, 2H), 5.37 (s, 1H), 6.68 (s, 1H),
6.94–7.08 (m, 4H), 7.10–7.43 (m, 8H); 13C NMR
(CDCl3) d 28.18, 40.55, 48.68, 54.86, 60.33, 67.02,
82.59, 115.11, 115.54, 120.49, 123.64, 124.42, 126.31,
128.43, 128.59, 129.70, 137.81, 141.53, 143.35, 144.49,
159.70, 164.59. Anal (C30H30F2N2O) C, H, N.
4.8.1. Chemicals. [3H]DA (specific activity 60 Ci/mmol)
was obtained from New England Nuclear (Boston,
MA, USA). For uptake experiments, drugs were dis-
solved in DMSO. DMSO concentration in the different
assays never exceeded 0.1% (v/v) and was without effect
on uptake.
4.8.2. Animals. Male Sprague–Dawley albino rats
weighting 150–200 g (Charles River, Calco, LC, Italy)
were housed three or four per cage and given free access
to food and water under controlled conditions of tem-
perature (22 1 ꢁC) and humidity (65 5%) with a
12 h light/dark cycle. All experimental procedures were
performed in strict accordance with the EC regulation
for care and use of experimental animals (EEC Nꢁ86/
609).
4.7.2. 3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-[3-(2-
methylphenyl)prop-2-enyl]-3,6-diazabicyclo[3.1.1]heptane
(2e). Purified by FC (eluent: CHCl3/MeOH 9.5:0.5);
yield 62%; Rf 0.36 (CHCl3/MeOH 9.5:0.5); mp 104–
1
107 ꢁC (as difumarate); IR: 1609; H NMR (CDCl3) d
4.8.3. Tissue preparation. Rats were killed by decapita-
tion and the brains were rapidly removed and placed
on an ice-cold plate. The striatum was quickly dissected
over ice and homogenized using a Teflon-glass homoge-
nizer (clearance approximately 0.009 in.) with 10 up-
and-down strokes at 850 rpm in 10 volumes (w/v) of
ice-cold 0.32 M sucrose containing 10 mM glucose and
2.05–2.20 (m, 1H), 2.32 (s, 3H), 2.67–2.82 (m, 1H),
2.98–3.14 (m, 4H), 3.22–3.38 (m, 2H), 3.40–3.55 (m,
2H), 3.61 (t, 2H, J = 5.6 Hz), 3.80–3.88 (m, 2H), 5.37
(s, 1H), 6.03–6.22 (m, 1H), 6.82 (d, 1H, J = 16.2 Hz),
6.95–7.05 (m, 4H), 7.10–7.47 (m, 8H); 13C NMR
(CDCl3) d 19.81, 27.70, 48.98, 54.62, 60.93, 66.99,

G. Loriga et al. / Bioorg. Med. Chem. 15 (2007) 3748–3755
3755
10 mM Tris (pH 7.4). The homogenate was then centri-
fuged at 1000g for 10 min at 4 ꢁC and the resulting
supernatant centrifuged at 12,000g for 20 min at 4 ꢁC
to obtain a crude synaptosomal fraction (P2). The syn-
aptosomes were resuspended in the assay buffer immedi-
ately before use.
curves and converted to Ki values as previously
described.16
References and notes
1. Dewick, Paul M. In Medicinal Natural Products-A
Biosynthetic Approach, Second ed.; John Wiley, Inc.:
New York, USA, 2002; pp 302–304.
2. Hser, Y. I.; Chou, C. P.; Hoffman, V.; Anglin, M. D. Sex.
Transm. Dis. 1999, 26, 82.
3. Ritz, M. C.; Lamb, R. C.; Goldberg, S. R.; Kuhar, M. J.
Science 1987, 237, 1219.
4. Withers, N. W.; Pulvirenti, L.; Koob, G. F.; Gillin, J. C.
J. Clin. Psychopharmacol. 1995, 15, 63.
5. Carrera, M. R. A.; Meijler, M. M.; Janda, K. D. Bioorg.
Med. Chem. 2004, 12, 5019.
4.8.4. Assay of [3H]DA uptake. The synaptosomes-con-
taining pellet was resuspended in a modified Krebs–
Henseleit buffer and assayed according to Schoemaker
and Nickolson.15 The standard uptake medium (pH
7.4 a 37 ꢁC) contained 140 mM NaCl, 5 mM KCl,
1 mM MgCl2, 10 mM glucose, 1.2 mM CaCl2, 10 mM
Hepes, 1 mM ascorbic acid, 0.054 mM EDTA, and
0.1 mM pargyline.
6. Newman, A. H.; Grundt, P.; Nader, M. A. J. Med. Chem.
2005, 48, 3663.
7. Zhang, Y.; Rothman, R. B.; Dersch, C. M.; de Costa, B.
R.; Jacobson, A. E.; Rice, K. C. J. Med. Chem. 2000, 43,
4840.
8. Goodman, M. M.; Kung, M. P.; Kabalka, G. W.;
Kung, H. F.; Switzer, R. J. Med. Chem. 1994, 37,
1535.
9. Emond, P.; Garreau, L.; Chalon, S.; Baozi, M.;
Caillet, M.; Briccard, J.; Frangin, Y.; Mauclaire, L.;
Besnard, J. C.; Guilloteau, D. J. Med. Chem. 1997,
40, 1366.
10. Loriga, G.; Manca, I.; Murineddu, G.; Chelucci, G.; Villa,
S.; Gessi, S.; Toma, L.; Cignarella, G.; Pinna, G. A.
Bioorg. Med. Chem. 2006, 14, 676.
11. Arnold, Z. Collect. Czech. Chem. Commun. 1965, 30,
2783.
Triplicate aliquots (90–150 lg protein) of the P2 frac-
tion were preincubated for 10 min at 37 ꢁC in the
above-mentioned buffer prior to the initiation of
uptake. The uptake was started by adding 50 nM
[3H]DA. After incubation at 37 ꢁC for 5 min the
uptake was stopped by rapid filtration under vacuum
through glass-fiber filter (Whatman GF/C) using a
single filter holder (Millipore). Filters were then
washed three times with 5 ml of ice-cold 0.9% NaCl
solution. The total time for the three washes was
about 5 s, after which the filters were removed
immediately and placed into vials containing 10 ml
of scintillation liquid (Ultima Gold MV, Packard,
Meridien, USA) and the filter-bound radioactivity
was measured in a liquid scintillation counter (Tricarb
2900, Packard, Meridien, USA).
12. Battistuzzi, G.; Cacchi, S.; Fabrizi, G. Org. Lett. 2003, 5,
777.
The values of [3H]DA uptake obtained from the synap-
tosomal fraction incubated over ice (blanks) were
subtracted from the corresponding samples at 37 ꢁC.
Inhibition curves were plotted and analyzed by
computer with appropriate software (Kell 6.0, Biosoft,
Cambridge, UK; graph Software, San Diego, CA,
USA). IC50 values were derived from the calculated
13. Israelashvili, S.; Gottlieb, Y.; Imber, M.; Habas, A.
J. Org. Chem. 1951, 16, 1519.
14. Ruiu, S.; Marchese, G.; Saba, P. L.; Gessa, G. L.; Pani, L.
Mol. Psychiatry 2000, 5, 673.
15. Schoemaker, H.; Nickolson, V. J. J. Neurochem. 1983, 40,
922.
16. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099.