6-(3,4-dichlorophenyl)-1-[(methyloxy)methyl]-3-azabicyclo[4.1.0]heptane: A new potent and selective triple reuptake inhibitor

Article abstract of DOI:10.1021/jm100481d

A pharmacophore model for triple reuptake inhibitors and the new class of 1-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes were recently reported. Further investigation in this area led to the identification of a new series of potent and selective trip

Full text of DOI:10.1021/jm100481d

J. Med. Chem. 2010, 53, 4989–5001 4989
DOI: 10.1021/jm100481d
6-(3,4-Dichlorophenyl)-1-[(Methyloxy)methyl]-3-azabicyclo[4.1.0]heptane: A New Potent and
Selective Triple Reuptake Inhibitor
Fabrizio Micheli,*,† Paolo Cavanni,† Daniele Andreotti,† Roberto Arban,† Roberto Benedetti,† Barbara Bertani,†
Michela Bettati,† Letizia Bettelini,† Giorgio Bonanomi,† Simone Braggio,† Renzo Carletti,† Anna Checchia,† Mauro Corsi,†
Elettra Fazzolari,† Stefano Fontana,† Carla Marchioro,‡ Emilio Merlo-Pich,† Michele Negri,† Beatrice Oliosi,‡
Emiliangelo Ratti,† Kevin D. Read, Maja Roscic,§ Ilaria Sartori,† Simone Spada,† Giovanna Tedesco,‡ Luca Tarsi,†
Silvia Terreni,† Filippo Visentini,‡ Alessandro Zocchi,† Laura Zonzini,† and Romano Di Fabio*,†
†Neurosciences Centre of Excellence for Drug Discovery, and ‡Molecular Discovery Research, GlaxoSmithKline Medicines Research Centre,
Via Fleming 4, 37135 Verona, Italy, §GlaxoSmithKline Research Centre Zagreb Ltd., Prilaz Baruna Filipovica 29, 10000 Zagreb,
Croatia, and Biological Chemistry and Drug Discovery, College of Life Sciences, Sir James Black Centre, University of Dundee,
Dundee, DD1 5EH, Scotland, U.K.
Received April 20, 2010
A pharmacophore model for triple reuptake inhibitors and the new class of 1-(aryl)-6-[alkoxyalkyl]-3-
azabicyclo[3.1.0]hexanes were recently reported. Further investigation in this area led to the identifica-
tion of a new series of potent and selective triple reuptake inhibitors endowed with good developability
characteristics. Excellent bioavailability and brain penetration are associated with this series of 6-(3,4-
dichlorophenyl)-1-[(methyloxy)methyl]-3-azabicyclo[4.1.0]heptanes together with high in vitro potency
and selectivity at SERT, NET, and DAT. In vivo microdialysis experiments in different animal models
and receptor occupancy studies in rat confirmed that derivative 17 showed an appropriate profile to
guarantee further progression of the compound.
Introduction
DA transporters at the same time comes from both preclinical
and clinical studies.3 The concurrent blockade of the uptake
of the three aminergic neurotransmitters, and therefore the
increased DA levels with respect to the “dual” drugs, might
address the anhedonic component of unipolar depression as
well as the shortening of the time to onset of clinical efficacy.
We have recently reported3,4 the rational design of two new
classes of TRUI endowed with excellent potency and in vivo
activity. The use of a TRUI pharmacophore model (Figure 2,
left panel) was imperative during medicinal chemistry ex-
ploration to appropriately address these three targets simul-
taneously. In particular, it was crucial in achieving the desired,
balanced ratio among the three transporters. In fact, as pre-
viously reported,3 many of the disclosed TRUI show similar
levels of inhibition at the three monoamine transporters
(SERTa ∼ NET ∼ DAT), while our target was to identify
molecules with an alternative profile such that SERT g NET
> DAT. The key characteristics of this model are represented
by the following features: a positive ionizable region, three
hydrophobicareas, a regionin whichan aromatic ringmay sit,
and an H-bond acceptor zone. As previously described, the
Unipolar depression is a terrible illness characterized by
low mood, low self-esteem, and loss of interest or pleasure in
usually enjoyable activities. Various drugs have been used for
decades to treat depressed patients. In particular, chemicals
able to interfere with either the uptake or with the metabolism
of aminergic neurotransmitters have found substantial appli-
cation. Monoamine oxidase (MAO) inhibitors and tricyclic
antidepressants achieved large diffusion in the early days,
but unfortunately they are associated with some side effects
that may hamper their efficacy.1 In recent times, drugs that
selectively block neurotransmitter reuptake in either seroto-
ninergic neurons (SSRI, e.g., paroxetine, Figure 1) or nora-
drenergic neurons (SNRI, e.g., reboxetine, Figure 1) became
the “gold standard” therapies. Additionally, drugs blocking
reuptake at both the serotoninergic and the noradrenergic
transporters (e.g., venlafaxine, Figure 1) or at both the nora-
drenergic and the dopaminergic neurons (e.g., bupropion,
Figure 1) also demonstrated clinical efficacy and acceptable
tolerability;1 these structures are also referred to as “dual”
reuptake inhibitors.
Quite recently, the structures of new compounds that are
“triple” reuptake inhibitors2 (TRUI) were disclosed; some of
these, such as indatraline, SEP-225289, and DOV derivatives
are reported in Figure 1. The theory on the efficacy of such
moleculesabletoinhibit amine reuptake ofthe 5-HT, NE, and
a Abbreviations: hERG, human ether-a go-go related gene Kþ chan-
nel; NCE, new chemical entity; PK, pharmacokinetic; P450, cytochrome
P450; hCli, human intrinsic clearance; F, bioavailability; B/B, brain/
blood ratio; Clb, blood clearance; Vd, distribution volume; SPA, scin-
tillation proximity assay; 5-HT, serotonin; NE, norepinephrine or
noradrenaline; DA, dopamine; SERT, serotonin transporter; NET,
noradrenaline transporter; DAT, dopamine transporter; MD, micro-
dialysis; pKi, inhibition constant from binding assays; fpKi, functional
inhibition constant for an antagonist from functional assays, e.g.,
functional inhibition constant obtained from functional inhibition
assays using the Cheng-Prusoff equation.
*To whom correspondence should be addressed. For F.M.: phone,
þ39-045-8218515; fax, þ39-045-8118196; e-mail, Fabrizio.E.Micheli@
gsk.com. For R.D.F.: phone, þ39-045-8218879; fax, þ39-045-8118196;
e-mail, Romano.M.Di-Fabio@gsk.com.
r
2010 American Chemical Society
Published on Web 06/09/2010
pubs.acs.org/jmc

4990 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Micheli et al.
Figure 1. Structures of known monoaminergic reuptake inhibitors.
Figure 2. Left: Triple reuptake inhibitors pharmacophore. Coding of features are as follows: blue sphere, positive ionizable; pink spheres,
H-bond acceptor; green spheres, hydrophobic; orange sphere, aromatic ring. Distances among the pharmacophoric points are shown in
angstroms. Middle: Derivative 1 fitted in the pharmacophore model. Right: Derivative 17 fitted in the pharmacophore model.
presence of a secondary/tertiary amine in the scaffold is
fundamental for achieving the primary activity at the three
transporters, while the decoration of the aromatic region
(hydrophobic areas) provides an appropriate “titration” of
the SERT/NET/DAT components. Finally, as will be further
exemplified below, the role of the H-bond acceptor in this
specific model seems to be critical in determining the DAT
affinity.
regioisomeric products 1-phenyl-3-azabicyclo[4.1.0]heptane
(3) and 6-phenyl-3-azabicyclo[4.1.0]heptane (10) were syn-
thesized exploiting the Beckmann rearrangement of the
1-phenylbicyclo[3.1.0]hexan-3-one oxime prepared from the
corresponding ketone in analogy to literature reports.5 The
enantiomers were separated by chiral HPLC, and the results
of their biological evaluation are reported in Tables 1 and 2. It
was quite evident that, also on these nonsubstituted [4.1.0]-
heptane scaffolds, despite the lack of interaction with one of
the key pharmacophoric features in the model, interesting pKi
values could be achieved at the three transporters and that the
6-phenyl-3-azabicyclo[4.1.0]heptane template 10 was slightly
superior to the regioisomeric 1-phenyl scaffold 3. The introduc-
tion of an appropriate side chain was the next step in the process.
Suitable synthetic schemes were devised to regioselectively place
Results and Discussion
Given the optimal fitting of 1 in the pharmacophore model
(Figure 2, middle panel), it was decided to identify alternative
bicyclic templates that maintained similar distances between
the key points of interaction, namely, the basic nitrogen, the
aromatic system, and the side chain. For this reason, the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4991
a
Table 1. Binding at the Three Transporters (SERT, NET, DAT) Expressed as pKi
entry
stereochemistry
R1
NA
NA
R
hSERT SPA pKi
hNET SPA pKi
hDAT SPA pKi
DOV 21,947
DOV 102,677
NA
NA
NA
NA
rac
NA
NA
NA
NA
H
6.85
7.08
9.80
9.50
6.90
7.10
6.60
7.50
6.80
7.90
7.50
6.83
7.23
9.30
8.40
6.80
7.20
6.40
6.70
6.00
7.00
7.20
7.10
7.42
8.10
7.90
7.10
7.40
7.00
6.90
6.20
7.20
6.90
1
2
3
4
5
6
7
8
9
NA
NA
H
se
H
H
se
rac
H
H
CH2OMe
CH2OMe
CH2OMe
H
H
se
H
se
exo (rac)
H
CH2OMe
a Only relative stereochemistry is shown. SEM for hSERT/NET/DAT data sets is (0.1. rac = racemate. se = single enantiomer. NA = not
applicable.
a
Table 2. Binding at the Three Transporters (SERT, NET, DAT) Expressed as pKi
entry
stereochemistry
R1
R
hSERT SPA pKi
hNET SPA pKi
hDAT SPA pKi
10
11
12
13
14
15
16
17
18
19
20
rac
se
H
H
H
H
H
H
H
H
H
H
H
H
7.60
7.40
7.90
7.70
7.40
8.10
8.80
9.20
7.70
7.30
8.00
6.60
6.60
7.00
6.80
6.40
7.00
7.90
8.10
6.90
5.70
6.50
7.30
7.30
7.60
6.70
7.00
7.10
7.60
8.00
7.00
5.90
6.40
se
rac
CH2OH
CH2OH
CH2OH
CH2OMe
CH2OMe
CH2OMe
H
se
se
rac
se
se
endo (rac)
exo (rac)
CH2OMe
CH2OMe
H
a Only relative stereochemistry is shown. SEM for hSERT/NET/DAT data sets is (0.1. rac = racemate. se = single enantiomer.
the alkyl alkoxy side chain in different positions on the new
scaffolds. Two of these pathways, illustrated for scaffold 10, are
reported in Schemes 1 and 2, while other specific schemes for
this template or for the regioisomeric scaffold 3 are reported in
the patent literature.6,7 The results of this exploration are
described in Tables 1 and 2. As predicted by the pharmacophore
model, the introduction of the methoxymethyl side chain on
derivative 3 in positions 6 (derivatives 6-8) and 7 (derivative 9)
determined a substantial improvement of affinity at the three
transporters, with 6 being equipotent to 9 at SERT. Because of
synthetic difficulties, only the exo derivative of this latter
compound could be tested. The introduction of the same side
chain on the regioisomeric template 10 (derivatives 16-20)
showed a significant difference between the two branching
positions on the scaffold. The introduction in position 1
(derivative 16) determined an approximately 10-fold increase in
affinity for the three transporters with respect to the best results
achieved with the side chain in position 7 (exo derivative 20).
Furthermore, a significant difference between the endo and exo
isomer (19 and 20, respectively) was also noticed, with the latter
being about 10-fold more potent than the former on the three
human transporters. Derivative 16 was separated by chiral
HPLC into its enantiomers (17, 18), and derivative 17 proved
to have nanomolar affinity at SERT and excellent affinity at
NET and DAT. As previously described,3 to ensure that
appropriate developability characteristics were present in the
new scaffolds since the inception of the exploration, generic
developability screens such as CYPEX bactosome P450 inhibi-
tion and rat and human in vitro clearance in liver microsomes
were added early in the biological screening cascade. Also in this
case, the methoxymethyl side chain proved to be a stable group.
The IC50 values for all major P450 isoforms tested (CYP1A2,
CYP2C9, CYP2C19, CYP2D6, and CYP3A4) were greater
than 10 μM with the exception of CYP2D6 (3 μM), and intrinsic

4992 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Scheme 1. General Synthetic Proceduresa
Micheli et al.
a (i) BOC2O, TEA, CH2Cl2, from 0 °C to room temp, 4 h; (ii) (a) NaH, DMF, 0 °C; (b) PhN(SO2CF3)2, DMF, room temp; (iii) ArB(OH)2, Na2CO3,
Pd(PPh3)4, toluene/EtOH; (iv) LiAlH4 in Et2O (1 M), -20 °C; (v) TFA, CH2Cl2, 0 °C; (vi) CH2I2, ZnEt2, CH2Cl2; (vi) (a) NaH, DMF, 0 °C; (b) RX,
DMF, room temp.
Scheme 2. General Synthetic Proceduresa
a (i) (a) NaH, DMF, 0 °C;(b) PhN(SO2CF3)2, DMF, room temp; (ii) 3,4-dichlorophenyl-B(OH)2, Na2CO3, Pd(PPh3)4, toluene/EtOH; (iii) N2CH2COOMe,
Rh2(OAc)4, CH2Cl2, 60 °C; (iv) LiAlH4 in Et2O (1 M), -20 °C; (v) (a) NaH, DMF, 0 °C; (b) RX, DMF, room temp; (c) TFA, CH2Cl2, 0 °C.
clearance (Cli) values both in human and in rat were moderate
(1.4 and 1.8 (mL/min)/g of protein). Interestingly enough, the
removal of the methoxy group led to compounds (13-15) with
good affinity at the three transporters. Derivative 15, in parti-
cular, showed a profile superior to the nondecorated template,
suggesting that the hydrophilic moiety was well tolerated. Given
this premise, it was decided to further explore the role of the side
chain and the pattern of substitution of the aromatic ring.
Results are reported in Table 3. The presence of a single chlorine
atom in position 4 of the phenyl group (21) led to a 10-fold
decrease of affinity vs SERT/NET/DAT, while the introduction
in the same position of a trifluoromethoxy group (22) with its
slightly larger volume and its lack of mesomeric effect led to
the identification of a selective SSRI. Another similar profile
with 100-fold selectivity over NET and DAT was achieved by
compound 23, while the replacement of a single chlorine atom
with a trifluoromethyl group (24, 25) gave a profile similar to
that of the monochloro derivative 21. Considering the overall
dimensions of a -CF3 group, these results might be interpreted,
once again, as a consequence of the lack of the retrodonating
mesomeric effect. The introduction of a 2-naphthyl substituent
(26) determined a substantial increase in SERT affinity with
respect to derivative 16, while the effect on NET and DAT was
comparable. The pure enantiomer 28 showed a subnanomolar
affinity at SERT and low nanomolar affinity at NET and DAT,
making this compound extremely interesting for further char-
acterization. The IC50 values for all major P450 isoforms tested
were greater than 8 μM; the Cli value in human was moderate
(1.2 (mL/min)/g of protein), while the Cli value in rat suffered
from the nonsubstituted nature of the naphthyl ring (17 (mL/
min)/g of protein), suggesting that an appropriate decoration/
protection of the group would be necessary before proceeding to
preclinical testing in vivo. As far as the exploration of the side
chain is concerned, a limited number of examples are reported in
Table 3. The introduction of a further methylene (29) to the
methoxymethyl group led to a superimposable in vitro affinity
profile, both as a racemate and as single enantiomers. The
replacement of the methyl group with the electron-withdrawing
-CF3 moiety (32) caused a slight decrease in affinity at the three
targets, while the introduction of a cyclopropyl methyl sub-
stituent (33-35) determined a variation in the primary in vitro
profile. In fact, derivative 34, when compared to 17, showed a
more “dual” SERT/DAT profile, making this compound an
interesting tool for further exploring the role of a differently
balanced reuptake inhibitor.
To probe the role of the ethereal oxygen in the new
template, a series of superior H-bond acceptors were pre-
pared; the results are reported in Table 4. The introduction of
the ethyl ester in 36 caused a slight increase in NET affinity, a
more substantial increase in DAT affinity, and a 10-fold

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4993
a
Table 3. Binding at the Three Transporters (SERT, NET, DAT) Expressed as pKi
entry
stereochemistry
Ar
4-Cl Ph
4-OCF3 Ph
R
hSERT SPA pKi
hNET SPA pKi
hDAT SPA pKi
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
rac
rac
rac
rac
rac
rac
se
Me
Me
Me
Me
Me
Me
Me
Me
Et
7.80
8.50
8.80
7.80
7.80
9.50
9.00
9.90
8.90
8.00
9.00
8.20
8.90
9.40
8.40
6.70
5.50
6.50
6.70
6.90
7.90
7.40
8.00
8.00
7.00
8.10
7.10
7.30
7.70
6.90
6.60
5.10
6.80
5.50
5.80
7.50
7.00
7.90
8.20
7.20
8.40
7.40
7.90
8.50
7.20
4-F, 3-Cl Ph
4-Cl, 3-CF3 Ph
4-CF3, 3-Cl Ph
2-naphthyl
2-naphthyl
2-naphthyl
se
rac
se
3,4- dichloro Ph
3,4- dichloro Ph
3,4- dichloro Ph
3,4- diChloro Ph
3,4- dichloro Ph
3,4- dichloro Ph
3,4- dichloro Ph
Et
Et
se
rac
rac
se
CH2CF3
CH2cPr
CH2cPr
CH2cPr
se
a SEM for hSERT/NET/DAT data sets is (0.1. Only relative stereochemistry is shown. rac = racemate. se = single enantiomer.
Table 4. Binding at the Three Transporters (SERT, NET, DAT)
Expressed as pKi
influenceonthe roleofthe nitrogen. These results are reported
in Table 5. A small hindrance (Me, 40) R to the nitrogen
(position 4) was well tolerated, witha slight decreaseinaffinity
at the three targets. The introduction of a small electron-
withdrawing group β to the nitrogen and therefore theoreti-
cally able to change its basicity was then tested. Both stereo-
isomers of the 5-fluoro derivative (41, 42) demonstrated the
same profile at SERT/NET/DAT; considering that, in theory,
at least one of the two should have been able to interact with
the antibonding orbitals of the C-N bond to influence its
basicity, it might be possible to conclude that no major
influence of this parameter was observed. On the other hand,
the introduction of a methoxy group (43, 44) demonstrated a
major influence on the primary in vitro affinity profile at the
three transporters. Considering the lower electronegativity of
the oxygen atom, it might be reasonable to conclude that this
effect was probably more linked to an overall steric effect of
the substituent in position 5 rather than to its effect on the
overall basicity of the system. Finally, the introduction of a
further substituent on the methoxymethyl side chain had a
positive effect on the primary in vitro profile of the com-
pounds (45, 46). Both derivatives showed a nanomolar profile
at the three targets; in particular 45 demonstrated a subnano-
molar profile at SERT and, despite the presence of an allyl
group, the IC50 values for all major P450 isoforms tested were
greater than 3 μM.
The final part of this preliminary exploration on this new
template was related to the understanding of the substitu-
tion of the nitrogen atom itself. The results are reported in
Table 6. The introduction of a methyl group (47) caused a
decrease in affinity at SERT and NET of about 10-fold and
50-fold, respectively, while almost no effect was observed
on DAT affinity. The introduction of a bulkier system
(i-Pr, 48) had a profound effect at the three transporters
with a strong reduction of affinity. We postulated that this
was potentially due to the hindrance of the Me and i-Pr
groups and not related to the presence of a tertiary nitrogen
in the scaffold. To prove this working hypothesis, we set up
an appropriate synthetic strategy to prepare a further
cyclized scaffold (a 6-azatricyclo[4.2.1.01,3]nonane template).
a
hSERT
SPA pKi SPA pKi SPA pKi
hNET
hDAT
entry stereochemistry
R
36
37
38
39
rac
rac
rac
se
COOEt
CONH2
7.80
7.00
7.00
9.00
8.10
6.10
6.30
7.80
8.30
6.60
6.80
8.40
CONHC3H3
COCH3
a Only relative stereochemistry is shown. SEM for hSERT/NET/
DAT data sets is (0.1. rac = racemate. se = Only one single enantiomer
was pure enough for testing.
decrease in the SERT activity, while the more hydrophilic
primary amide 37 showed a general decrease at all three
targets. This profile did not change with the introduction a
lipophilic substituent (38), suggesting that the presence of an
H-bond donor (NH) in this region might be poorly tolerated;
alternatively, it might be hypothesized that a conformational
change is caused by the presence of the amide bond and that
the potentialH-bondacceptor is no longer abletoproperly act
because it is spatially directed elsewhere. The introduction of
the methyl ketone 39 (only one enantiomer was tested because
neither the racemate nor the other enantiomer could be iso-
lated pure enough for primary testing) led to a profile similar
to that of 16 but with a higher DAT component. Accordingly,
in agreement with what was previously reported,3 it might be
hypothesized that the potential H-bond acceptor region
mapped by the pharmacophore model has lower importance
for the SERT and NET transporters, while it might have a
greater role in determining the DAT affinity.
The next step in the exploration was related to the investi-
gation of the substitution tolerated by the scaffold and its

4994 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Micheli et al.
a
Table 5. Binding at the Three Transporters (SERT, NET, DAT) Expressed as pKi
a SEM for hSERT/NET/DAT data sets is (0.1. Only relative stereochemistry is shown. Stereo. = stereochemistry. rac. = racemate. / =
diastereoisomer 1. // = diastereoisomer 2.
a
Table 6. Binding at the Three Transporters (SERT, NET, DAT) Expressed as pKi
a SEM for hSERT/NET/DAT data sets is (0.1. Only relative stereochemistry is shown. Stereo. = stereochemistry. s.e. = single enantiomer.
As evident from the two separate enantiomers (49, 50), good
levels of potency were achieved on the nondecorated scaffold.
Further investments are being made to appropriately decorate
this new promising scaffold.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4995
Figure 3. Time course of the effect of 17 (3 mg/kg, ip) on norepinephrine (A), dopamine (B), and serotonin (C) outflow in the rat medial
prefrontal cortex. Arrow shows injection, and asterisk (/) highlights significant difference vs respective vehicle time point (ANOVA with
repeated measure of time followed, when appropriate, by planned comparison). Data are the mean ( SEM of neurotransmitter concentration
expressed as percentage of basal values.
Further Characterization of Compound 17. The stereo-
chemistry of this molecule was assigned using vibrational
circular dichroism (VCD) as previously reported,3 and deri-
vative 17 was identified as the (1S,6R) isomer in accordance
to the method. This result was subsequently confirmed by
X-ray experiments which will be reported in due course.
Subsequently, derivative 17 was tested using filtration
assay binding conditions, and the values obtained were in
good agreement with the SPA binding (pKi = 8.98 ( 0.02
(n = 5), 7.92 ( 0.02 (n = 5), 7.92 ( 0.03 (n = 5) on SERT,
NET, DAT, respectively). Furthermore, the ability of 17 to
block [3H]5-HT, [3H]NE, and [3H]DA uptake was evaluated in
functional uptake SPA assays using LLCPK cells stably trans-
fected with human SERT, NET, or DAT. The compound
inhibited monoamine uptake with pIC50 = 8.4 ( 0.28 (SERT),
8.7 ( 0.30 (NET), 8.1 ( 0.05 (DAT) (n = 3), confirming that it
binds to human SERT, NET, and DAT but also demonstrating
that it can block the function of the transporters. The functional
pIC50 observed in the DAT assay was in line with the affinity of
the compound in the respective binding assay, while the func-
tional pIC50 observed at NET was higher than the binding
affinity for NET. In contrast, the functional potency at SERT
was lower than its binding affinity for this transporter. In this
latter case, a difference was expected, since the SERT uptake
SPA assay is known to be particularly sensitive to test condi-
tions (e.g., cell numbers) and, from previous experience,3,4 most
SERT blockers tested in uptake assays showed pIC50 lower
than the corresponding affinity in the binding assay.
An additional critical step before performing in vivo
experiments in preclinical models on this compound was
the evaluation of its affinity in rat and mice SERT, NET, and
DAT. The data obtained for the rat and the mouse (pKi of
8.9, 8.6, and 8.0 for rat and pKi of 8.8, 8.8, and 8.0 for mouse
on SERT, NET, and DAT, respectively) are in good agree-
ment with both the SPA and the filtration data generated on
human recombinant cell lines.
Another important passage was to verify the selectivity of
the compound on different targets. The compound demon-
strated at least a 30-fold window with respect to its DAT
affinity (i.e., its lower affinity among the three transporters)
over more than 100 different receptors, enzymes, ion chan-
nels, and transporters when tested at Cerep.8
The pharmacokinetics and oral bioavailability of 17 were
investigated in rat9 showing good bioavailability (F = 54%),
moderate distribution volume (Vd = 5.8 L/kg), and mode-
rate blood clearance (Clb = 48 (mL/min)/kg) resulting in a
half-life of 1.7 h. The average brain/blood (B/B) concentra-
tion ratio was equivalent to 6.3 at 1 h.
Considering its overall balanced profile in terms of pri-
mary activity and PK properties, we decided to explore the in
vivo behavior of the compound.
The first experiment was related to the increase in the
extracellular concentration of monoamines, and this was
measured in vivo;9 microdialysis probes were implanted in
the medial prefrontal cortex (mPFC) or nucleus accumbens
(NA) in freely moving rats, and samples were analyzed with

4996 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Micheli et al.
Figure 4. Time course of the effect of 17 (3 mg/kg, ip) on norepinephrine (A), dopamine (B), and serotonin (C) outflow in the rat nucleus
accumbens. Arrow shows injection. Asterisk (/) highlights significant difference vs respective vehicle time point (ANOVA with repeated
measure of time followed, when appropriate, by planned comparison). Data are the mean ( SEM of neurotransmitter concentration expressed
as percentage of basal values.
Table 7. 17 ex Vivo SERT Occupancy in Rat Cortex (as %)
dose (mg/kg)
mean
SEM
vehicle
0.03
0.1
0.3
1
0
4.6
0.0
0.0
12.6
36.3
58.2
82.8
98.2
2.9
3.0
10.5
1.4
3
20
0.1
monoamine transporters and therefore to produce an anti-
depressant-like effect.
In the first of these experiments (Figure 5, left) it was tested
in the 5-hydroxytryptophan (5-HTP) potentiation assay.10
The compound was administered orally 2 h before the
experiment. The results demonstrate that 17 significantly
increased, in a dose dependent manner, 5-HTP stereotyped
behavior at 3, 10, and 30 mg/kg. In the same study, the
standard SSRI drug citalopram significantly increased
5-HTP stereotyped behavior. Accordingly, these data con-
firm in vivo the inhibition of the 5-HT transporter.
In the second experiment (Figure 5, right), 17 was eval-
uated for its potential antidepressant-like effect in the forced
swimming test11 (FST), which is sensitive to known anti-
depressant drugs. Once again, the compound was adminis-
tered orally 2 h before the test in mice at 3, 10, and 30 mg/kg.
As clearly seen in the figure, the compound significantly
reduced immobility time at all doses as predicted by its in
vitro affinity and PK profile.
Figure 5. Effects of derivative 17 in the FST model (right) and in
the 5-HTP model (left) in mice.
HPLC coupled to electrochemical detection for the mea-
surement of NE, DA, and 5-HT concentrations. In the
mPFC, 17 (3 mg/kg ip) induced a significant increase in
NE, DA, and 5-HT levels that lasted throughout the
experiment (Figure 3). In the NA, the same dose of the
compound significantly augmented the outflow of NE, DA,
and 5-HT (Figure 4). These results demonstrate that the
acute administration of this molecule produced an increase
in the extracellular levels of all monoamines in both the
mPFC and NA in the rat.
Given these results, 17 was tested in two in vivo models in
mice to acquire further information on its ability to block the
Finally, two further experiments were performed to eval-
uate ex vivo occupancy of SERT elicited by 17. In the first

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4997
Figure 6. Autoradiographic study in rat brain: SERT occupancy after 10 mg/kg 17.
binding) or a final concentration of 10 μM fluoxetine (to define
study, the occupancy was determined 1 h after the adminis-
tration of different doses of the compound from the binding
of [3H]citalopram to rat brain cortex. The results are re-
ported in Table 7 and show a significant correlation between
the doses of 17 and the percent of occupancy at SERT as
expected by the PK and in vitro profile of the compound. In
the second experiment, the evaluation of ex vivo SERT
occupancy was performed using an autoradiographic tech-
nique, a method that allows quantification in many brain
regions with high anatomical resolution. The compound was
administered orally at 1, 3, and 10 mg/kg, and results for
the top dose are reported in Figure 6. 17 showed high levels of
SERT occupancy at the highest dose tested (10 mg/kg)
in almost every brain region (J95% occupancy in cortex,
striatum, substantia nigra, and superior coulliculus and
87.92% occupancy in dorsal raphe), as expected by the
SERT pKi value and in agreement with previous data
obtained with brain homogenates (Table 7).
nonspecific binding, NSB), 200 μL of 0.25 nM [N-methyl-3H]-
citalopram (Amersham Biosciences, 80 Ci/mmol), and 200 μL of
hSERT-LLCPK membranes (about 2.5 μg protein/well). After
2 h at room temperature the reaction was stopped by rapid
filtration through GF/B UniFilter 96 filter plate (Perkin-Elmer)
presoaked in 0.5% polyethyleneimine (PEI) using a Perkin-Elmer
FilterMat-196 harvester. The filter plate was washed 3 times with
1 mL/well ice-cold 0.9% NaCl solution, dried, and finally an
amount of 50 μL of Microscint 20 (Perkin-Elmer) were added to
each well. The radioactivity was counted using TopCount liquid
scintillation counter (Packard-Perkin-Elmer) and recorded as
counts per minute (CPM). Data analysis was performed by using
a four-parameter logistic equation with GraphPad PRISM soft-
ware, and pKi was calculated from pIC50 by using the Cheng-
Prusoff equation. Competition binding assay for human NET
(hNET) was conducted essentially as previously reported for SERT
except for the use of hNET-LLCPK cell membranes (4.8 μg/well),
1.5 nM [N-methyl-3H]nisoxetine as radioligand (Amersham Bio-
sciences, 84 Ci/mmol), and 10 μM desipramine for NSB. Finally,
the same procedure was also adopted for human DAT (hDAT)
competition binding assay but using hDAT/LLCPK cell mem-
branes (9.6 μg/well), 10 nM [N-methyl-3H]WIN-35,428 (Perkin-
Elmer, 85.6 Ci/mmol), and 10 μM GBR-12909 for NSB.
Finally, the safety profile of the compound was studied in
agreement with the requests of the regulatory authorities to
proceed to dosing in human volunteers. The results will be
provided in due course.
Filtration Binding for Rat Native SERT, NET, and DAT and
Mouse Native SERT. Filtration binding studies were carried out
on rat and mouse brain native tissue. The affinity of the com-
pound was determined on rat or mouse cortex for SERT, on rat
hippocampus for NET, and on rat striatum for DAT, using the
appropriate radioligand. Competition binding assays for rat
and mouse SERT, rat NET, and rat DAT were conducted
essentially as previously reported for human recombinant
monoamine transporters but using a different amount of mem-
branes (20 and 7 μg of protein/well for rat and mouse SERT
binding, 40 and 20 μg/well for rat NET and DAT binding,
respectively).
Conclusions
The discovery of the 6-aryl-1-[alkoxyalkyl]-3-azabicyclo-
[4.1.0]heptane template as a potent and selective class of triple
reuptake inhibitors is reported. The molecules belonging to
this class are endowed with an excellent developability profile.
They also perfectly fit the TRUI pharmacophore model
recently described.
Derivative 17, in particular, showed an excellent in vitro
and in vivo profile, with clear activity in preclinical animal
modelscorrelated tothe increase of the monoamines. MD and
ex vivo experiments performed on this molecule clearly in-
dicate its mode of action through monoamine uptake inhibi-
tion. Safety studies are in progress for a better characteriza-
tion of this new scaffold.
hERG-[3H]Dofetilide Binding Assay. hERG activity was
measured using [3H]dofetilide binding in a scintillation proxi-
mity assay (SPA) format. The activity was measured with a
PerkinElmer Viewlux imager.
SPA Binding for Human Recombinant SERT, NET, and DAT.
The affinity of the compounds to the human transporters has
been assessed with radioligand displacement binding of [3H]cita-
lopram, [3H]nisoxetine, and [3H]WIN-35,428 for SERT, NET,
and DAT in BacMam-recombinant human SERT, NET, and
DAT membranes with the SPA technology. Briefly, 0.3 μL of
test compound was added to 30 μL of the SPA mixture contain-
ing 1 mg/mL SPA (GE HealthCare, RPNQ0260) beads (SERT)
or 2 mg/mL SPA beads (NET and DAT), 6 or 40 or 20 μg/mL
SERT or NET or DAT BacMam membranes, 0.02% Pluronic
F-127, 3 nM [3H]citalopram or 10 nM [3H]nisoxetine or 10 nM
[3H]WIN-35,428 for SERT or NET or DAT binding SPA in the
Experimental Section
Biological Test Methods. In Vitro Studies. Filtration Binding
Assay for Human Recombinant SERT, NET, and DAT. Filtra-
tion binding assays were run using membranes prepared from
LLCPK cells stably transfected with each of the three human
transporters. Filtration [3H]citalopram binding assay for human
SERT (hSERT) was conducted in a deep-well 96-well plate in a
total volume of 404 μL/well by adding 4 μL of test compound
(100ꢀ solution in neat DMSO) or DMSO (to define total

4998 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Micheli et al.
assay buffer (20 mM HEPES, 145 mM NaCl, 5 mM KCl, pH
7.4). Incubation was performed overnight at room temperature.
Bound radioactivity was measured with the Viewlux instrument.
Data analysis was performed by using a four-parameter logistic
equation with ActivityBase software, and pKi was calculated
from pIC50 by using the Cheng-Prusoff equation.
Functional Uptake for SERT, NET, and DAT. The potency of
the compounds in blocking the [3H]serotonin, [3H]noradrenalin,
and [3H]dopamine uptake was evaluated in a functional uptake
SPA assay on LLCPK cells stably transfected with human SERT,
NET, and DAT. Briefly, 0.2 μL of test compound was added to
10 μL of the bead-cell SPA mixture containing 1.5 mg/mL SPA
beads (SERT and NET) or 2 mg/mL of SPA beads (DAT), 25 000
or 50 000 or 75 000 cell/well of SERT or NET or DAT LLCPK
cells, 0.02% Pluronic F-127 in the assay buffer (20 mM HEPES,
145 mMNaCl, 5 mM KCl, pH 7.4). The uptake was started bythe
substrate addition of 10 μL of 35 nM [3H]serotonin or 45 nM
[3H]noradrenalin or 75 nM [3H]dopamine for SERT or NET or
DAT uptake SPA. Incubation was performed at room tempera-
ture for 1 h. Uptake was measured with the Viewlux instrument,
and data analysis was performed as before.
P450 CYPEX Assay. Inhibition (IC50) of human CYP1A2,
2C9, 2C19, 2D6, and 3A4 was determined using Cypex Bacto-
somes expressing the major human P450s. A range of concen-
trations (0.1, 0.2, 0.4, 1, 2, 4, and 10 μM) of test compound were
prepared in methanol and preincubated at 37 °C for 10 min in 50
mM potassium phosphate buffer (pH 7.4) containing recombi-
nant human CYP450 microsomal protein (0.1 mg/mL; Cypex
Limited, Dundee, U.K.) and probe-fluorescent substrate. The
final concentration of solvent was between 3% and 4.5% of the
final volume. Following preincubation, NADPH regenerating
system (7.8 mg of glucose 6-phosphate, 1.7 mg of NADP, and
6 units of glucose 6-phosphate dehydrogenase/mL of 2% (w/v)
NaHCO3; 25 μL) was added to each well to start the reaction.
Production of fluorescent metabolite was then measured over a
10 min time course using a Spectrafluor Plus plate reader. The
rate of metabolite production (AFU/min) was determined at
each concentration of compound and converted to a percentage
of the mean control rate using Magellan (Tecan software). The
inhibition (IC50) of each compound was determined from the
slope of the plot using Grafit, version 5 (Erithacus software,
U.K.). Miconazole was added as a positive control to each plate.
CYP450 isoform substrates used were ethoxyresorufin (ER,
1A2, 0.5 μM), 7-methoxy-4-triflouromethylcoumarin-3-acetic
acid (FCA, 2C9, 50 μM), 3-butyryl-7-methoxycoumarin (BMC,
2C19, 10 μM), 4-methylaminomethyl-7-methoxycoumarin
(MMC, 2D6, 10 μM), diethoxyflourescein (DEF, 3A4, 1 μM),
and 7-benzyloxyquinoline (7-BQ, 3A4, 25 μM). The test was
performed in three replicates.
Intrinsic Clearance (Cli) Assay. Intrinsic clearance (Cli) values
were determined in rat and human liver microsomes. Test
compounds (0.5 μM) were incubated at 37 °C for 30 min in
50 mM potassium phosphate buffer (pH 7.4) containing 0.5 mg
of microsomal protein/mL. The reaction was started by addition
of cofactor (NADPH, 8 mg/mL). The final concentration of
solvent was 1% of the final volume. At 0, 3, 6, 9, 15, and 30 min
an aliquot (50 μL) was taken, quenched with acetonitrile con-
taining an appropriate internal standard, and analyzed by
HPLC-MS/MS. The intrinsic clearance (Cli) was determined
from the first-order elimination constant by nonlinear regres-
sion using Grafit, version 5 (Erithacus software, U.K.), cor-
rected for the volume of the incubation and assuming 52.5 mg of
microsomal protein/g of protein for all species. Values for Cli
were expressed as (mL/min)/g of protein. The lower limit of
quantification of clearance was determined to be when <15%
of the compound had been metabolized by 30 min, and this
corresponded to a Cli value of 0.5 (mL/min)/g of protein. The
upper limit was 50 (mL/min)/g of protein.
syndrome characterized by stereotypic behavior. The combina-
tion monoamine reuptake inhibitors with subthreshold doses of
5-HTP has been proposed as a model to evaluate in vivo effects
of drug acting at the serotonin transporter (Ortmann, 1980).
Mice were pretreated with 17 120 min before an intraperito-
neal (ip) injection of 5-HTP (100 mg/kg) and placed individually
into a perspex box (20.5 cm ꢀ 20.5 cm ꢀ 34 cm). Experiments
were videotaped, and the following behaviors over 30 min were
scored: aimless hyperlocomotor activity (aHLMA), abduction
of hind limbs (HLA), flat body posture (FBP), head twiches/
tremor (HT), body tremor (BT). Each behavior was scored
either present (scored = 1) or absent (scored = 0) by an obser-
ver blind to the drug treatment.
Forced Swimming Test. The mouse forced swimming test
(Porsolt et al., 1977) is widely used as an animal model for
detecting and screening antidepressant activity. Mice were
dropped individually into glass cylinders (height 25 cm, dia-
meter 10 cm) containing 10 cm of water maintained at 25 ( 1 °C
and left there for 6 min. A mouse was judged immobile when it
floated in an upright position and made only small movements
to keep its head above water. The duration of immobility was
recorded during the last 4 min of the 6 min testing period. All
experiments were videotaped, and the observer was unaware of
the drug treatment scores immobility.
Compound 17 was administered orally 120 min before test.
MD and Locomotor Activity Data. Animals. Male CD rats
(Charles River, Italy) weighing between 280 and 310 g at the
beginning of the experiment were housed in constant conditions
of temperature (21 ( 1 °C) and moisture under a 12 h light/dark
cycle with water and food available ad libitum. They were
handled everyday to get accustomed to manipulation and pre-
sence of researchers.
Intracerebral Microdialysis Surgery. Animals were anesthe-
tized with proper concentrations of medetomidine hydrochlor-
ide (Domitor) and xylazine (Zoletil), while carpofren (Rymadil)
was injected to induce analgesia. They were then placed on a
stereotaxic apparatus for small animals. A guide cannula was
lowered and positioned above the left medial prefrontal cortex
(anteroposterior 3.2 mm, lateral 0.5 mm, dorsoventral 1.8 mm
from bregma) or nucleus accumbens (anterior, þ1.7 mm; ventral,
-6.0 mm; mediolateral, 1.2 mm from bregma) and secured to the
skull using dental cement, anchored by four stainless steel screws.
A removable stainless steel stylet was placed inside the cannula
to maintain its patency throughout the experimental period.
Following surgery the animals were injected with atipamezole
hydrochloride (Antisedan) and Rubrocillin antibiotic and housed
in single cages for 1 week to recover.
Microdialysis Experiment. The day before the experiment the
stylette was removed from the guide cannula, and a micro-
dialysis probe with a 2 or 4 mm long membrane (depending on
the region) was inserted (MAB 4 Agntho’s, Sweden). The
following day microdialysis experiments were performed in
their home cage. Both inlet and outlet of the probe were
attached with FEP tubings (dead volume of 1.2 μL/10 cm) to
a dual quartz lined two-channel liquid swivel (model 375/D/
22QM Instech Labs, Plymouth Meeting, PA) mounted on a
low mass spring counterbalanced arm (MCLA, Instech). One
channel was connected to a gas tight syringe (Hamilton 1002
LTN 2.5 mL, Bonaduz, Switzerland) on a microinfusion pump
(The Univentor 802 syringe pump) to deliver artificial cere-
brospinal fluid (KCl 2.5 mM, NaCl 125 mM, CaCl2 1.3 mM,
MgCl2 1.18 mM, Na2HPO4 2 mM, pH 7.4, with H3PO4 85%)
at a steady flow rate of 1.1 μL/min, and the second channel
was connected to a refrigerated fraction collector (Univentor
820 microsampler) containing microtubes with 3 μL of 1 mM
oxalic acid solution. Length of the outlet tubing was calculated
to achieve a 20 min delay of sample collection. Samples were
collected every 20 min and frozen in dry ice for subsequent
HPLC analysis. Two hours of perfusion were allowed before
starting the experiment.
5-HTP Potentiation Test. The administration of the precursor
of 5-hydroxytryptophan (5-HTP) to mice leads to a serotonergic

Article
Probe Localization. The position of the probe in the brain
areas was verified at the end of each experiment. A blue dye was
injected through a probe without the dialysis membrane, and the
brains were then removed and coronally sectioned at the level of
the PFC or NAc. Trace localization was visually identified
through comparison with a stereotaxic atlas of rat brain (Paxinos
and Watson, 2005). Animals with incorrect probe position were
discarded from the final analysis.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4999
The enantiomeric purity of each single enantiomer obtained
after preparative chromatography on chiral columns was always
verified on analytical column.
The purity of the compounds reported in the manuscript was
established through HPLC methodology. All the compounds
reported in the manuscript have a purity of >95%.
General Synthetic Procedures. 1-(1,1-Dimethylethyl) 3-Meth-
yl-4-hydroxy-5,6-dihydro-1,3(2H)-pyridinedicarboxylate (54).
To a stirred solution of methyl 4-oxo-3-piperidinecarboxylate
hydrochloride in dry DCM at 0 °C and under a nitrogen atmo-
sphere, TEA was added dropwise over 5 min. The mixture was
stirred at 0 °C for 5 min, then allowed to reach room tempera-
ture. Bis(1,1-dimethylethyl) dicarbonate was then added, and
the solution was left stirring overnight at room temperature
under nitrogen. After quenching and workup, the crude product
was purified by flash chromatography (eluting with ethyl acet-
ate/cyclohexane 1:3) to give the title compound. NMR (1H,
CDCl3): δ 11.95-12.02 (m, 1 H), 4.07 (br s, 2 H), 3.77-3.82 (m,
3 H), 3.58 (t, 2 H), 2.34-2.43 (m, 2 H), 1.46-1.50 (m, 9 H).
1-(1,1-Dimethylethyl) 3-Methyl-4-{[(trifluoromethyl)sulfonyl]-
oxy}-5,6-dihydro-1,3(2H)-pyridinedicarboxylate (55). To a stirred
solution of 1-(1,1-dimethylethyl) 3-methyl-4-oxo-1,3-piperidine-
dicarboxylate (54) in dry DMF at 0 °C and under a nitrogen
atmosphere, NaH (60% on mineral oil) was added portionwise.
The reaction mixture was stirred at 0 °C for 10 min. Then a
solution of N-phenyl-bis(trifluoromethanesulfonimide) in dry
DMF was added dropwise, and stirring was continued for 0.5 h.
After quenching and workup, the solvent was removed under
reduced pressure and the compound was purified by flash chro-
matography to give the title compound. NMR (1H, CDCl3): δ
4.29 (br s, 2 H), 3.82-3.87 (m, 3 H), 3.64 (t, 2 H), 2.50-2.57 (m,
2 H), 1.46-1.52 (m, 9 H). MS (m/z): 390 [MH]þ, 412 [MNa]þ.
1-(1,1-Dimethylethyl) 3-Methyl-4-(3,4-dichlorophenyl)-5,6-di-
hydro-1,3(2H)-pyridinedicarboxylate (56). To a mixture of
1-(1,1-dimethylethyl) 3-methyl-4-{[(trifluoromethyl)sulfonyl]-
oxy}-5,6-dihydro-1,3(2H)-pyridinedicarboxylate (55), 3,4-di-
chlorophenylboronic acid, and Pd(PPh3)4 under nitrogen were
added toluene, ethanol, and Na2CO3 (aqueous 2 M solution) in
sequence. The mixture was stirred at 80 °C for 1 h, and then the
reaction mixture was allowed to reach room temperature. After
quenching and workup, the crude product was purified by flash
chromatography (eluting with ethyl acetate/cycloexane 1:3) to
give the title compound. NMR (1H, CDCl3): δ 7.42 (d, 1 H), 7.25
(d, 1 H), 6.98 (dd, 1 H), 4.26 (br s, 2 H), 3.61 (t, 2 H), 3.55-3.58
(m, 3 H), 2.47 (br s, 2 H), 1.50-1.54 (m, 9 H).
1,1-Dimethylethyl 4-(3,4-Dichlorophenyl)-5-(hydroxymethyl)-
3,6-dihydro-1(2H)-pyridinecarboxylate (57). To a stirred solu-
tion of 1-(1,1-dimethylethyl) 3-methyl-4-(3,4-dichlorophenyl)-
5,6-dihydro-1,3(2H)-pyridinedicarboxylate (56) in dry diethyl
ether under nitrogen atmosphere at -20 °C, LiAlH4 (1 M in
diethyl ether) was added dropwise over 1 min. The reaction
mixture was left stirring at -20 °C for 15 min, then quenched
with aqueous saturated NH4Cl solution and diethyl ether After
workup, the crude product was purified by flash chromatogra-
phy (eluting with ethyl acetate/cycloexane 1:3) to give the title
compound. NMR (1H, DMSO-d6): δ 7.62 (d, 1 H), 7.56 (d, 1 H),
7.26 (dd, 1 H), 4.90 (t, 1 H), 4.02 (br s, 2 H), 3.79 (d, 2 H), 3.49 (t,
2 H), 2.35 (br s, 2 H), 1.42-1.46 (m, 9 H).
6-(3,4-Dichlorophenyl) 1-[(Methyloxy)methyl]-3-azabicyclo-
[4.1.0]heptane-3-carboxylate (17). To a stirred solution of CH2I2
in dry CH2Cl2 under argon atmosphere at 0 °C ZnEt2 (1 M in
hexane) was added dropwise. The mixture was stirred at 0 °C
for 20 min and then cooled at -20 °C. A solution of 57 in
dry CH2Cl2 was added dropwise, and the reaction mixture
was stirred for an additional 30 min, then 40 min at 0 °C, and
overnight at roomtemperature. After quenchingand workup, the
crude product was quickly purified by flash chromatography
(eluting with ethyl acetate/cycloexane 1:3). This intermediate
was dissolved in dry CH2Cl2 under argon atmosphere and stirred
at room temperature. Bis(1,1-dimethylethyl) dicarbonate was
HPLC Procedure. NA, DA, and 5-HT concentrations were
determined in dialysate samples by HPLC with electrochemical
detection (Antec Decade). Separation was achieved using a
mobile phase consisting of 12 mM Na2HPO4, 88 mM NaH2PO4,
5 mM NaCl, 0.1 mM EDTA Na2, 20% MeOH, 10% MeCN,
pH 6.00 ( 0.05. Chromatographic data were acquired and
processed through Empower software.
3
Data Analysis. For each animal absolute neurochemical data
in picogram were transformed to percent values vs average of
the three basal values. They were preliminarily analyzed with a
2D Box & Whiskers plot to determine and eliminate outlier and
extreme values. Data eliminated were substituted with the mean
of the same group at the same time. Final data were analyzed
with two-way ANOVA for repeated measures with treatment
and time as factors. Whenever allowed, preplanned post hoc
multiple comparisons were applied to compare time points of
different treatments.
Concurrent general motor activity was measured through an
automated digital video analysis of animal movements (View-
Point). Data were analyzed as previously described for micro-
dialysis data.
Ex Vivo Autoradiography of SERT after Oral Administration
of 17 in Rat. Sprague-Dawley rats were acutely treated with 17
at 1, 3, and 10 mg/kg po (-90 min), and spontaneous locomotor
activity was recorded for 30 min. At the end of the test, animals
were sacrificed and brains were quickly removed and frozen
and processed for in vitro autoradiography of SERT (2nM
[3H]citalopram).
General Chemical Procedures. Proton magnetic resonance
(NMR) spectra are typically recorded either on a Varian instru-
ment at 300, 400, or 500 MHz or on a Bruker instrument at 300
and 400 MHz. Chemical shifts are reported in ppm (δ) using the
residual solvent line as internal standard. Splitting patterns are
designated as follows: s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; b, broad. The NMR spectra were recorded at a
temperature ranging from 25 to 90 °C. When more than one
conformer was detected, the chemical shifts for the most abun-
dant one was reported. Mass spectra (MS) are typically taken on
a 4 II triple quadrupole mass spectrometer (Micromass UK) or
on a Agilent MSD 1100 mass spectrometer, operating in ES (þ)
and ES (-) ionization mode or on an Agilent LC/MSD 1100
mass spectrometer, operating in ES (þ) and ES (-) ionization
mode coupled with an HPLC Agilent 1100 series instrument. In
the mass spectra only one peak in the molecular ion cluster is
reported. When HPLC walk-up retention time is reported, the
analysis is done on a HPLC Agilent 1100 series instrument with
the following conditions: column, Luna C18 100A 50 mm ꢀ
2 mm, 3 μm; mobile phase, (MeCN þ 0.05% TFA)/(H2O þ
0.05% TFA), gradient from0/100 to95/5in8 min; flux 1 mL/min.
Flash silica gel chromatography was carried out on silica gel
230-400 mesh (supplied by Merck AG Darmstadt, Germany) or
over Varian Mega Be-Si prepacked cartridges or over prepacked
Biotage silica cartridges.
SPE-SCX cartridges are ion exchange solid phase extraction
columns supplied by Varian. The eluent used with SPE-SCX
cartridges is methanol followed by 2 N ammonia solution in
methanol. In a number of preparations, purification was per-
formed using either Biotage manual flash chromatography
(Flashþ) or automatic flash chromatography (Horizon, SP1)
systems. All these instruments work with Biotage silica car-
tridges. SPE-Si cartridges are silica solid phase extraction
columns supplied by Varian.

5000 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Micheli et al.
added, and the mixture was left stirring overnight. After quench-
ing and workup the material was purified by flash chromato-
graphy (eluting with diethyl ether/n-hexane 1:2 to 1:1) to give
the 1,1-dimethylethyl 4-(3,4-dichlorophenyl)-5-(hydroxymethyl)-
3,6-dihydro-1(2H)-pyridinecarboxylate intermediate. This mate-
rial was dissolved in dry THF under argon atmosphere at 0 °C.
NaH (60% on mineral oil) was added in one portion, and the
stirring continued for 30 min. After this period CH3I was added
dropwise and the mixture was allowed to reach roomtemperature
and stirred for 1.5 h. After quenching and workup, the mate-
rial was purified by flash chromatography (eluting with diethyl
ether/n-hexane 40:60) to give the 1,1-dimethylethyl 4-(3,4-
dichlorophenyl)-5-[(methyloxy)methyl]-3,6-dihydro-1(2H)-pyri-
dinecarboxylate intermediate. Finally, deprotection with TFA in
CH2Cl2 was performed at 0 °C to give the title compound. NMR
(1H, DMSO-d6): δ 8.71 (br s, 2 H), 7.73 (d, 1 H), 7.59 (d, 1 H), 7.41
(dd, 1 H), 3.45 (d, 1 H), 3.09-3.16 (m, 2 H), 3.04 (s, 3 H), 2.92 (d,
1 H), 2.73-2.82 (m, 1 H), 2.66 (d, 1 H), 2.01-2.17 (m, 2 H),
1.22-1.29 (m, 2 H); MS (m/z): 286 [MH]þ
1,1-Dimethylethyl 4-{[(Trifluoromethyl)sulfonyl]oxy}-3,6-di-
hydro-1(2H)-pyridinecarboxylate (61). To a stirred solution of
diisopropylamine in dry THF at -78 °C and under a nitrogen
atmosphere, butyllithium (2.5 M in hexane) was added. The
reaction mixture was stirred at -78 °C for 15 min. DMPU
(1.8 mL) and a solution of 1,1-dimethylethyl 4-oxo-1-piperidi-
necarboxylate (60) in THF were added, and the reaction mixture
was stirred at -78 °C for 2 h. Then a solution of N-phenyl-
bis(trifluoromethanesulfonimide) in THF was added and stir-
ring was continued at 0 °C for 9 h and at room temperature per
16 h. The solvent was removed under reduced pressure and the
crude purified by flash chromatography (eluting with ethyl
acetate/cycloexane 2:8) to give the title compound. NMR (1H,
CDCl3): δ 5.79 (br s, 2 H), 4.07 (m, 2 H), 3.65 (m, 2 H), 2.47 (m,
2 H), 1.50 (s, 9 H).
1-[(Methyloxy)methyl]-6-{4-[(trifluoromethyl)oxy]phenyl}-3-
azabicyclo[4.1.0]heptane Hydrochloride (22). NMR (1H, MeOH-
d4): δ ppm 7.52 (d, 2 H), 7.24 (d, 2 H), 3.72 (d, 1 H), 3.27 (d, 1 H),
3.25 -3.21 (m, 1 H), 3.16 -3.12 (m, 3 H), 3.07 (d, 1 H), 2.97-
2.84 (m, 1 H), 2.74 (d, 1 H), 2.32-2.12 (m, 2 H), 1.31 (d, 1 H), 1.22
(d, 1 H).
6-(3-Chloro-4-fluorophenyl)-1-[(methyloxy)methyl]-3-azabi-
cyclo[4.1.0]heptane Hydrochloride (23). NMR (1H, DMSO-d6):
δ ppm 8.28 (s, 1 H), 7.66 (d, 1 H), 7.25-7.47 (m, 2 H), 3.41 (d, 1
H), 3.01-3.14 (m, 5 H), 2.92 (d, 1 H), 2.75 (d, 1 H), 2.68 (d, 1 H),
1.98-2.10 (m, 2 H), 1.17-1.26 (m, 2 H). MS (m/z): 270 [MH]þ.
6-[4-Chloro-3-(trifluoromethyl)phenyl]-1-[(methyloxy)methyl]-
3-azabicyclo[4.1.0]heptane (24). NMR (1H, CDCl3): δ 7.72 (d,
1 H), 7.48-7.53 (m, 1 H), 7.42-7.46 (m, 1 H), 3.42 (d, 1 H),
3.08-3.14 (m, 4 H), 3.04 (d, 1 H), 2.81-2.90 (m, 1 H), 2.66-2.76
(m, 2 H), 1.97-2.06 (m, 1 H), 1.85-1.93 (m, 2 H), 1.11 (d,1 H),
1.04 (d, 1 H). MS (m/z): 320 [MH]þ.
6-[3-Chloro-4-(trifluoromethyl)phenyl]-1-[(methyloxy)methyl]-
3-azabicyclo[4.1.0]heptane (25). NMR (1H, CDCl3): δ ppm 7.71
(s, 1 H), 7.46 (dd, 2 H), 3.40 (d, 1 H), 3.12 (s, 3 H), 2.99-3.10 (m,
2 H), 2.76-2.91 (m, 1 H), 2.65-2.76 (m, 2 H), 1.91-2.07 (m,
2 H), 1.79-1.91 (m, 1 H), 1.10 (d, 1 H), 1.02 (d, 1 H). MS(m/z):
320 [MH]þ.
1-[(Methyloxy)methyl]-6-(2-naphthalenyl)-3-azabicyclo[4.1.0]-
heptane (26). NMR (1H, CDCl3): δ 7.77-7.86 (m, 3 H),
7.72-7.76 (m, 1 H), 7.42-7.52 (m, 3 H), 3.37 (d, 1 H), 3.18 (d,
1 H), 3.10 (s, 3 H), 3.02 (d, 1 H), 2.80-2.89 (m, 2 H), 2.70-2.79
(m, 1 H), 1.96-2.08 (m, 2 H), 1.25 (d, 1 H), 1.11 (d, 1H). MS(m/z):
268 [MH]þ.
6-(3,4-Dichlorophenyl)-1-[(ethyloxy)methyl]-3-azabicyclo[4.1.0]-
heptane Hydrochloride (29). NMR (1H, MeOH-d4): δ 7.58 (d, 1 H),
7.38 (d, 1 H), 7.25 (dd, 1 H), 3.64 (d, 1 H), 3.15 (m, 5 H), 2.79 (m,
1 H), 2.57 (d, 1 H), 2.11(m, 2H), 1.19 (d, 1 H), 1.12(d, 1H), 0.98 (t,
3 H). MS (m/z): 300 [MH]þ.
3-(1,1-Dimethylethyl) 7-Ethyl 6-(3,4-dichlorophenyl)-3-azabi-
cyclo[4.1.0]heptane-3,7-dicarboxylate (63). To a mixture of 61,
3,4-dichlorophenylboronic acid, and Pd(PPh3)4 under nitrogen
were added toluene, ethanol, and Na2CO3 in sequence. The
mixture was stirred at 80 °C for 2 h. Then it was allowed to reach
room temperature. After quenching and workup, the intermedi-
ate was purified by flash chromatography (eluting with ethyl
acetate/cycloexane 1:9) to give intermediate 62. The compund
was dissolved in CH2Cl2, and rhodium acetate was added. The
mixture was heated at 40 °C. A solution of ethyl diazoacetate
in CH2Cl2 was added with a syringe pump in 4 h, maintaining
the internal temperature at 50 °C during the addition. The
solvent was removed under reduced pressure and the crude
purified by flash chromatography (eluting with ethyl acetate/
cycloexane 2:8) to give the intermediate title compound (MS
(m/z): 414 [MH]þ).
6-(3,4-Dichlorophenyl)-7-[(methyloxy)methyl]-3-azabicyclo-
[4.1.0]heptane (20). To a stirred solution of 63 in dry toluene
under a nitrogen atmosphere at -20 °C, LiAlH4 (1 M in diethyl
ether) was added dropwise. The reaction mixture was left stirring
at -20 °C for 1 h. After quenching and workup, the crude
intermediate 64 was obtained (MS (m/z): 372 [MH]þ). To a
stirred solution of this product in dry DMF under nitrogen
atmosphere at 0 °C, NaH (60% on mineral oil) was added. The
mixture stirred for 30 min at 0 °C. Methyl iodide was added. The
mixture was allowed to reach room temperature, and it was
stirred for an additional 2 h. After quenching and workup, the
crude was treated with TFA in CH2Cl2 at 0 °C to give the title
compound after careful column chromatography. NMR (1H,
CDCl3): δ 7.37 (d, 1 H), 7.33 (d, 1 H), 7.13 (dd, 1 H), 4.01 (dd,
1 H), 3.78 (dd, 1 H), 3.44 (s, 3H), 3.32 (m, 1H), 3.17 (m, 1H), 2, 84
(m, 1H), 2.59 (m, 1H), 2.04 (m, 1H), 1.88 (m, 1H), 1.37 (m, 2H).
6-(4-Chlorophenyl)-1-[(methyloxy)methyl]-3-azabicyclo[4.1.0]-
heptane (21). NMR (1H, CDCl3): δ ppm 7.25 (s, 4 H), 3.30 (d,
1 H), 3.08-3.14 (m, 4 H), 2.85-2.91 (m, 2 H), 2.63-2.80 (m,
2 H), 1.81-1.99 (m, 2 H), 0.99-1.05 (m, 2 H).
6-(3,4-Dichlorophenyl)-1-{[(2,2,2-trifluoroethyl)oxy]methyl}-3-
azabicyclo[4.1.0]heptane (32). NMR (1H, CDCl3): δ 7.5 (s, 1 H)
7.45 (d, 1 H) 7.35 (m, 1 H), 3.9 (d, 2 H) 3.7 (d, 1 H) 3.6 (m, 2 H)
3.25 (d, 1 H) 3.15 (m, 2 H) 2.3 (m, 1 H) 2.2 (m, 1 H) 1.25 (m, 2 H).
6-(3,4-Dichlorophenyl)-4-methyl-1-[(methyloxy)methyl]-3-
azabicyclo[4.1.0]heptane (40). NMR (1H, CDCl3): δ 7.46 (d,
1 H), 7.33 (d, 1 H), 7.20 (dd, 1 H), 3.54 (d, 1 H), 3.06-3.17 (m,
3 H), 2.89-3.01 (m, 2 H), 2.72 (d, 1 H), 2.27-2.53 (m, 1 H),
1.91-2.07 (m, 1 H), 1.27-1.46 (m, 1 H), 1.04 (d, 3 H), 0.87-0.98
(m, 2 H). MS (m/z): 300 [M þ H]þ.
6-(3,4-Dichlorophenyl)-1-[1-(methyloxy)-3-buten-1-yl]-3-azabi-
cyclo[4.1.0]heptane (45). The previously described 1,1-dimethyl-
ethyl 6-(3,4-dichlorophenyl)-1-(hydroxymethyl)-3-azabicyclo-
[4.1.0]heptane-3-carboxylate was dissolved in dry CH2Cl2 and
stirred under a nitrogen atmosphere at 0 °C. After 10 min,
Dess-Martin periodinane was added portionwise and the mix-
tureslowly warmedtoroomtemperature andstirred for 1 h. After
quenching and workup, the intermediate formyl derivative (1,1-
dimethylethyl)
6-(3,4-dichlorophenyl)-1-formyl-3-azabicyclo-
[4.1.0]heptane-3-carboxylate) was obtained as a yellow oil (314
[MH - 56]þ). The compound was dissolved in THF at 20 °C, and
to the solution bromo(2-propen-1-yl)magnesium was added.
After quenching and workup, the residue was purified by chro-
matography (c-Hex/EtOAc 0-30%, 100 g silica column) to give
two diastereomers as oils. One diastereoisomer was dissolved in
dry DMF, and NaH was added at 0 °C, followed by the addition
of methyl iodide. After quenching and workup, the residue was
purified by chromatography (CyH/EtOAc from 0% EtOAc to
30%), and it was dissolved in CH2Cl2. TFA was added, and the
mixture was stirred at 0 °C for 30 min. After quenching and
workup the compound was purified by SCX to give the title
compound. NMR(1H, CDCl3): δ 7.53 (s, 1H), 7.38 (d, 1H), 7.27
(m, 1H), 5.73 (m, 1H), 5.04 (d, 1H), 4.99 (d, 1H), 3.50(d, 1H), 2.99
(s, 3H), 2.97 (d, 1H), 2.85 (m. 1H), 2.59 (m, 1H), 2.40 (m, 1H),
2.25(m, 1H), 2.17(m, 1H), 1.94 (t, 2H), 1.00 (m, 2H). MS(m/z) =
326 [MH]þ.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 5001
6-(3,4-Dichlorophenyl)-3-methyl-1-[(methyloxy)methyl]-3-
Finally we thank Dr. C. P. Leslie for careful proofreading of
the manuscript.
azabicyclo[4.1.0]heptane (47). NMR (1H, CDCl3): δ 7.44 (d, 1 H),
7.35 (d, 1 H), 7.18 (dd, 1 H), 3.15 (s, 3 H), 2.90 (dd, 2 H), 2.78 (d,
1 H), 2.70 (d, 1 H), 2.23-2.30 (m, 5 H), 1.96-2.13 (m, 2 H), 1.05
(d, 1 H), 1.01 (d, 1 H). MS (m/z): 300 [M þ H]þ.
References
3-(3,4-Dichlorophenyl)-6-azatricyclo[4.2.1.01,3]nonane (49, 50).
The previously described 1,1-dimethylethyl 6-(3,4-dichloro-
phenyl)-1-(hydroxymethyl)-3-azabicyclo[4.1.0]heptane-3-carbo-
xylate was transformed into the corresponding ethyl ester
according to WO2008/031772. The solution was treated with
triethylsilane, followed by TFA. The reaction mixture was
stirred for 2 h at room temperature. After quenching and
workup, the obtained compound was dissolved in dry CH2Cl2.
The solution was treated with Boc2O and TEA. After quench-
ing and workup, the intermediate compound was hydrolyzed
with potassium tert-butoxide to give the corresponding car-
boxylic acid. This compound was dissolved in dry CH2Cl2.
TEA, oxalyl chloride, and DMF were added at 0 °C under a
nitrogen atmosphere. The mixture was stirred at 0 °C for 30
min and then at room temperature for 1 h. The reaction
mixture was evaporated under reduced pressure. The residue
was dissolved in a mixture of anhydrous THF and anhydrous
acetonitrile, and to this mixture was added TMS-diazo-
methane (2 M in diethyl ether) at 0 °C under a nitrogen
atmosphere. The mixture was stirred at 0 °C for 1 h and then
for an additional 2 h at room temperature. After quenching
and workup, this compound was dissolved in THF/water
(3:1). Silver benzoate was added and the obtained mixture
sonicated using an ultrasound bath. After quenching and
workup, the compound was taken up in methanol and
CH2Cl2. Then TMS-diazomethane (2 M in hexane) was added
dropwise under an atmosphere of nitrogen at room tempera-
ture and the mixture was stirred for 2 h. The resulting methyl
ester was dissolved in dry toluene and the obtained solution
cooled to -20 °C. Then LiAlH4 (2 M solution in THF) was
added dropwise and the reaction mixture was stirred at -20 °C
for 1 h to give the primary alcohol. The compound was
dissolved in dry CH2Cl2. Mesyl-Cl and DIPEA were added
at 0 °C. The reaction mixture was stirred at room temperature
for 1 h. After quenching and workup, the new compound was
dissolved in dry CH2Cl2. TFA was added at 0 °C and the
reaction mixture stirred at room temperature for 5 h. Finally,
after quenching and workup, the obtained derivative was
dissolved in dry DMF, sodium iodide was added, and the
reaction mixture was stirred at 80 °C. The mixture was then
purified by chromatography using a SCX cartridge to give the
title compound as a yellow oil. 1H NMR, CDCl3: δ ppm
1.08-1.14 (m, 1 H), 1.49 (d, 1 H), 1.51-1.57 (m, 1 H), 1.65-
1.71 (m, 1 H), 1.95 (d, 1 H), 2.10 (dd, 1 H), 2.30-2.36 (m, 2 H),
2.63 (d, 1 H), 2.84-2.91 (m, 1 H), 3.35-3.41 (m, 1 H), 3.66 (dd,
1 H), 7.03 (dd, 1 H), 7.27 (overlapped with CDCl3, 1 H) 7.33 (d,
1 H). MS (m/z): 268 [MH]þ.
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The two enantiomers were separated by chiral HPLC:
column, Chiralcel OD-H; mobile phase, n-hexane/ethanol þ
0.1% isopropylamine 90/10% v/v; flow rate, 14 mL/min; UV,
230 nm.
^
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review performed by the GlaxoSmithKline Committee on Animal
Research and Ethics (CARE) and to the Company Policy on the
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flow rate, 1 mL/min; DAD, 210-340 nm; retention times, 8.34
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Acknowledgment. We thank all the colleagues who helped
in generating the data reported in this manuscript, in parti-
cular A. Nalin, Dr. F. Ferroni, Dr. G. Tamborrino, and Dr.
Doug Minick. We also thank Dr. D. Donati, Dr. J. Hagan,
and Dr. T. Rossi for their support during the program life.