1-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanesand6-(aryl) -6-[alkoxyalkyl]-3azabicyclo[3.1.0]hexanes: A new series of potent and selective triple reuptake inhibitors

2534 J. Med. Chem. 2010, 53, 2534–2551

DOI: 10.1021/jm901818u

1-(Aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes and 6-(Aryl)-6-[alkoxyalkyl]-3-

azabicyclo[3.1.0]hexanes: A New Series of Potent and Selective Triple Reuptake Inhibitors

Fabrizio Micheli,*^,†Paolo Cavanni,^†Roberto Arban,^†Roberto Benedetti,^†Barbara Bertani,^†Michela Bettati,^†

Letizia Bettelini,^†Giorgio Bonanomi,^†Simone Braggio,^†Anna Checchia,^†Silvia Davalli,^‡Romano Di Fabio,^†

Elettra Fazzolari,^†Stefano Fontana,^†Carla Marchioro,^‡Doug Minick,^‡,§Michele Negri,^†Beatrice Oliosi,^‡Kevin D. Read,

Ilaria Sartori,^†Giovanna Tedesco,^‡Luca Tarsi,^†Silvia Terreni,^†Filippo Visentini,^‡Alessandro Zocchi,^†and Laura Zonzini^†

^†Neurosciences Centre of Excellence for Drug Discovery, and ^‡Molecular Discovery Research, GlaxoSmithKline Medicine Research Centre,

Via Fleming 4, 37135 Verona, Italy, ^§Molecular Discovery Research, GlaxoSmithKline, Five Moore Drive, Research Triangle Park,

North Carolina, and Biological Chemistry and Drug Discovery, College of Life Sciences, Sir James Black Centre, University of Dundee,

Dundee DD1 5EH, Scotland, U.K.

Received December 9, 2009

The discovery of new highly potent and selective triple reuptake inhibitors is reported. The new classes of

1-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes and 6-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes

are described together with detailed SAR. Appropriate decoration of the scaffolds was achieved with the

help of a triple reuptake inhibitor pharmacophore model detailed here. Selected derivatives showed

good oral bioavailability (>30%) and brain penetration (B/B > 4) in rats associated with high in vitro

potency and selectivity at SERT, NET, and DAT. Among these compounds, microdialysis and in vivo

experiments confirm that derivative 15 has an appropriate developability profile to be considered for

further progression.

Introduction

reported in Figure 1. The idea behind a “broad-spectrum”^2e

antidepressant that is capable of inhibiting amine uptake of

the 5-HT,^aNE, and DA transporters comes from both

preclinical^2d-gand clinical studies. It is known that dopa-

mine agonists themselves (e.g., pergolide, bromocriptine)

showed efficacy as augmenting agents with antidepressant in

clinical studies.²ⁱAccordingly, the contemporary block of the

three aminergic neurotransmitters may produce, in addition

to the elevation of 5-HT and NE, an increase in the CNS levels

of DA. It has been hypothesized^2b,e,hthat such specific

increase in DA levels might address the anhedonic component

of depression as well as shorten the time to onset. Therefore,

the TRUI might result in improved efficacy toward a broader

range of the depressed population.

Drugs which are able to interfere with either the uptake or

with the metabolism of aminergic neurotransmitters have

been used for decades to treat depressed patients. The earliest

medications, like monoamino oxidase (MAO) inhibitors or

tricyclic antidepressants, gained a wide diffusion but are

unfortunately linked to some side effects that may limit their

efficacy.¹More recently, drugs which act by selectively block-

ing neurotransmitter reuptake in either serotoninergic neu-

rons (SSRI, e.g., paroxetine, Figure 1) or noradrenergic

neurons (SNRI, e.g., reboxetine, Figure 1) have become

established as effective antidepressants. Furthermore, drugs

which block reuptake at both serotoninergic and noradrener-

gic transporters (e.g., venlafaxine, Figure 1) or at both

noradrenergic and dopaminergic neurons (e.g., bupropion,

Figure 1), also known as dual uptake inhibitors, provide good

efficacy and tolerability too.¹

Many of the TRUI templates reported to date show level of

inhibition of the monoamine transporter such as SERT =

NET = DAT.

In the quest for the identification of potent and selective

TRUI, we report the discovery of a new series of 1-(aryl)-

6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes, together with a

new series of 6-(Aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]-

hexanes, which are endowed with excellent pharmacokinetic

(PK) properties in preclinical species and which work very

effectively in different animal models related to the aminergic

system.

During the past decade, part of GSK research focus in CNS

drug discovery has aimed at the discovery of novel chemical

entities able to influence the reuptake of aminergic neuro-

transmitters by their transporters and the rational design was

followed to achieve this task and the development of two new

potent and selective series based on azabicyclo[3.1.0]hexanes

is reported here.

An important recent development has been achieved with

the discovery of triple reuptake inhibitors²(TRUI) like

indatraline, SEP-225289, or DOV derivatives, which are

*To whom correspondence should be addressed. Phone: þ39-045-

8218515. Fax: þ39-045-8118196. E-mail: Fabrizio.E.Micheli@gsk.com.

^aAbbreviations: hERG, human ether-a go-go K^þchannel; NCE,

novel chemical entity; PK, pharmacokinetic; P450, cytochrome P450;

hCli, human intrinsic clearance; F%, bioavailability; B/B, brain/blood;

Clb, blood clearance; Vd, distribution volume; SPA, scintillation proxi-

mity assay; 5-HT, serotonin; NE, norepinephrine or noradrenaline; DA,

dopamine; SERT, serotonin transporter; NET, noradrenaline transpor-

ter; DAT, dopamine transporter; MD, microdialysis; pK_i, inhibition

constant from binding assays; fpK_i, functional inhibition constant for an

antagonist from functional assays e.g. functional inhibition constant

obtained form inhibition functional assays using Cheng-Prusoff equa-

tion.

r

pubs.acs.org/jmc

Published on Web 02/19/2010

2010 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2535

Figure 1. Structures of known monoaminergic reuptake inhibitors.

reuptake inhibitor pharmacophore. Average coordinates for

the common pharmacophoric features were taken to deter-

mine their 3D arrangement, as illustrated pictorially in Figure 2.

Further details can be found in the Supporting Information

available.

Results and Discussion

Given the features exemplified in the pharmacophore

model (Figure 2) and the relative distances among the

pharmacophoric points there represented, it was decided to

introduce an alkoxyalkyl side chain to potentially increase

the point of interactions of the template with the transporter;

in particular it was hypothesized that the ideal region to

probe for a substituted azabicyclo[3.1.0]hexane was repre-

sented by the position 6 of the same scaffold, having been the

position 5 previously explored in house in the past without

any substantial improvement in the potency of the scaffold.

It is also important to notice that, in this specific pharma-

cophore model, some uncertainty is related to the role of the

H-bond acceptor mapped by the ether motif. An ether

oxygen in general is not a particularly strong H-bond

acceptor, and it may well be that this pharmacophoric point

is an artifact, as this atom is present in topologically equiva-

lent positions in all the compounds used to derive the

pharmacophore.

The compounds belonging to this new series were prepared

in agreement to the synthetic route reported in Scheme 1 and

results are reported in Table 1. In particular, in our quest for

the “ideal” triple reuptake inhibitor the desired profile was

defined as a compound having relative affinities in the order

SERT g NET > DAT, thus allowing the study in vivo of an

alternative profile to the currently existing derivatives where

SERT = NET = DAT. A different in vitro profile may

theoretically account for a different in vivo read-out either

in terms of desired activity or potential side effects.

Figure 2. The triple reuptake inhibitors pharmacophore. Coding of

features: blue sphere, positive ionizable; pink spheres, H-bond

acceptor; green spheres, hydrophobic; orange sphere, aromatic ring.

Distances among the pharmacophoric points are shown in ang-

stroms.

Considering the in house expertise and the general informa-

tion available in the literature, it was hypothesized that an

appropriate decoration of the azabicyclo[3.1.0]hexane scaf-

fold would have led to an increased affinity for the three

aminergic transporters.

A TRUI pharmacophore model was developed in house

exploiting both “mixed-activity” and selective transporter

inhibitors, and more precisely, three specific pharmacophore

models for SERT, NET, and DAT were built using structu-

rally rigid and selective derivatives. These compounds derived

both from GlaxoSmithKline’s proprietary collection and

from the public domain (e.g., the examples reported in

Figure 1 or in ref 3). The structures were modeled within the

Maestro^4amodeling environment. Conformational analyses

were carried out for the three structures with BatchMin^4b

using the following parameters: 1000 steps/rotatable bond,

OPLS_2005 FF, implicit water model of solvation, 5000

minimization steps with PRCG. Conformations lying within

a 3 kcal/mol energy window were kept. A single-ligand

pharmacophore was then built over these structures using

the phase^4cmodule within Maestro and selecting the most

relevant pharmacophoric features. The three pharmaco-

phores were finally merged together to create the triple

All thecompounds preparedwere assayedfor theircapacity

to bind to the three monoamine transporters (SERT, NET,

and DAT) in SPA-binding primary assays on membranes

from cells transiently transduced with BacMam virus. Addi-

tionally, the ability of the most interesting compounds to

block [³H]5-HT, [³H]NE, and [³H]DA uptake were evaluated

in functional uptake SPA assays using LLCPK cells stably

2536 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6

Micheli et al.

Table 1. Binding at the Three Transporters (SERT, NET, DAT) Expressed as pK_i^a

entry

stereochemistry

R

R₁

Ar

hSERT SPA pK_i

hNET SPA pK_i

hDAT SPA pK_i

DOV 21,947

DOV 102,677

NA

H

Me

H

NA

7.70

6.70

7.29

7.81

8.82

7.14

9.12

7.59

9.02

8.30

9.50

7.50

9.30

8.02

9.60

9.40

9.80

7.60

8.90

7.25

9.20

9.70

7.40

9.90

9.20

9.50

7.60

9.30

9.60

8.10

9.00

8.90

8.40

9.10

9.40

7.60

9.00

9.80

7.90

10.10

7.20

6.10

6.74

7.54

8.50

5.89

8.76

6.65

8.71

7.80

9.20

6.90

9.30

7.50

9.20

8.80

9.30

6.70

7.80

8.40

6.46

8.80

9.40

6.70

9.60

8.90

9.30

6.80

9.10

9.80

7.90

8.70

7.40

7.50

8.30

8.80

6.50

8.70

9.00

6.50

9.20

6.90

6.70

6.32

6.34

6.96

6.18

7.25

6.29

7.60

6.70

7.90

6.30

7.70

6.60

7.90

7.60

8.10

6.70

7.40

7.30

6.20

7.60

8.10

6.60

8.30

7.90

8.40

6.60

8.20

8.60

7.00

7.70

7.10

6.20

7.10

7.40

7.20

7.60

7.30

6.40

7.60

1

endo (rac)

exo (rac)

endo (rac)

exo (se)

exo (rac)

exo (se)

endo (rac)

exo (rac)

exo (se)

exo (rac.)

exo (se)

exo (rac)

exo (se)

exo (rac)

exo (se)

exo (rac)

exo (se)

exo (rac)

exo (se)

exo (rac)

exo (se)

exo (rac)

exo (se)

CH₂OH

CH₂OMe

3,4-di Cl Ph

2-naphthyl

2

3

4

CH₂OMe

5

6

7

CH₂OCH₂c-Pr

CH₂On-Pr

CH₂OEt

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

CH₂OEt

CH₂OCH₂CF₃

CH₂Oi-Pr

CH₂Oc-butyl

CH₂Oc-pentyl

CH₂Oc-hexyl

CH₂O(4-F-phenyl)

CH₂NMe₂

CH₂SMe

n-Pr

CH₂OMe

^aSEM for hSERT/NET/DAT data sets is (0.1. (rac) = racemate. (se) = single enantiomer. NA = Not applicable.

Scheme 1. General Synthetic Procedures for the Preparation of Compounds 1-39^a

^aX = O, S, C; Ar = napthyl for derivatives 37, 38, 39, and 3,4-dichlorophenyl for the other mentioned compounds. (i) ArNH₂, CuCl₂, tBuNO₂,

CH3CN, 0 °C; (ii) Me₂SCH₂CO₂Et^þBr^-, NaH, or ethyldiazoacetate/THF 0 °C to rt; (iii) BH₃-THF in THF at reflux; (iv) (a) BOC₂O, TEA, CH₂Cl₂,

(b) NaH, RX in THF/DMF, (c) TFA, CH₂Cl₂0 °C to rt; (v) (a) BOC₂O, CH₂Cl₂, (b) MsCl/Et₃N followed by RONa or RSNa or RMgBr, (c) TFA,

CH₂Cl₂0 °C to rt.

transfected with human SERT, NET, or DAT. These more

interesting compounds also underwent filtration binding as-

says on membranes prepared from LLCPK cells stably trans-

fected with each of the three human transporters.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2537

Figure 3. The exo derivatives 15 and 16 fitted in the pharmacophore model in comparison with the potential endo isomers. It can be clearly

seen, both in terms of visual overlapping and in terms of phase Fitness (ranging from 0 to 1, the higher the better), that the triple reuptake

inhibitors pharmacophore superimposes much better with the exo isomers with respect to the corresponding endo isomers.

Furthermore, to ensure that the selected templates were

endowed with appropriate developability characteristics from

the beginning of the exploration, the NCEs went through

generic developability screens such as CYPEX bactosome

P450 inhibition and rat and human in vitro clearance in liver

microsomes early in the screening cascade.

assay binding conditions, and the values obtained were in

good agreement with the SPA binding (pK_i= 9.13/8.34/7.07

on SERT/NET/DAT respectively); finally, the rat-SERT

native tissue binding in filtration was also verified, leading

to a pK_i= 8.75. To verify, since the beginning, if the

introduction of the alkoxyalkyl side chain was appropriate

in terms of developability of the potential new class, 5 was

further assayed in agreement to the above-described screening

cascade. IC₅₀values for all major P450 isoforms tested

(CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4)

were greater than 6 μM and intrinsic clearance (Cl_i) values

both in human and in rat resulted moderately low (<0.5 and

3.8 mL/min/g of protein). Considering this positive in vitro

data, the pharmacokinetic (PK) profile in vivo in rat was

evaluated;⁵derivative 5 showed a relatively low blood clear-

ance (Cl_b= 16 mL/min/kg), leading to an appropriate half-

life for in vivo testing in appropriate disease models (t_1/2=2.4h).

The distribution volume was also relatively low (V_d= 2.9

L/kg) and the bioavailability was excellent (F = 94%).

Considering that central nervous system (CNS) is the final

target for this class of molecules, brain penetration was

evaluated too. The compound demonstrated a brain/blood

(B/B) ratio of 4, with a brain fraction unbound of 2.7%

showing excellent penetration properties and low tissue bind-

ing values. Very encouraged by these initial results, which

could be considered as the discovery of an interesting dual

SERT/NET derivative, the investigation on this class was

further expanded. The major objectives of the exploration

Given the route described in Scheme 1, the first compound

to be tested was the intermediate alcohol prepared with the

3,4-dichlorophenyl aryl derivative so to directly compare with

literature standard derivatives. This compound was separated

into its endo (1) and exo (2) derivatives, and both compounds

were tested as racemate. Interestingly enough, the introduc-

tion of the hydroxymethyl group had no major effect on the

affinity for the transporters with respect to the unsubstituted

derivative; nonetheless, a significant difference was observed

between the endo and exo compound toward both SERT and

NET affinity, while no major difference was noticed over

DAT activity. The following alkylation of the alcohol to the

corresponding methyl ether further increased this gap, with

the exo derivative 3 much more active than the corresponding

endo one (4); furthermore, in agreement with the original

design, the introduction of this group greatly increased both

SERT and NET affinity and also led to a moderate DAT

affinity increase. The separation of the racemate 3 by chiral

chromatography led to the single enantiomers 5 and 6.

Derivative 5 showed a nanomolar affinity at SERT and

similar affinity over NET, while the DAT affinity was about

70-fold lower. The compound was also tested using filtration

2538 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6

Micheli et al.

Scheme 2. General Synthetic Procedures for the Preparation of Compounds 40-44^a

^aPG = H or BOC (i) (a) BOC₂O, TEA, CH₂Cl₂, (b) Dess-Martin periodinane; (ii) Me(Ph)₃P^þBr^-, BuLi, THF; (iii) (a) BH₃-THF, THF, (b) NaOH,

H₂O₂30%, (c) TFA, CH₂Cl₂0 °C to rt; (iv) NaH, RX in THF/DMF; (c) TFA, CH₂Cl₂0 °C to rt.

were related to the dimension of the pocket available for

binding within the transporter, to its sensitivity to polarity,

and to the role played by the oxygen bridge. The introduction

of a bulky cyclopropyl methyl derivative (7-9) was well

tolerated, leading to a very potent molecule (9) on each of

the three transporters; in particular, the greater relative

increase was obtained on the dopamine transporter. The

bulkiness was well tolerated also in terms of developability

properties with all of the P450 isoforms inhibited with IC₅₀

greater than 10 μM with the exception of CYP2C19 and

CYP2D6 (IC₅₀= 3 and 1 μM, respectively). Human and

rat Cl_iwere comparable to derivative 5 despite a higher

lipophilicity of the molecule (<0.5 and 2.6 mL/min/g of

protein, respectively). The in vivo PK evaluation in rat of

derivative 9 demonstrated that the good results achieved by 5

were not related to a singleton derivative; actually, the com-

pound, despite a higher lipophilicity, was still endowed with

acceptable PK parameters: Cl_bwas high (72 mL/min/kg) with

a high volume of distribution (5.2 L/kg), leading to a reduced

half-life (t_1/2= 1 h). Bioavailability was moderate (F = 32%)

and the brain penetration very high (B/B = 10), although this

was driven by higher nonspecific binding as the key para-

meter, fraction unbound, was very low (F_ub= 0.1%). As

observed for the previous compounds, also in this case the

endo derivative was much less potent (10). Therefore, con-

sidering the superiority of the exo substitution with respect to

the endo one, the exploration was continued on the former

template. This experimental result was also in agreement with

the predictions of the pharmacophore model used during the

planning of the series. Actually, when all the four potential

stereoisomers of the [3.1.0] scaffold were fitted in the model,

the exo derivatives showed a better fit than the corresponding

endo ones as exemplified in Figure 3.

The introduction of a linear and slightly less lipophilic

moiety (n-Pr, 11-13) led to a superimposable in vitro profile

of derivative 13 with 9, both from the primary binding at the

transporters and from the DMPK point of view. The further

reduction by one carbon atom (Et, 14-16) led to similar

affinity properties (15) but greatly improved in vitro DMPK

characteristics. All of the major P450 isoforms were inhibited

with IC₅₀greater than 9 μM with one exception (CYP2D6

IC₅₀= 4 μM) and human and rat Cl_iwere relatively low (1.2

and 1.3 mL/min/g of protein, respectively). The in vivo PK

profile in rat was excellent too: 15 showed low Cl_b(18 mL/

min/kg) and distribution volume (V_d= 1.4 L/kg), with

acceptable half-life (t_1/2= 1.2 h) and very good bioavailability

(F = 61%). Brain penetration was good (B/B = 4.6) as well as

the fraction unbound (1.4%).

observed in the DAT assay was in line with the affinity of 15 in

the respective binding assay, the functional pIC₅₀observed at

NET was higher than the binding affinity for NET. On the

contrary, functional potency of 15 at SERT was lower than its

binding affinity for this transporter. With the latter, it is

important to notice that a difference was expected because

the SERT uptake-SPA assay is known to be particularly

sensitive to test conditions (e.g., cell numbers) and, from

previous in house experience, most SERT blockers tested in

uptake assays showed pIC₅₀values lower than the corre-

sponding affinity in the binding assay. However, because

pIC₅₀values are a relative measure of compounds potency,

being dependent on assay conditions, reliable comparison of

the functional potency of the compounds at the three mono-

amine transporters is not possible. Therefore, the results of

these functional uptake studies are to be considered only

indicative of the likely functional ratio of effect in vivo,

whereas the binding affinities must be considered a reliable

basis on which to predict a potential in vivo effect.

N-Methylation of the secondary amine, 17, led to approxi-

mately a 10-fold decrease in affinity at the three transporters,

while the introduction of a dipole in the system (18-20) gave a

5-fold decrease (20). Moreover, the replacement of the term-

inal methyl group with a CF₃derivative, despite a higher

lipophilicity, did not substantially change the in vitro DMPK

parameters and gave a similar in vivo PK profile (Cl_b= 42

mL/min/kg; V_d= 3.2 L/kg; t_1/2= 1.4 h; F = 41%); the major

differences were observed was in terms of brain penetration

and fraction unbound (B/B = 18; F_ub= 0.6%). The intro-

ductionof a branching alpha tothe oxygenatom (i-Pr, 21-23)

further increased the affinity for the three transporters (23 vs

13) by about 2.5-fold and was not detrimental for the in vitro

developability profile (IC₅₀greater than 8 μM on all major

P450 isoforms; human and rat Cl_i=1.4 and <0.5 mL/min/

kg, respectively). A further increase in bulkiness had no major

impact on affinity (c-butyl, 24-26; c-pentyl 27-29) with

slightly higher NET values for 28 and started to become

detrimental with the introduction of the bulkiest c-hexyl

derivative 30, still maintaining high levels of affinity on all

the three transporters. The introduction of an aromatic

moiety, 31, had no major impact on SERT affinity but led

to a more marked decrease in terms of NET and DAT when

compared to 30.

Compared to the parent racemic compound 4, the intro-

duction of a strong polar component (NMe₂, 32) had no

major impact in terms of loss of affinity; actually, the NET

affinity decreased 10-fold while the SERT and DAT affinity

decreased approximately 5-fold. The original question relat-

ing to the real existence of an H-bond acceptor feature in the

pharmacophore model was potentially answered in the fol-

lowing steps. The replacement of the oxygen with a sulfur

atom (32-34) potentially demonstrates that the ether probably

Considering its overall profile, 15 was further characterized

and shown to efficiently inhibit the uptake of all of the three

monoamines (pIC₅₀= 8.90/9.80/8.30 on SERT/NET and

DAT, respectively). However, whereas the functional pIC₅₀

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2539

Table 2. Binding at the Three Transporters (SERT, NET, DAT) Expressed as pK_i^a

entry

stereochemistry

R

hSERT SPA pK_i

hNET SPA pK_i

hDAT SPA pK_i

40

41

42

43

44

45

46

47

48

49

exo (rac)

exo (se)

exo (rac)

exo (se)

CH₂CH₂OH

CH₂CH₂OMe

CH₂CH₂OEt

8.60

9.10

9.60

7.50

9.10

7.80

9.00

9.70

10.10

7.80

8.10

8.50

9.00

6.00

8.40

7.70

8.00

9.50

9.90

7.10

7.20

7.30

6.90

7.10

6.90

7.50

7.70

7.30

5-methyl-1,3-oxazol-2-yl

4-methyl-1,3-thiazol-2-yl

CH₂(4-methyl-1,3-thiazol-2-yl)

^aSEM for hSERT/NET/DAT data sets is (0.1. (rac) = racemate. (se) = single enantiomer.

Scheme 3. General Synthetic Procedures for the Preparation of Compounds 45-49^a

^aPG = H or BOC, R = OH or NHAlk, HET = heterocycle (i) (a) Jones reagent at 0 °C in CH₂Cl₂, (b) HOBT, EDC, NH₄R; (ii) (a) heating with

appropriate halo-ketone, (b) TFA, CH₂Cl₂0 °C to rt; (iii) steps from (i) to (iii) in Scheme 2; (iv) (a) Jones reagent at 0 °C in CH₂Cl₂, (b) HOBT, EDC,

NH₄OH, (c) Lawesson’s reagent, (d) heating with appropriate halo-ketone, (e) TFA, CH₂Cl₂0 °C to rt.

does not constitute a weak hydrogen-bond acceptor in the

transporter. The slightly higher lipophilicity of 34 compared to

5 has a slight impact on SERT but leaves NET and DAT

affinity almost unaltered within experimental error. This evi-

dence is also potentially confirmed by the replacement of the

oxygen with a carbon atom where the racemate 36 is not

different from the corresponding racemate 14 as far as NET

and DAT are concerned and show a 2-fold decrease in terms of

SERT affinity. Finally, among the different aryl derivative

tested in the exploration (data not shown), in this specific series

of derivatives, the 2-naphthyl group (37-39) seems to act well

as 3,4-dichlorophenyl bioisoster. Actually, in this specific series,

39 showed a 10-fold higher SERT affinity with respect to 5 and

approximately a 5-fold affinity increase over NET and DAT,

leading to a picomolar affinity compound on serotonin trans-

porter. In this case, human and rat Cl_iwere 2.2 and 3.9 mL/min/

kg, respectively, and the CYP450 inhibition profile showed

IC₅₀greater than 4 μM for all the major P450 isoforms except

CYP1A2 (0.9 μM).

In the second part of the exploration, the ether position was

moved one position further down the apical side chain and the

compounds were prepared in agreement with the general

Scheme 2, and the results are reported in Table 2. Also in this

case, the first compound to be tested as racemate was the exo

intermediate alcohol 40. Compared to the corresponding

racemic derivative 2, this compound showed an average

increase of 5-fold in terms of affinity on the three transporters.

Methylation of the alcohol led to derivatives 41-43; the

increased chain length of 42 compared to derivative 5 led to

a 5-fold increase in the SERT activity, and a 2-3-fold increased

affinity in terms of NET and DAT. The compound was tested

for its ability to inhibit the uptake of the monoamines giving a

pIC₅₀of 8.80/9.40/7.80 on SERT, NET, and DAT respec-

tively. Once again, the lower activity observed on SERT

compared to the binding results has to be considered in light

of the explanation previously reported. The increase in length

of the ether chain from methyl to ethyl group (from 41 to 44)

did not show the same increase observed moving from 3 to 14,

possibly suggesting that the side chain had already reached the

best fitting or explored the maximum length tolerated by the

binding pocket. To probe if this was the case, some specific

heterocycles were prepared according to the synthetic

Scheme 3 and the results are reported in Table 2.

The oxazolyl derivative 45 was probably able to pick up no

more interactions than derivative 2, but its introduction was

not detrimental for activity, confirming the availability of

enough space in that region of the transporter; the slightly

more lipophilic thiazolyl derivative 46 actually acted in the

expected direction, increasing the affinity on the transporters

by about 5-fold, confirming the general trend achieved up to

that point of the exploration. Interestingly enough, the elon-

gation of such a molecule (47-49) led to the very potent

derivative 48; differently from the 2-naphthyl derivative 39, in

this case not only the SERT affinity was picomolar but also

the NET affinity was in the low nanomolar range with the

DAT comparable and about 100-fold lower, thus leading to

the identification of another potential dual activity derivative.

For confirmation, experiments of reuptake were performed

2540 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6

Micheli et al.

Table 3. Binding at the Three Transporters (SERT, NET, DAT) Expressed as pK_i^a

entry

stereochemistry

R

hSERT SPA pK_i

hNET SPA pK_i

hDAT SPA pK_i

50

51

52

53

54

55

56

57

58

59

60

exo

CH₂OEt

9.30

5.60

9.20

9.40

9.00

9.60

9.50

9.30

9.50

9.70

7.90

4.60

8.10

8.60

7.60

8.70

8.10

8.80

8.40

7.70

5.20

8.00

8.20

7.60

8.10

8.00

7.80

8.20

8.30

8.20

endo

exo

CH₂OCH₂c-Pr

CH₂Oc-butyl

exo

CH₂OCH₂CF₃

CH₂Osec-butyl

CH₂CH₂OMe

CH₂CH₂OEt

exo (rac)

exo

exo (rac)

exo

CH₂CH₂OiPr

CH₂CH₂Osec-butyl

CH₂CH₂Oc-butyl

^aSEM for hSERT/NET/DAT data sets is (0.1. (rac) = racemate, referred to the nature of the sec-butyl alcohol used.

Scheme 4. General Synthetic Procedures for the Preparation of Compounds 50-55^a

^aPG = H or BOC; (i) toluene, reflux; (ii) BH₃-THF, refluxing THF; (iii) (a) RX, NaH, DMF from 0 °C to rt, (b) TFA, CH₂Cl₂0 °C to rt.

Scheme 5. General Synthetic Procedures for the Preparation of Compounds 56-60^a

^aPG = H or BOC; (i) (a) BOC₂O, TEA, CH₂Cl₂, (b) Dess-Martin periodinane; (ii) Me(Ph)₃P^þBr^-, BuLi, THF; (iii) (a) BH₃-THF, THF, (b)

NaOH, H₂O₂30%; (iv) (a) or MsCl/TEA in CH₂Cl₂0 °C to rt followed by RONa in DMF, (b) TFA, CH₂Cl₂0 °C to rt.

and 48 gave pIC₅₀of 9.00/9.60/7.70 on SERT, NET, and

DAT, respectively.

onlytothe nature of thesec-butylalcohol used inthe coupling.

By comparing derivatives 50 and 51, it is possible to infer that

the extended exo derivatives appear to provide a better fitting

within the three transporters binding site, leading to a com-

pound with high affinity for all of them. Once again, this was

in agreement with the pharmacophore model predictions as

reported in Figure 4. Derivative 50 also showed a positive

P450 profile with IC₅₀values greater than 5 μM on all the

isoforms. Unfortunately, it was not as metabolically stable,

with Cl_ivalues slightly higher than in the previous template

(h-and r-Cli being 3.4 and 5.1 mL/min/g of protein, respectively).

The introduction of the cyclopropyl methyl substituent (52)

led to a slight DAT affinity increase, while a bulkier alpha

branched cyclo-butyl moiety (53) led to a substantial increase

To complete the exploration, given the new information

provided for the pharmacophore model, it was decided to

keep the side chain in position 6 of the bicyclic template and to

move also the 3,4-dichlorophenyl derivative in the same

position as a potential positive overlap might also have arisen

from this combination. The results of the exploration are

reported in Table 3, and the compounds were prepared

according to Scheme 4 for the alkoxy methylene substituent

and following Scheme 5 for the alkoxyethylene derivatives.

On this new scaffold, the presence of a plane of symmetry

leads to a pro-chiral situation in which the aryl ring can sit exo

or endo. In Table 2 the definition (rac) for racemate is referred

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2541

Figure 4. The exo derivative 50 fitted in the pharmacophore model in comparison with the endo isomer 51.

compound showed at least 100-fold selectivity over a wide

of both NET and DAT components, resulting in a very

balanced triple reuptake inhibitor. These modifications led

to a slight change in the P450 profile of the derivatives, with 52

having IC₅₀values greater than 10 μM on all the isoforms with

the exception of CYP2D6 and CYP3A4 DEF (IC₅₀= 1 and

4 μM, respectively) and 53 having IC₅₀values greater than

10 μM onall the isoforms with the exception of CYP3A4DEF

(IC₅₀= 3 μM). The introduction of a slight dipole (54,

-CH₂CF₃) with respect to 50 had no major effect on the

affinity at the three transporters but introduced a potential

liability in the P450 profile with the IC₅₀of direct inhibition of

CYP2D6 = 0.3 μM. The presence of an R branching (s-Butyl,

55) endowed with higher conformational freedom when

compared to 53 did not alter neither the primary affinity

profile nor the positive P450 profile. The introduction of a

further methylene to extend the position of the ether in the

space is represented by derivatives 56-60. For derivatives 56

and 57, no major effect on the primary affinity profile was

observed, but unfortunately 57 showed a potential liability in

the P450 profile (CYP2D6, IC₅₀< 0.1 μM). The introduction

of the R branching (58-60) had a positive effect on the

affinity, in particular on DAT being 60, another potent and

selective triple reuptake inhibitor, with no major effect on the

previously observed P450 profile.

Among the potent dual and triple reuptake inhibitors

identified in the exploration, it was decided to select some of

the best derivatives to be further characterized along the

screening cascade, and derivative 15 is here reported as a

model.

Further Characterization of Derivative 15. The first step for

a more complete characterization of 15 was the determina-

tion of its absolute stereochemistry. The stereochemistry of

this molecule and its enantiomer 16 were assigned using

vibrational circular dichroism (VCD),⁶and derivative 15

was identified as the (1S,5S,6S) isomer in accordance to the

methodology reported in the Experimental Section. Inter-

estingly enough, as reported in Figure 3, the pharmacophore

model was also able to correctly identify the chirality of best

fitting isomer 15.

range of receptors, with the most notable results here re-

ported (adrenergic R_1A, fpK_i= 6.7; 5-HT_2A, fpK_i= 6.8; H1,

fpK_i= 6.0; muscarinic acetylcholine receptor: M1, fpK_i=

6.8; M2, fpK_i= 6.7; M3, fpK_i= 7.2; M4, fpK_i= 6.6; M5,

fpK_i= 7.0). The compound was also submitted to a hERG

electrophysiology assay⁷to assess its overall activity for this

K^þchannel, and the results (IC₅₀4.2 μM) confirmed the

hERG binding affinity (pK_i= 4.9).

Considering the possibility to test 15 in vivo in preclinical

species, the compound was tested in rat native tissues

(filtration binding) where it showed an excellent profile with

pK_i= 9.17/9.87/7.87 on SERT, NET, and DAT, respec-

tively. The higher affinity on rat NET was also observed for

other compounds of the same series and was also common to

some standard derivatives. This is in line with a generally

increased affinity for rat NET observed for other compounds

in comparison to the human transporter and might support

the existence of a species difference in NET pharmacology.

Mouse SERT filtration binding data were also generated,

and the compound showed a pK_i= 9.07, in good agreement

with the above-reported rat data.

Considering its overall balanced profile in terms of pri-

mary activity and PK properties, and considering the desired

ratio among the human transporters with SERT g NET >

DAT (ratio ≈ 1/3/50), it was decided to explore the in vivo

behavior of the compound. Nonetheless, considering that

the rat profile showed a NET g SERT > DAT, a further

preliminary step was considered necessary to fully interpret

the final results. Because of its mode of action, 15 was

expected to increase the extracellular concentration of

monoamines as measured by in vivo microdialysis.

To test this hypothesis, microdialysis probes were im-

planted in the medial prefrontal cortex (MPC) in freely

moving rats with concurrent locomotor activity measure-

ment and quantification of NE, DA, and 5-HT brain levels⁸

by HPLC analysis and electrochemical detection. Derivative

15 was tested at 0.1, 0.3, and 1 mg/kg (ip) and, as reported in

Figure 5, at the highest dose tested, it induced a slow and

sustained increase in the levels of the monoamines that lasted

throughout the experiment (3 h post treatment); this was

particularly significant for DA levels. At the same time, an

increased locomotor activity was observed in rat at the same

The second step of the characterization, in agreement with

the screening cascade, was related to increasing the informa-

tion on the selectivity of the compound using a panel

of binding and functional assays available in house. The

2542 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6

Micheli et al.

Figure 5. Time course of the effect of 15 on norepinephrine (diamond), dopamine (square), and serotonin (triangle) outflow in the rat medial

prefrontal cortex. Arrow shows injection. Data are mean ( SEM of neurotransmitter concentration expressed as percentage of basal values.

Doses tested = 1 mg/kg, ip.

Figure 6. Time course of the effect of 15 on rat locomotor activity at 1 mg/kg ip.

dose (Figure 6). These results demonstrate that the acute

administration of 15 produced a slow onset/long lasting

increase in the extracellular levels of all monoamines in

MPC in rats, in agreement with the triple reuptake inhibitor

profile.

At this point, 15 was tested in three in vivo models in mice

to further confirm its ability to inhibit the monoamine

transport and produce antidepressant-like effects.

In the first of these experiments (Figure 7, right), it was

tested in the 5-hydroxytryptophan (5-HTP) potentiation

assay.⁹The compound was administered orally 2 h before

the experiment. The results demonstrate that 15 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 confirm

in vivo the inhibition of the 5-HT transporter.

In the second experiment (Figure 7, center), 15 was

evaluated for its effects on spontaneous locomotor activity

in the rat.¹⁰The compound was administered orally 2 h

before the experiment at 3, 10, and 30 mg/kg and it was

found to significantly increase locomotor activity at 10 mg/

kg., while the lack of effect at 30 mg/kg was unforeseen.

The data are consistent with an increased dopaminergic

neurotransmission due to the inhibition of the DA trans-

porter by 15.

In the last experiment, the potential antidepressant-like

effect of 15 was evaluated in the forced swimming test¹¹

(FST), which is sensitive to known antidepressant drugs

(Figure 7, left). Once again, the compound was administered

orally 2 h before the test in mice at 1, 3, and 10 mg/kg. As

clearly seen in the figure, 15 significantly reduced immobility

time at 3 and 10 mg/kg as predicted by its in vitro and

PK profile. However, the possibility that the increase in

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2543

Figure 7. Effects of derivative 15 in the FST model (left panel) on the spontaneous locomotor activity (middle panel) and in the 5-HTP model

(right panel) in mice.

locomotor activity (in rats) in the same dose range could have

affected the results of the FST cannot be ruled out.

using the Cheng-Prusoff equation. Competition binding assay

for human NET (hNET) was conducted essentially as pre-

viously reported for SERT except for the use of hNET-LLCPK

cell membranes (4.8 μg/well), 1.5 nM of [N-methyl-³H]-

nisoxetine as radioligand (Amersham Biosciences, 84 Ci/mmol),

and 10 μM desipramine for NSB. Finally, the same procedure

was adopted also for human DAT (hDAT) competition binding

assay but using hDAT/LLCPK cell membranes (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.

Conclusions

The planned introduction of an appropriate side chain into

a 1-(aryl)-3-azabicyclo[3.1.0]hexane template and its transfor-

mation into a 6-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]-

hexane scaffold led to the identification of two different

classes of potent and selective triple reuptake inhibitors. These

moleculesareendowedwithan excellent developabilityprofile

and showed activity in preclinical animal models related to the

increase of the monoamines.

Derivative 15, in particular, was used to probe the role of a

system in which SERT, NET, and DAT are appropriately

chosen in a given ratio. MD and in vivo experiments per-

formed on this molecule clearly indicate its mode of action

through monoamine uptake inhibition.

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

compound was determined on rat or mouse cortex for SERT,

on rat hippocampus for NET, and on rat striatum for DAT by

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).

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 hu-

man SERT (hSERT) was conducted in 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

binding) or a final concentration of 10 μM fluoxetine (to

define nonspecific binding, NSB), 200 μL of 0.25nM [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% polyethylenimmine

(PEI) using a Perkin-Elmer FilterMat-196 harvester. Filterplate

was washed 3 times with 1 mL/well ice-cold 0.9% NaCl solution,

dried, and finally 50 μL of Microscint 20 (Perkin-Elmer) were

added to each well. The radioactivity was counted using Top-

Count liquid scintillation counter (Packard-Perkin-Elmer) and

recorded as counts per minute (CPM). Data analysis was

performed by using a 4-parameter logistic equation with Graph-

Pad PRISM software and pK_ihas been calculated from pIC₅₀by

hERG-[³H] Dofetilide Binding Assay. hERG activity was

measured using ³H dofetilide binding in a scintillation proximity

assay (SPA) format. The activity was measured with a Perki-

nElmer 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]citalopram, [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 were added by 30 μL of the SPA mixture,

containing 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 of 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 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 View-

lux instrument. Data analysis was performed by using a four-

parameter logistic equation with ActivityBase software and pK_i

has been calculated from pIC₅₀by using the Cheng-Prusoff

equation.

2544 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6

Micheli et al.

Figure 8. Upper panel: signal-to-noise and raw VCD spectra of 15 (red) and 16 (blue). Lower panel: baseline corrected VCD spectra of 15 (red)

and 16 (blue). Bands in the experimental assignment set have been labeled in raw and baseline corrected VCD data.

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 were

added by 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), 25000 or 50000 or 75000 cell/well of SERT or NET or

DAT LLCPK cells, 0.02% Pluronic F-127 in the assay buffer

(20 mM HEPES, 145 mM NaCl, 5 mM KCl, pH 7.4). The

uptake was started by the 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. Incuba-

tion was performed at room temperature 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, UK) 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 glucose 6-phosphate, 1.7 mg NADP and 6 units

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 v5 (Erithacus software, UK).

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 (Cl_i) Assay. Intrinsic clearance (CL_i)

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

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

containing an appropriate internal standard, and analyzed by

HPLC-MS/MS. The intrinsic clearance (CL_i) was determined

from the first-order elimination constant by nonlinear regres-

sion using Grafit v5 (Erithacus Software, UK), corrected for the

volume of the incubation and assuming 52.5 mg microsomal

protein/g of protein for all species. Values for CL_iwere ex-

pressed as mL/min/g of protein. The lower limit of quantifica-

tion of clearance was determined to be when <15% of the

compound had been metabolized by 30 min and this corre-

sponded to a CL_ivalue of 0.5 mL/min/g of protein. The upper

limit was 50 mL/min/g of protein.

VCD. Experimental VCD and IR spectra were acquired in the

2000-800 cm^-1frequency range using a BioTools ChiralIR FT-

VCD spectrometer operating at 4 cm^-1resolution. Samples

were analyzed in CDCl₃at approximate concentrations of

10 mg/125μL. Harmonic frequencies and corresponding vibra-

tional intensities were calculated using Gaussian 98 ((Gaussian

‘98, Gaussian, Inc., Pittsburgh, PA (www.gaussian.com/index.

htm)). Predicted gas-phase harmonic frequencies were shifted to

the condensed phase region using a uniform scaling factor of

0.98.

The observed and baseline-corrected VCD data for 15 and

16 are shown in Figure 8. The signs of the 15 marker bands

identified in these spectra were used to assign the configura-

tions. The VCD and IR spectra observed for 15 are compared

in Figure 9a with VCD and IR spectra calculated for a full

structure model with (1S,5S,6S) configuration. In the upper

panel of this figure, marker bands in the observed VCD

spectrum have the same signs as corresponding marker

bands in the calculated VCD spectrum, indicating that this

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2545

Figure 9. (a) Upper panel: VCD spectrum of 15 (red) vs calculated VCD spectrum (black). Lower panel: IR spectrum of 15 (red) compared

with calculated IR spectrum (black). Bands in the experimental assignment set have been labeled in both experimental and calculated VCD

spectra. (b) Upper panel: VCD spectrum of 16 (blue) vs calculated VCD spectrum (black). Lower panel: IR spectrum of 16 (blue) compared

with calculated IR spectrum (black). Bands in the experimental assignment set have been labeled in both experimental and calculated VCD

spectra.

enantiomer has the same absolute configuration as the mod-

el. Therefore, 15 was assigned as the (1S,5S,6S)-isomer. The

VCD and IR spectra observed for 16 are compared in

Figure 9b with the same calculated and VCD and IR spectra.

In this case, marker bands in the experimental VCD spectrum

are oppositely signed relative to the corresponding marker

bands in the calculated VCD spectrum, indicating that 16 is

the mirror image enantiomer of the model, and therefore was

assigned as the (1R,5R,6R)-isomer. Close overall agreement

between calculated and observed VCD and IR spectra in

Figures 9a and 9b indicates that these assignments are highly

reliable.

guidelines of the “Principles of Laboratory Animal Care” (NIH

publication no. 86-23, revised 1985), and with a project license

that was obtained according to Italian law (Art. 7, Legislative

Decree no. 116, 27 January 1992), which acknowledges Eur-

opean Directive 86/609/EEC on the care and welfare of labora-

tory animals.

Naive male CD-1 mice (Charles River, Italy) weighing 25-30 g

were used in the 5-HTP potentiation test, in the forced swim-

ming test, and for the assessment of spontaneous locomotor

activity.

5-HTP Potentiation. The administration of the precursor of

5-hydroxytryptophan (5-HTP) to mice leads to a serotonergic

syndrome characterized by stereotypic behavior. The combina-

tion monoamine reuptake inhibitors with subthreshold doses of

In Vivo Studies. All experiments were prereviewed and ap-

proved by a local animal care committee in accordance with the

2546 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6

Micheli et al.

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 15 120 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 were scored for the

following 30 min: aimless hyper locomotor 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

observer 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 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 an observer who was una-

ware of the drug treatment scored immobility.

Compound 15 was administered orally 120 min before test.

Spontaneous Locomotor Activity. Spontaneous locomotor

activity was measured by using activity boxes equipped with

infrared monitoring sensors (AccuScan model RXYZXCM-16,

Columbus, OH). Each box (40 cm ꢀ 40 cm ꢀ 30.5 cm) was made

of clear plexiglas and covered with a plexiglas lid with air-holes

with infrared sensors located along the perimeter 2.5 cm above

the floor. Data were collected and analyzed (AccuScan model

CDA-8, Columbus, OH). Animals were treated orally with

compound 15 120 min before test, and spontaneous locomotor

activity quantified as total distance traveled, was recorded over

30 min.

mM, CaCl₂1.3 mM, MgCl₂1.18 mM, Na₂HPO₄2 mM, pH 7.4

with H₃PO₄85%) at a steady flow rate of 1.1 μL/min, and the

second channel was connected to a refrigerated fraction collec-

tor (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 al-

lowed before starting the experiment.

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.

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 pg were transformed in % values vs average of the three basal

values. They were preliminary analyzed with 2D Box and

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 com-

parisons were applied to compare time points of different

treatments.

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 every day to get accustomed to manipulation and

presence 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: þ 1.7 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 ru-

brocillin antibiotic and housed in single cages for one week to

recover.

Microdialysis Experiment. The day before the experiment the

stylette was removed from the guide cannula and a microdialysis

probe with a 4-mm-long membrane was inserted (MAB 4

Agntho’s, Sweden). The following day microdialysis experi-

ments were performed in their home cage. Both inlet and outlet

of the probe were attached with FEP tubings (dead volume 1.2

μL/10 cm) to a dual quartz lined two-channel liquid swivel

(model 375/D/22QM Instech Laboratories, Plymouth Meeting

PA) mounted on a low mass spring counterbalanced arm

(MCLA, Instech). One channel was connected to a gastight

syringe (Hamilton 1002 LTN 2.5 mL, Bonaduz, Switzerland) on

a microinfusion pump (the Univentor 802 syringe pump) to

deliver artificial cerebrospinal fluid (KCl 2.5 mM, NaCl 125

Concurrent general motor activity was measured through an

automated digital video analysis of animal movements

(ViewPoint). Data were analyzed as previously described for

microdialysis data.

Chemical Procedures. General. Experimental Section. Proton

magnetic resonance (NMR) spectra are typically recorded either

on Varian instruments at 300, 400, or 500 MHz or on a Bruker

instrument at 300 and 400 MHz. Chemical shifts are expressed

in δ (ppm) units, and peak multiplicity are expressed as follows:

singlet (s), doublet (d), doublet of doublets (dd), triplet (t),

multiplet (m), broad singlet (br s), broad multiplet (br m).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 abundant 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 spectro-

meter, operating in ES (þ) and ES (-) ionization mode coupled

with HPLC instrument Agilent 1100 series [LC/MS-ES (þ):

analysis performed on a Supelcosil ABZ þPlus (33 mm ꢀ 4.6

mm, 3 μm) (mobile phase: 100% [water þ 0.1% HCO₂H] for 1

min, then from 100% [water þ 0.1% HCO₂H] to 5% [water þ

0.1% HCO₂H] and 95% [CH₃CN ] in 5 min, finally under these

conditions for 2 min; T = 40 °C; flux = 1 mL/min; LC/MS-ES

( ): analysis performed on a Supelcosil ABZ þPlus (33 mm ꢀ 4.6

mm, 3 μm) (mobile phase: 100% [water þ 0.05% NH₃] for 1 min,

then from 100% [water þ 0.05% NH₃to 5% [water þ 0.05%

NH₃] and 95% [CH₃CN ] in 5 min, finally under these condi-

tions for 2 min; T = 40 °C; flux = 1 mL/min]; in the mass

spectra, only one peak in the molecular ion cluster is reported.

DAD chromatographic traces, mass chromatograms, and

mass spectra may be taken on a on a UPLC/MS Acquity system

coupled with a Micromass ZQ mass spectrometer operating in

ESI positive or negative. The phases used are: (A) H₂O/ACN 95/

5 þ 0.1% TFA; (B) H₂O/ACN 5/95 þ 0.1% TFA. The gradient

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2547

is: t = 0 min, 95%A 5%B; t = 0.25, 95%A 5%B; t = 3.30,

100%B, t = 4.0) 100%B, followed by 1 min of reconditioning.

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 Var-

ian. The eluent used with SPE-SCX cartridges is methanol

followed by 2N ammonia solution in methanol. In a number

of preparations, purification was performed using either Bio-

tage manual flash chromatography (Flashþ) or automatic flash

chromatography (Horizon, SP1) systems. All these instruments

work with Biotage Silica cartridges. SPE-Si cartridges are silica

solid phase extraction columns supplied by Varian.

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 >95%.

General Synthetic Procedures. 3-(3,4-Dichlorophenyl)-1H-

pyrrole-2,5-dione (62). To a stirred slurry of maleimide (61),

anhydrous CuCl₂and tert-butyl nitrite in CH₃CN at 0 °C a

solution of 3,4-dichloro aniline in CH₃CN was added dropwise.

The reaction mixture was stirred at room temperature for 1 h,

and 20% aqueous HCl was added. The mixture was extracted

with ethyl acetate, and the organic layer was washed with

saturated aqueous NaCl and dried over Na₂SO₄. To a solution

of the crude obtained in 2-propanol, 2,6-lutidine was added and

the mixture was warmed at reflux for 30 min. After elimination

of the solvent under vacuum, the crude was dissolved in ethyl

acetate and the organic phase washed with water and dried over

sodium sulfate. The organic phase was concentrated under

vacuum and the crude treated with diethyl ether. The solid

was filtrated and dried under vacuum to give the title compound

as brown solid. MS m/z 241 [M - H]^-.

Ethyl-1-(3,4-dichlorophenyl)-2,4-dioxo-3-azabicyclo[3.1.0]hexane-

6-carboxylate (63). Sodium hydroxide 60% in mineral oil was

added in small portions to a stirred solution of (ethoxy-

carbonylmethyl)-dimethylsulfonium bromide in anhydrous

DMSO (20 mL). The resulting mixture was allowed to stir at

room temperature for 1.5 h and then 3-(3,4-dichlorophenyl)-

1H-pyrrole-2,5-dione (62) dissolved in DMSO (20 mL) was

added dropwise, and the resulting mixture was stirred at room

temperature for 1 min. Reaction temperature was brought to

0 °C, and aqueous saturated NH₄Cl was slowly added, followed

by Et₂O. After separation of the two phases, the organic layer was

washed twice with water and brine, and dried over Na₂SO₄.

Evaporation of the solvent under vacuum gave a crude com-

pound which was purified by flash chromatography (eluting

with ethyl acetate/cycloesane 20:80) to give the title compound.

MS m/z 326 [M - H]^-.

[1-(3,4-Dichlorophenyl)-3-azabicyclo[3.1.0]hex-6-yl]methanol

(2). To a stirred solution of 63 in of dry THF, BH₃-THF

complex in THF was slowly added at 0 °C under N₂. The

reaction mixture was refluxed for 6 h and then cooled to 0 °C,

and aqueous HCl was added cautiously and the reaction mixture

stirred for 1 h. The solvent was partially removed under vacuum,

and the residue was loaded on mixed-mode strong cation

exchange (MCX) column eluting with NH₃/MeOH (2M). The

methanolic phase was evaporated under vacuum and the crude

was purified by flash chromatography and separated through

chiral HPLC to give the title compound. NMR (¹H, CDCl₃): δ

7.40 (m, 2H), 7.38 (d, 1H), 7.14 (dd, 1H), 3.51 (dd, 1H), 3.41 (dd,

1H), 3.32 (d, 1H), 3.12 (m, 2H), 2.91 (d, 1H), 1.69 (m, 1H), 1.39

(m, 1H). MS m/z 258 [MH]^þ.

then the reaction mixture was concentrated under vacuum and

the crude product treated with dichloromethane and bicarbo-

nate. The organic phase was dried over sodium sulfate and the

solvent evaporated under vacuum to give a crude product. To a

stirred solution of this crude material in dry DMF, sodium

hydride was added at 0 °C and the reaction mixture stirred at

room temperature for 1 h. Methyl iodide was added, and the

reaction mixture was stirred at room temperature for 4 h. Ethyl

acetate and water were added and the organic phase separated,

washed with brine, dried over sodium sulfate, and the solvent

evaporated under vacuum to give a crude product. To a solution

of this crude in DCM, TFA was added. The reaction mixture

was stirred at room temperature for 1 h, it was concentrated in

vacuo and the crude product was loaded on SCX column eluting

with MeOH/NH₃. The crude material obtained was purify by

flash chromatography (eluting with dichloromethane/methanol/

ammonia aq 95:5:0.5) to give 5 mg of the title compound as

white oil. This was submitted to semipreparative HPLC to give

the separated enantiomers by using a chiral column chiralpak

AD-H, eluent A: n-hexane; B: ethanol, gradient isocratic 18% B,

flow rate 14 mL/min, detection UV at 230 nm. Retention times

given were obtained using an analytical HPLC using a chiral

column chiralpak AD-H, 25 cm ꢀ 4.6 cm, eluent A: n-hexane; B:

ethanol, gradient isocratic 20% B, flow rate 0.8 mL/min, detec-

tion UV at 230 nm. NMR (¹H, CDCl₃): δ 7.41 (d, 1H), 7.39 (d,

1H), 7.16 (dd, 1H), 3.30 (d, 1H), 3.21 (s, 3H), 3.20-3.10 (m, 4H),

3.90 (d, 1H), 1.74 (m, 1H), 1.37 (m, 1H). MS m/z 272 [MH]^þ.

Retention time = 8.54 min.

Other compounds were prepared similarly. Further details

can be found in refs 12 and 13.

6-{[(Cyclopropylmethyl)oxy]methyl}-1-(3,4-dichlorophenyl)-

3-azabicyclo[3.1.0]hexane (9). NMR (¹H, DMSO): δ 9.86-8.13

(br s, 2H), 7.73.7.70 (d, 1H), 7.62-7.58 (d, 1H), 7.42-7.36 (dd,

1H), 3.81-3.70 (d, 1H), 3.52-3.45 (dd, 1H), 3.41-3.36 (d, 1H),

3.28-3.22 (dd, 1H), 3.20-3.14 (d, 1H), 3.09-3.02 (dd, 1H),

2.95-2.80 (m, 2H), 2.24-2.14 (m, 1H), 1.68-1.54 (m, 1H),

0.88-0.74 (m, 1H), 0.45-0.20 (m, 2H), -0.06-0.09 (m, 2H).

Retention time = 7.54 min.

1-(3,4-Dichlorophenyl)-6-[(propyloxy)methyl]-3-azabicyclo-

[3.1.0]hexane (13). NMR (¹H, CDCl₃): δ 9.24 (br s 2H), 7.72 (d,

1H), 7.60 (d, 1H), 7.39 (dd, 1H), 3.78 (d, 1H), 3.50 (dd, 1H), 3.40

(d, 1H), 3.23 (m, 1H), 3.16 (m, 2H), 2.97 (m, 1H), 2.91 (dd, 1H),

2.22 (m, 1H), 1.66 (m, 1H), 1.34 (m, 2H), 0.73 (t, 3H). MS m/z

300 [MH]^þ. Retention time = 5.49 min.

1-(3,4-Dichlorophenyl)-6-[(ethyloxy)methyl]-3-azabicyclo-

[3.1.0]hexane (15). NMR (¹H, CDCl₃): δ 7.43 (s, 1H), 7.37 (d,

1H), 7.18 (d, 1H), 3.41-3.20 (m, 4H), 3.15 (s, 2H), 3.07 (m, 1H),

2.91 (d, 1H), 1.94 (m, 1H), 1.63 (m, 1H), 1.38 (m, 1H), 1.13 (t,

3H), NH not observed. MS m/z 286 [MH]^þ. Retention time =

7.0 min.

6-[(Ethyloxy)methyl]-3-methyl-3-azabicyclo[3.1.0]hexane (18).

NMR (¹H, CDCl₃): δ 7.42 (s, 1H), 7.35 (d, 1H), 7.15 (d, 1H),

3.38-3.28 (m, 2H), 3.26-3.12 (m, 3H), 3.05-2.97 (m, 1H), 2.54

(m, 1H), 2.33 (s, 3H), 2.32 (m, 1H), 1.98 (m, 1H), 1.61 (m, 1H),

1.07 (t, 3H). MS m/z 300 [MH]^þ.

1-(3,4-Dichlorophenyl)-6-{[(2,2,2-trifluoroethyl)oxy]methyl}-

3-azabicyclo[3.1.0]hexane (20). NMR (¹H, CDCl₃) δ 7.41 (m,

2H), 7.16 (d, 1H), 3.67-3.51 (m, 3H), 3.35 (d, 1H), 3.27 (t, 1H),

3.16 (m, 2H), 2.92 (d, 1H), 1.67 (s, 1H), 1.41 (m, 1H). MS m/z 340

[MH]^þ. Retention time = 6.0 min.

1-(3,4-Dichlorophenyl)-6-{[(1-methylethyl)oxy]methyl}-3-aza-

bicyclo[3.1.0]hexane (23). NMR (¹H, CDCl₃): δ ppm 7.48 (1 H,

d), 7.39 (1 H, d), 7.19 (1 H, dd), 3.36-3.46 (2 H, m), 3.24-3.35 (3

H, m), 2.94-3.06 (2 H, m), 1.65-1.71 (1 H, m), 1.44-1.53 (1 H,

m), 1.08 (3 H, d), 1.01 (3 H, d). MS m/z 300 [MH]^þ. Retention

time = 8.6 min.

1-(3,4-Dichlorophenyl)-6-[(methyloxy)methyl]-3-azabicyclo-

[3.1.0]hexane (4). To a stirred solution of 2 in dichloromethane

at room temperature, triethylamine and bis(1,1-dimethylethyl)

dicarbonate were added. Stirring was continued for 6 h, and

6-[(Cyclobutyloxy)methyl]-1-(3,4-dichlorophenyl)-3-azabicy-

clo[3.1.0]hexane Hydrochloride (25). NMR (¹H, CDCl₃): δ ppm

7.42-7.49 (m, J = 1.26 Hz, 1 H), 7.16 (dd, J = 8.15, 1.45 Hz,

1 H), 3.65-3.78 (m, 1 H), 3.33 (d, J = 11.49 Hz, 1 H), 3.13-3.23

2548 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6

Micheli et al.

(m, 3 H), 2.87-3.04 (m, 2 H), 1.97-2.20 (m, 4 H), 1.80-1.92 (m,

1 H), 1.58-1.72 (m, 3 H), 1.33-1.51 (m, 2 H). MS m/z 312

[MH]^þ. Retention time = 7.9 min.

6-[(Cyclopentyloxy)methyl]-1-(3,4-dichlorophenyl)-3-azabicy-

clo[3.1.0]hexane (28). NMR (¹H, CDCl₃): δ ppm 7.46 (d, 1 H),

7.37 (d, 1 H), 7.17 (dd, 1 H), 3.60-3.68 (m, 1 H), 3.30-3.39 (m, 2

H), 3.13-3.17 (m, 2 H), 2.87-2.97 (m, 2 H), 1.30-1.86 (m, 11

H). MS m/z 326 [MH]^þ. Retention time = 5.2 min.

6-[(Cyclohexyloxy)methyl]-1-(3,4-dichlorophenyl)-3-azabicy-

clo[3.1.0]hexane (30). NMR (¹H, CDCl₃): δ ppm 10.43 (br s,

1 H), 9.72 (br s, 1 H), 7.51 (d, 1 H), 7.44 (d, 1 H), 7.20 (dd, 1 H),

3.69-3.79 (m, 1 H), 3.54-3.69 (m, 2 H), 3.31-3.42 (m, 2 H),

2.95-3.09 (m, 2 H), 1.98 (t, 1 H), 1.57-1.86 (m, 5 H), 1.44-1.53

(m, 1 H), 1.11-1.23 (m, 5 H). MS m/z 340 [MH]^þ.

The enantiomers of the title compound were separated by

chiral HPLC by using a chiral column Chiralcel OJ-H (25 cm ꢀ

0.46 cm), eluent A: n-hexane; B: 2-propanol þ 0.1% isopropyl-

amine, isocratic 4% B, 10% B from 27 min, flow rate 1 mL/min,

detection UV at 210-340 nm, CD 230 nm. Retention times

given were obtained using an analytical HPLC using a chiral

column Chiralcel OJ-H (25 cm ꢀ 0.46 cm), eluent A: n-hexane;

B: 2-propanol þ 0.1% isopropylamine, isocratic 4% B, flow rate

1 mL/min, detection UV at 210-340 nm, CD 230 nm. NMR

(¹H, CDCl₃): δ ppm 7.42-7.32 (m, 2 H), 7.11 (m, 1 H), 3.32 (d, 1

H), 3.17 (s, 2H), 2.95 (d, 1 H), 2.40 (m, 1 H), 2.20-2.10 (m, 1 H),

2.10 (s, 3 H), 2.06 (bs, 1 H), 1.68 (m, 1 H), 1.32 (m, 1 H). MS m/z

288 [MH]^þ. Retention time = 17.2 min.

1-(3,4-Dichlorophenyl)-6-propyl-3-azabicyclo[3.1.0]hexane (36).

To a stirred solution of 2 in dichloromethane at room tempera-

ture, triethylamine and bis(1,1-dimethylethyl) dicarbonate were

added. Stirring was continued for 6 h and then the reaction

mixture was diluted with dichloromethane and quenched with a

saturated solution of NH₄Cl. The organic phase was dried, and

the solvent evaporated under vacuum. The compound was

dissolved in THF, and triethylamine and methanesulfonyl

chloride were added at 0 °C. The reaction mixture was stirred

at 25 °C for 1 h, and then water was added and the mixture was

extracted with dichloromethane. The organic phase was washed

with an aqueous saturated NH₄Cl solution, dried over Na₂SO₄,

and concentrated in vacuo. The compound was dissolved in

THF and added to a stirred solution of copper(I) bromide-

dimethylsulfide complex and EtMgBr 3 M in diethyl ether

kept at -78 °C for 30 min. The mixture was heated to room

temperature and stirred for 3 h. The reaction mixture was

quenched saturated NH₄Cl aqueous solution and filtered off

through celite. The filtrate was washed with an ammonia

solution and extracted with ethyl acetate. The extract was dried

over Na₂SO₄and concentrated in vacuo. The residue dissolved

in CH₂Cl₂, and TFA was added at 0 °C. The reaction was left for

2 h at rt, and it was concentrated in vacuo; the residue was

purified by a SCX cartridge. NMR (¹H, CDCl₃): δ ppm 9.66 (bs,

1H), 7.45 (d, 1 H), 7.30 (d, 1H), 7.09 (m, 1 H), 3.70 (d, 1 H), 3.56

(m, 2H), 3.36 (bs, 1H), 1.8 (s, 1 H), 1.48 (s, 1 H), 1.38-1.28 (m, 3

H), 0.90-0.78 (m, 3 H). MS m/z 271 [MH]^þ. Retention time =

17.2 min.

1-(3,4-Dichlorophenyl)-6-{[(4-fluorophenyl)oxy]methyl}-3-aza-

bicyclo[3.1.0]hexane (31). NMR (¹H, CDCl₃): δ ppm 7.43 (s, 1

H), 7.36 (d, 1 H), 7.18 (d, 1 H), 6.92 (m, 2 H), 6.70 (m, 2 H), 3.82

(m, 1 H), 3.60 (m, 1 H), 3.36 (d, 1 H), 3.19 (s, 2 H), 2.95 (d, 1 H),

2.15 (bs, 1 H) 1.75 (m, 1 H), 1.58 (m, 1 H). MS m/z 352 [MH]^þ.

1-(3,4-Dichlorophenyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl}-

dimethylamine (32). To a stirred solution of 2 in dichloro-

methane at room temperature, triethylamine and bis(1,1-

dimethylethyl) dicarbonate were added. Stirring was continued

for 6 h and then the reaction mixture was diluted with dichlor-

omethane and quenched with a saturated solution of NH₄Cl.

The organic phase was dried and the solvent evaporated under

vacuum. Dess-Martin periodinane was added at 25 °C, and

after 30 min, sodium thiosulfate and a saturated NaHCO₃

aqueous solution were added and the mixture was stirred at

room temperature for 30 min. The mixture was then extracted

with dichloromethane; the organic phase was washed with

brine, dried over Na₂SO₄, and concentrated in vacuo. The

compound was dissolved in dry tetrahydrofuran, and dimethyl-

amine (0.349 mL), acetic acid (0.043 mL), and sodium triace-

toxyborohydride (173 mg) were added at 25 °C. After 1 h stirring

at room temperature, the mixture was concentrated in vacuo.

Dichloromethane was then added, and the organic phase was

washed with an aqueous saturated NaHCO₃solution and brine,

dried over Na₂SO₄, and concentrated in vacuo. The crude

material was purified by flash chromatography (Biotage Si

12S column, gradient cyclohexane/ethyl acetate from 90/10 to

0/100 and then ethyl acetate/methanol 100/1) to give the title

compound as a colorless oil. NMR (¹H, CDCl₃): δ ppm

7.40-7.32 (m, 2 H), 7.09 (m, 1 H), 3.30 (d, 1 H), 3.15 (s, 2H),

2.96 (d, 1 H), 2.40 (m, 1 H), 2.20 (s, 6 H), 2.19 (bs, 1 H),

1.75-1.62 (m, 2 H), 1.20 (m, 1 H). MS m/z 285 [MH]^þ.

1-(3,4-Dichlorophenyl)-6-[(methylthio)methyl]-3-azabicyclo-

[3.1.0]hexane (34). To a stirred solution of 2 in dichloromethane

at room temperature, triethylamine and bis(1,1-dimethylethyl)

dicarbonate were added. Stirring was continued for 6 h and then

the reaction mixture was diluted with dichloromethane and

quenched with a saturated solution of NH₄Cl. The organic

phase was dried and the solvent evaporated under vacuum.

The compound was dissolved in THF, and triethylamine and

methanesulfonyl chloride were added at 0 °C. The reaction

mixture was stirred at 25 °C for 1 h, and then water was added

and the mixture was extracted with dichloromethane. The

organic phase was washed with an aqueous saturated NH₄Cl

solution, dried over Na₂SO₄, and concentrated in vacuo. It was

dissolved in dry N,N-dimethylformamide, and sodium thio-

methoxide was added. The mixture was stirred overnight at

room temperature. An aqueous saturated NaHCO₃solution

was then added, and the mixture was extracted with dichloro-

methane. The organic phase was washed with brine, dried over

Na₂SO₄, and concentrated in vacuo and dissolved in dry di-

chloromethane; trifluoroacetic acid was added at room tem-

perature. After 30 min, the reaction mixture was concentrated in

vacuo and the residue was purified by a SCX cartridge to give

the title compound.

6-[(Methyloxy)methyl]-1-(2-naphthalenyl)-3-azabicyclo[3.1.0]-

hexane (39). The racemate was separated by SFC HPLC using a

chiral column Chiralcel OD-H, 25 cm ꢀ 0.46 cm, eluent ethanol

þ 0.1% isopropylamine 25%, flow rate 2 mL/min, detection UV

at 230 nm. Retention time = 12.7 min. NMR (¹H, CDCl₃): δ

7.86-7.76 (m, 4H), 7.52-7.45 (m, 3H), 3.44 (d, 1H), 3.28-3.25

(m, 3H), 3.19 (s, 3H), 3.15-3.04 (m, 2H), 1.84 (m, 1H), 1.45

(m, 1H).

1-(3,4-Dichlorophenyl)-3-azabicyclo[3.1.0]hex-6-yl]ethanol (40).

To a stirred solution of 2 in dichloromethane at room tempera-

ture, triethylamine and bis(1,1-dimethylethyl) dicarbonate were

added. Stirring was continued for 6 h and then the reaction

mixture was diluted with dichloromethane and quenched with a

saturated solution of NH₄Cl. The organic phase was dried and

the solvent evaporated under vacuum. Dess-Martin period-

inane was added at 25 °C, and after 30 min, sodium thiosulfate

and a saturated NaHCO₃aqueous solution were added and the

mixture was stirred at room temperature for 30 min. The

mixture was then extracted with dichloromethane; the organic

phase was washed with brine, dried over Na₂SO₄, and concen-

trated in vacuo to give the crude 64. To a stirred suspension

of methyl(triphenyl)phosphonium bromide in THF at 0 °C,

butyllithium (2.5 M in hexane) was added dropwise. The dark-

yellow reaction mixture was allowed to reach room tempera-

ture and stirred for 20 min and then cooled to 0 °C, and the

above-described crude 64 in THF was added dropwise. The ice-

bath was removed and the reaction mixture stirred for 6 h

at room temperature. Diethyl ether and water were added,

the organic phase was dried on sodium sulfate, the solvent

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2549

evaporated under vacuum, and the crude product was purified by

flash chromatography to achieve intermediate 65. The inter-

mediate was dissolved in THF at 0 °C, and under a nitrogen

atmosphere, BH₃-THF complex (1 M in THF) was added

dropwise. The ice-bath was removed and the reaction mixture

stirred for 3.5 h at room temperature. The mixture was cooled to

0 °C and quenched by adding water. Then 3 M NaOH and 30%

H₂O₂were added and the resulting mixture stirred for 0.5 h. The

reaction mixture was diluted with water and extracted with ethyl

acetate; the organic phase was dried over Na₂SO₄and the

solvent removed under reduced pressure. The compound was

dissolved in CH₂Cl₂, and TFA was added at room temperature.

After 1 h, the reaction mixture was evaporated under vacuum,

the residue dissolved in methanol, and 1 M NaOH was added.

After 0.5 h, the solvent was removed under vacuum and the

residue dissolved in CH₂Cl₂. The organic phase was separated

through a separator phase cartridge and the solvent evaporated

under reduced pressure to give the title compound.

sulfate. The solvent was evaporated under reduced pressure to

give a crude product, which was purified by flash chromatog-

raphy. This compound was dissolved in CH₂Cl₂, TFA was

added, and the reaction mixture was stirred at rt for 1 h. The

reaction mixture was concentrated under reduced pressure and

the residue was partitioned between DCM and saturated aqu-

eous NaHCO₃. The mixture was passed through a phase

separator cartridge and the organic phase evaporated to give

crude title compound. NMR (¹H, CDCl₃): δ ppm 7.30-7.40 (m,

2 H) 7.05-7.10 (m, 1 H) 6.58-6.61 (m, 1 H) 2.91-3.45 (m, 4 H)

2.40-2.61 (m, 2 H) 2.20-2.30 (m, 3 H) 1.72-1.79 (m, 1 H)

1.48-1.59 (m, 1 H)). MS m/z 323.05 [MH]^þ.

1-(3,4-Dichlorophenyl)-6-(4-methyl-1,3-thiazol-2-yl)-3-azabi-

cyclo[3.1.0]hexane (46). To a stirred solution of 2 in dichlor-

omethane at room temperature, triethylamine and bis(1,1-

dimethylethyl) dicarbonate were added. Stirring was continued

for 6 h and then the reaction mixture was diluted with dichlor-

omethane and quenched with a saturated solution of NH₄Cl.

The organic phase was dried and the solvent evaporated under

vacuum. The compound was dissolved in acetone, and at 0 °C,

Jones reagent was added dropwise. After quenching and work

up, the compound was dissolved in AcOEt and N,N⁰-carbonyl-

diimidazole was added. The mixture was stirred for 1.5 h at rt.

The mixture was then cooled to 0 °C, and concentrated NH₄OH

was added. After quenching and recovery of the compound,

Lawesson’s reagent was used to prepare the corresponding

thioamide, which was subsequently dissolved in toluene and

chloroacetone was added. The reaction mixture was heated at

80 °C for 3 h. On cooling to room temperature, the solvent was

removed under vacuum and the residue taken up in CH₂Cl₂.

TFA was added, and the reaction mixture was stirred for 2 h at

rt. The reaction mixture was concentrated under reduced pres-

sure, the residue dissolved in CH₂Cl₂, and purified using a SCX

cartridge. NMR (¹H, CDCl₃): δ ppm 7.19-7.36 (m, 2 H) 7.01

(dd, 1 H) 6.47-6.61 (m, 1 H) 3.05-3.54 (m, 4 H) 2.67-2.73 (m, 1

H) 2.47-2.54 (m, 1 H) 2.30-2.34 (m, 3 H). MS m/z 325 [MH]^þ.

1-(3,4-Dichlorophenyl)-6-[(4-methyl-1,3-thiazol-2-yl)methyl]-

3-azabicyclo[3.1.0]hexane (47). Derivative N-BOC protected

derivative 40 was dissolved in acetone, and Jones reagent was

added dropwise 0 °C. After quenching and work up, the

compound was dissolved in EtOAc and N,N⁰-carbonyldiimida-

zole followed by concentrated NH₄OH at 0 °C. To a stirred

solution of the crude amide in THF at rt, Lawesson’s reagent

was added portion wise. The mixture was heated to 80 °C and

stirred for 3 h. The intermediate thioamide obtained was reacted

with toluene, and the mixture was stirred for 3 h at 80 °C. The

solution was concentrated under reduced pressure to give a

residue. This product was dissolved in CH₂Cl₂, TFA was added,

and the reaction mixture was stirred at rt for 1 h. The reaction

mixture was concentrated under reduced pressure and purified

to give the title product. NMR (¹H, CDCl₃): δ ppm 7.31-7.41

(m, 2 H) 7.05-7.12 (m, 1 H) 6.70-6.74 (m, 1 H) 3.30-3.40 (m, 1

H) 3.12-3.19 (m, 2 H) 2.86-3.01 (m, 2 H) 2.56-2.66 (m, 1 H)

2.36-2.44 (m, 3 H) 1.74-1.79 (m, 1 H) 1.47-1.54 (m, 1 H). MS

m/z 339 [MH]^þ.

NMR (¹H, CDCl₃): δ ppm 7.38 (d, 1 H) 7.32-7.34 (m, 1 H)

7.08 (dd, 1 H) 3.59-3.63 (m, 2 H) 3.31 (d, 1 H) 3.10-3.13 (m, 2

H) 2.93 (d, 1 H) 1.68 (none, 18 H) 1.50-1.61 (m, 2 H) 1.08-1.30

(m, 3 H). MS m/z 272 [MH]^þ.

1-(3,4-Dichlorophenyl)-6-[2-(methyloxy)ethyl]-3-azabicyclo-

[3.1.0]hexane (42). To a stirred solution of 40 in THF, at room

temperature, NaH (60% in oil) was added portion wise. After 20

min, methyl iodide was added dropwise and the resulting

reaction mixture was stirred overnight. Diethyl ether and aqu-

eous saturated NH₄Cl were added, the organic phase washed

with brine, dried on Na₂SO₄, and evaporated under reduced

pressure to give the crude N-Boc intermediated. This product

was dissolved in CH₂Cl₂, TFA was added, and the reaction

mixture was stirred at room temperature for 1 h. After this

period of time, the mixture was evaporated under reduced

pressure and the residue dissolved in CH₂Cl₂, washed with 1

M NaOH, and separated through a phase separator cartridge.

The organic phase was evaporated under reduced pressure to

give the title compound as an oil which was submitted to

semipreparative chiral chromatography (column: Chiralcel

OD-H (25 cm ꢀ 0.46 cm), mobile phase: n-hexane/2-propanol

95/5% v/v; flow rate: 1.0 mL/min; DAD: 210-340 nm; CD: 230

nm). NMR (¹H, CDCl₃): δ ppm 7.37 (dd, 1 H) 7.31-7.34 (m, 1

H) 7.07 (dd, 1 H) 3.25-3.36 (m, 6 H) 3.06-3.12 (m, 2 H) 2.91 (d,

1 H) 1.49-1.59 (m, 2 H) 1.23-1.30 (m, 2 H). MS m/z 286 [MH]^þ.

Retention time = 18.8 min.

1-(3,4-Dichlorophenyl)-6-[2-(ethyloxy)ethyl]-3-azabicyclo[3.1.0]-

hexane (44). Prepared in analogy to 42. NMR (¹H, CDCl₃): δ

ppm 7.37 (d, 1 H) 7.32 (d, 1 H) 7.04-7.10 (m, 1 H) 3.39-3.47 (m,

2 H) 3.28-3.39 (m, 3 H) 3.10-3.15 (m, 2 H) 2.93 (d, 1 H)

1.55-1.60 (m, 2 H) 1.15-1.21 (m, 4 H) 0.89-0.94 (m, 1 H). MS

m/z 300 [MH]^þ.

1-(3,4-Dichlorophenyl)-6-[(5-methyl-1,3-oxazol-2-yl)methyl]-

3-azabicyclo[3.1.0]hexane (45). To a stirred solution of 2 in

dichloromethane at room temperature, triethylamine and bis-

(1,1-dimethylethyl) dicarbonate were added. Stirring was con-

tinued for 6 h, and then the reaction mixture was diluted with

dichloromethane and quenched with a saturated solution of

NH₄Cl. The organic phase was dried and the solvent evaporated

under vacuum. The compound was dissolved in acetone, and at

0 °C, Jones reagent was added dropwise. After quenching and

recovery of the product, the compound was dissolved in

CH₂Cl₂; 1-hydroxybenzotriazole monohydrate and N-(3-

6-(3,4-Dichlorophenyl)-6-[(ethyloxy)methyl]-3-azabicyclo[3.1.0]-

hexane (60). To a solution of methyl diazo(3,4-dichlorophe-

nyl)acetate in toluene, maleimide was added and the reaction

mixture was refluxed for 4 h. The solvent was removed under

reduce pressure, and the residue was purified via flash chroma-

tography. To a solution of the compound thus obtained in dry

dimethylaminopropyl)-N⁰-ethylcarbodiimide

hydrochloride

THF, at 0 °C, BH₃THF complex 1 M in THF was added and

3

then the reaction mixture was refluxed for 4 h. MeOH and HCl

1 M in Et₂O were added, and the solution was stirred at rt

overnight. The mixture was concentrated in vacuo, and the

residue was purified by SCX cartridge. To a solution of this

intermediate in CH₂Cl₂at 0 °C, di-tert-butyl dicarbonate was

added and the reaction mixture was stirred at rt overnight. After

quenching and work up, the crude was purified by flash chro-

matography (Si 25M) to afford two isomers. The exo (NMR

were added. Subsequently, propargyl amine was added drop-

wise and the reaction mixture was stirred at rt overnight. After

quenching and work up, the crude product was dissolved in

glacial acetic acid, mercury(II) acetate was added, and the

reaction mixture was heated to 110 °C and stirred for 2.5 h.

The solvent was removed under vacuum and the residue dis-

solved in ethyl acetate. The organic phase was washed with

saturated NaHCO₃and then brine and dried over sodium

2550 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6

Micheli et al.

(¹H, CDCl₃): δ ppm: 7.48 (s, 1H), 7.41 (d, 1H), 7.23 (d, 1H),

3.91-3.84 (m, 2H), 3.75-3.60 (m, 4H), 2.03 (s, 2H), 1.49 (s, 9H),

1.32 (t, 1H)) one was in dry CH₂Cl₂and at 0 °C, TEA (40 μL)

and methansulfonylchloride (28.6 μL) were added and the

reaction mixture was stirred at rt overnight. After quenching

and work up, the compound was dissolved in dry DMF and

EtONa in dry DMF was added at 0 °C. The reaction mixture

was stirred at rt for 3 h and then 1 h at 60 °C. Aqueous NH₄Cl

saturated solution was added and then extracted with Et₂O. The

combined organic phases were washed with aqueous NaCl

saturated solution, dried, and concentrated in vacuo. To a

solution of the compound in CH₂Cl₂, at 0 °C, TFA was added

and then the reaction mixture was stirred at rt for 2 h. The

solution was concentrated in vacuo, and the residue was purified

by SCX cartridge to give the title compound. NMR (¹H,

CDCl₃): δ ppm 7.39 (d, 1 H) 7.31 (d, 1 H) 7.14 (dd, 1 H) 3.78

(s, 2 H) 3.41-3.42 (m, 2 H) 3.27-3.33 (m, 2 H) 3.21-3.26 (m, 2

H) 1.87-1.94 (m, 2 H) 1.13 (t, 3 H). MS m/z 286 [MH]^þ.

6-(3,4-Dichlorophenyl)-3-azabicyclo[3.1.0]hexane (52). NMR

(¹H, MeOD) δ ppm 7.59 (s, 1 H) 7.47 (d, 1 H) 7.31 (d, 1 H)

3.74-3.81 (m, 4 H) 3.49 (d, 2 H) 3.40-3.28 (m, 2 H) 2.42 (s, 2 H)

1.02 (t, 1 H) 0.52-0.50 (m, 2 H) 0.19-0.18 (m, 2 H). MS m/z 312

[MH]^þ.

6-[(Cyclobutyloxy)methyl]-6-(3,4-dichlorophenyl)-3-azabicy-

clo[3.1.0]hexane (53). NMR (¹H, MeOD) δ ppm7.52-7.59 (m, 1

H) 7.48 (d, 1 H) 7.30 (dd, 1 H) 3.86-4.00 (m, 1 H) 3.78 (d, 2 H)

3.61 (s, 2 H) 3.37-3.49 (m, 2 H) 2.41 (s, 2 H) 0.78-2.25 (m, 6 H).

MS m/z 312 [MH]^þ.

6-(3,4-Dichlorophenyl)-6-{[(2,2,2-trifluoroethyl)oxy]methyl}-

3-azabicyclo[3.1.0]hexane (54). NMR (¹H, DMSO-d₆) δ ppm

9.70 (bs, 1 H) 9.43 (bs, 1 H) 7.58 (m, 2 H) 7.29 (d, 1 H) 4.09 (q, 2

H) 3.94 (s, 2 H) 3.64 (m, 2 H) 3.31 (m, 2 H) 2.39 (s, 2 H). MS m/z

340 [MH]^þ.

2 H) 3.24-3.30 (m, 5 H) 2.24-2.36 (m, 2 H) 1.86 (t, 2 H). MS m/z

286 [MH]^þ.

6-(3,4-Dichlorophenyl)-6-[2-(ethyloxy)ethyl]-3-azabicyclo[3.1.0]-

hexane (57). NMR (¹H, CDCl₃) δ ppm 10.35 (s, 1 H) 9.72 (s, 1 H)

7.33-7.44 (m, 2 H) 7.07-7.15 (m, 1 H) 3.70-3.84 (m, 2 H) 3.64

(d, 2 H) 3.40 (q, 2 H) 3.29 (t, 2 H) 2.17 (s, 2 H) 1.89 (t, 2 H)

1.08-1.27 (m, 3 H). MS m/z 300 [MH]^þ.

6-(3,4-Dichlorophenyl)-6-{2-[(1-methylethyl)oxy]ethyl}-3-aza-

bicyclo[3.1.0]hexane (58). NMR (¹H, DMSO-d₆)

δ ppm

9.47-9.79 (m, 1 H) 8.81-9.08 (m, 1 H) 7.50-7.67 (m, 2 H)

7.16-7.37 (m, 1 H) 3.52-3.65 (m, 2 H) 3.36-3.47 (m, 1 H)

3.23-3.32 (m, 2 H) 3.13-3.23 (m, 2 H) 2.15-2.27 (m, 2 H)

1.73-1.85 (m, 2 H) 1.03 (d, 6 H). MS m/z 314 [MH]^þ.

Acknowledgment. We thank all the colleagues who helped

in generating the data reported in this manuscript, in particular

Dr. A. Feriani and Dr. D. Dal Ben. We also thank Dr. D. Donati,

Dr. J. Hagan, and Dr. T. Rossi for their support during the

programme life.

Supporting Information Available: An example of TRUI

minimal pharmacophore construction. This material is avail-

able free of charge via the Internet at http://pubs.acs.org.

References

(1) (a) Kulkarni, S. K.; Dhir, A. Current investigational drugs for

major depression. Exp. Opin. Investig. Drugs 2009, 18, 767–788. (b)

Wong, M. L.; Licinio, J. From monoamines to genomic targets: a

paradigm shift for drug discovery in depression. Nat. Rev. Drug

Discovery 2004, 3, 136–151. (c) Millan, M. J. The role of monoamines

in the actions of established and “novel” antidepressant agents: a critical

review. Eur. J. Pharmacol. 2004, 500, 371–384. (d) Nemeroff, C. B.

The burden of severe depression: a review of diagnostic challenges and

treatment alternatives. J. Psychiatry Res. 2007, 41, 189–206.

6-(3,4-Dichlorophenyl)-6-{[(1-methylpropyl)oxy]methyl}-3-aza-

bicyclo[3.1.0]hexane (55). NMR (¹H, CDCl₃)) δ ppm 7.46 (s, 1

H) 7.36 (d, 1 H) 7.18 (d, 1 H) 3.72-3.87 (dd, 2 H) 3.22-3.36 (m, 5

H) 1.95 (s, 2 H) 1.35-1.60 (m, 2 H) 1.09 (d, 3 H) 0.85 (t, 3 H).

6-(3,4-Dichlorophenyl)-6-[2-(methyloxy)ethyl]-3-azabicyclo-

[3.1.0]hexane Hydrochloride (56). To a solution of methyl diazo-

(3,4-dichlorophenyl)acetate in toluene, maleimide was added

and the reaction mixture was refluxed for 4 h. The solvent was

removed under reduced pressure, and the residue was purified

via flash chromatography. To a solution of the compound thus

(2) (a) Arnt, J.; Christensen, A. V.; Hyttel, J. Pharmacology in vivo of

the phenylindan derivative, Lu-19,005, a new potent inhibitor of

dopamine, noradrenaline, and 5-hydroxytryptamine uptake in

rat brain. Arch. Pharmacol. 1985, 329, 101–107. (b) Chen, Z.;

Skolnick, P. Triple uptake inhibitors: therapeutic potential in depres-

sion and beyond. Expert Opin. Investig. Drugs 2007, 16, 1365–1377.

(c) Rakofsky, J. J.; Holtzheimer, P. E.; Nemeroff, C. B. Emerging

targets for antidepressant therapies. Curr. Opin. Chem. Biol. 2009,

13, 291–302. (d) Liang, Y.; Shaw, A. M.; Boules, M.; Briody, S.;

Robinson, J.; Oliveros, A.; Blazar, E.; Williams, K.; Zhang, Y.;

Carlier, P. R.; et al. Antidepressant like pharmacological profile of a

novel triple reuptake inhibitor, (1S,2S)-3-(methylamino)-

2-(naphthalen-2-yl)-1-phenylpropan-1-ol (PRC200-SS). J. Pharma-

col. Exp. Ther. 2008, 327, 573–583. (e) Skolnick, P.; Popik, P.;

Janowsky, A.; Beer, B.; Lippa, A. S. ``Broad spectrum'' antidepres-

sants: is more better for the treatment of depression? Life Sci. 2003,

73, 3175–3179. (f) Aluisio, L.; Lord, B.; Barbier, A. J.; Fraser, I. C.;

Wilson, S. J.; Boggs, J.; Dvorak, L. K.; Letavic, M. A.; Maryanoff, B.

E.; Carruthers, N. I. In vitro and in vivo characterization of JNJ-

7925476, a novel triple monoamine uptake inhibitor. Eur. J. Phar-

macol. 2008, 587, 141–146. (g) Bannwart, L. M.; Carter, D. S.; Cai,

H. Y.; Choy, J. C.; Greenhouse, R.; Jaime-Figueroa, S.; Iyer, P. S.; Lin,

C. J.; Lee, E. K.; Lucas, M. C. Novel 3,3-disubstituted pyrrolidines as

selective triple serotonin/norepinephrine/dopamine reuptake inhibi-

tors. Bioorg. Med. Chem. Lett. 2008, 18, 6062–6066. (h) Papakostas,

G. I. Dopaminergic-based pharmacotherapies for depression. Eur.

Neuropsychopharmacol. 2006, 16, 391–402. (i) Skolnick, P.; Popik,

P.; Janowsky, A.; Beer, B.; Lippa, A. S. Antidepressant-like actions of

DOV21,947: a “triple” reuptake inhibitor. Eur. J. Pharmacol. 2003, 461,

99–104.

obtained in dry THF, at 0 °C, BH₃THF complex 1 M in THF

3

was added and then the reaction mixture was refluxed for 4 h.

MeOH and HCl 1 M in Et₂O were added and the solution was

stirred at rt overnight. The mixture was concentrated in vacuo,

and the residue was purified by SCX cartridge. To a solution of

this intermediate in CH₂Cl₂at 0 °C, di-tert-butyl dicarbonate

was added and the reaction mixture was stirred at rt overnight.

After quenching and work up, the crude was purified by flash

chromatography (Si 25M) to afford two isomers. The exo

isomer in dry CH₂Cl₂at 0 °C, was added with Dess-Martin

periodinane portion wise, and then the reaction mixture was

stirred at rt overnight. The compound thus obtained was

dissolved in and added dropwise to a stirred solution of the

preformed ylide obtained by addition of BuLi to methyltriphe-

nylphosphonium bromide in dry THF at 0 °C. After quenching

and work up, the compound was dissolved in dry THF at 0 °C,

and borane THF complex 1 M in THF was added dropwise. The

ice-bath was removed, and the reaction mixture was stirred for

3.5 h at rt. The solution was then quenched at 0 °C with water,

and NaOH 3 M and H₂O₂were added; the resulting mixture was

stirred at 0 °C for 0.5 h. After quenching and work up, the

compound was dissolved in DMF, NaH was added at 0 °C, and

the reaction mixture was stirred at rt for 30 min. Iodomethane

was added to the reaction mixture, and then the temperature was

increased to 40 °C. After quenching and work up, the crude was

purified to give the title compound. NMR (¹H, MeOD) δ ppm

7.40-7.60 (m, 2 H) 7.27 (dd, 1 H) 3.72 (d, 2 H) 3.42-3.57 (m,

(3) (a) Scheel-Kruger, J.; Moldt, P; Watjen, F. Tropane-derivatives,

their preparation and use. U.S. Patent 6395748, 2002; (b) Scheel-

Kruger, J.; Moldt, P; Watjen, F. Tropane-derivatives, their preparation

and use. U.S. Patent 6288079, 2001; (c) Scheel-Kruger, J.; Moldt, P;

Watjen, F. Tropane-derivatives, their preparation and use. Patent WO

9730997, 1997; (d) Scheel-Kruger, J.; Olsen, G. M.; Nielsen, E. O.;

Dahl, B. H.; Jensen, L. H. Fused tropane derivatives as neurotransmitter

reuptake inhibitors. Patent WO 9716451, 1997.

(4) (a) Maestro, version 8.5.111; Schrodinger LLC: New York, 2006; (b)

BatchMin, version 9.6; Schrodinger LLC: New York, 2008; (c) Phase,

version 3.0.110; Schrodinger LLC: New York, 2006.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2551

(5) The research complied with national legislation and with company

policy on the Care and Use of Animals and with related codes of

practice.

(10) Martin, G. E.; Bendesky, R. J. Mouse locomotor activity: an in

vivo test for dopamine autoreceptor activation. J. Pharmacol. Exp.

Ther. 1984, 229, 706–711.

(6) Minick, D. J.; Rutkowske, R. D.; Miller, L. A. D. Strategies for

successfully applying vibrational circular dichroism in a pharmaceu-

tical research environment. Am. Pharmaceut. Rev. 2007, 10, 118–123.

(7) Aptuit Limited, 3 Simpson Parkway, Livingston, West Lothian,

EH54 7BH, Scotland; www.aptuit.uk.

(8) Zocchi, A.; Fabbri, D.; Heidbreder, C. A. Aripiprazole increases

dopamine but not noradrenaline and serotonin levels in the mouse

prefrontal cortex. Neurosci. Lett. 2005, 387, 157–161.

(9) Burchall, C. J.; Soroko, F. E.; Rigdon, G. C. Potentiation of the

behavioral effects of 5-hydroxytryptophan by BW 1370U87, a

selective monoamine oxidase-A inhibitor. Drug Dev. Res. 1992,

25, 209–213.

(11) Bourin, M.; Chenu, F.; Prica, C.; Hascoet, M. Augmentation effect

of combination therapy of aripiprazole and antidepressants on

forced swimming test in mice. Psychopharmacology (Heidelberg,

Ger.) 2009, 206, 97–107.

(12) Bonanomi, G.; Micheli, F.; Terreni, S. Azabicyclic compounds as

serotonin, dopamine and norepinephrine re-uptake inhibitors and

their preparation, pharmaceutical compositions and use in the

treatment of depression. Patent WO 2008074716, 2008.

(13) Bertani, B.; Bonanomi, G.; Di Fabio, R.; Fazzolari, E.; Micheli, F.;

Tarsi, L. Preparation of 3-azabicyclo[3.1.0]hexane compounds as

monoamine neurotransmitter re-uptake inhibitors. Patent WO

2009027293, 2009.

Article Doi

1-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanesand6-(aryl) -6-[alkoxyalkyl]-3azabicyclo[3.1.0]hexanes: A new series of potent and selective triple reuptake inhibitors

DOI: 10.1021/jm901818u

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