1,2,4-Triazolyl azabicyclo[3.1.0]hexanes: A new series of potent and selective dopamine D3 receptor antagonists

Article abstract of DOI:10.1021/jm901319p

The discovery of new highly potent and selective dopamine (DA) D ₃ receptor antagonists has recently allowed the characterization of the DA D₃ receptor in a range of preclinical animal models of drug addiction. A novel series of 1,2,4-triazol-3-yl-azabicyclo[3.1.0]hexanes, members of which showed a high affinity and selectivity for the DA D₃ receptor and excellent pharmacokinetic profiles, is reported here. Members of a group of derivatives from this series showed good oral bioavailability and brain penetration and very high in vitro affinity and selectivity for the DAD₃ receptor, as well as high in vitro potency for antagonism at this receptor. Several members of this series also significantly attenuate the expression of conditioned place preference (CPP) to nicotine and cocaine. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901319p

374 J. Med. Chem. 2010, 53, 374–391
DOI: 10.1021/jm901319p
1,2,4-Triazolyl Azabicyclo[3.1.0]hexanes: A New Series of Potent and Selective Dopamine D3 Receptor
Antagonists
Fabrizio Micheli,*,†,§ Luca Arista,þ Giorgio Bonanomi,†,§ Frank E. Blaney,^,3 Simone Braggio,†,§ Anna Maria Capelli,‡,§
Anna Checchia,†,§ Federica Damiani,∧ Romano Di-Fabio,†,§ Stefano Fontana,†,§ Gabriella Gentile,†,§ Cristiana Griffante,†,§
Dieter Hamprecht,z Carla Marchioro,‡,§ Manolo Mugnaini,†,§ Jacqui Piner,#,3 Emiliangelo Ratti, ,3 Giovanna Tedesco,‡,§
Luca Tarsi,†,§ Silvia Terreni,†,§ Angela Worby,^,3 Charles R. Ashby Jr.,O and Christian Heidbreder[
†Neurosciences Centre of Excellence, ‡Molecular Discovery Research, § GlaxoSmithKline Medicines Research Centre, Via Fleming 4, 37135
Verona, Italy, Neurosciences Centre of Excellence, ^Molecular Discovery Research, #Safety Assessment, 3GlaxoSmithKline Medicines Research
Centre, NFSP, Harlow, U.K., ODepartment of Pharmaceutical Sciences, Saint John’s University, Jamaica, New York 11439, [Reckitt Benckiser
Pharmaceuticals, Richmond, Virginia 23235, zBoehringer Ingelheim, Milan, Italy, þNovartis Institute Research, Basel, Switzerland, and
∧European Patent Office, Munich, Germany
Received September 4, 2009
The discovery of new highly potent and selective dopamine (DA) D3 receptor antagonists has recently
allowed the characterization of the DA D3 receptor in a range of preclinical animal models of drug
addiction. A novel series of 1,2,4-triazol-3-yl-azabicyclo[3.1.0]hexanes, members of which showed a
high affinity and selectivity for the DA D3 receptor and excellent pharmacokinetic profiles, is reported
here. Members of a group of derivatives from this series showed good oral bioavailability and brain
penetration and very high in vitro affinity and selectivity for the DA D3 receptor, as well as high in vitro
potency for antagonism at this receptor. Several members of this series also significantly attenuate the
expression of conditioned place preference (CPP) to nicotine and cocaine.
Introduction
promising developability characteristics; a detailed structure
activity relationship (SAR) and a series of in vitro and in vivo
experiments are provided to characterize this series of highly
potent and selective DA D3 receptor antagonists.
The location of the dopamine (DAa) D3 receptor in the
rodent and human brain, changes in the expression of these
receptors upon exposure to drugs of abuse, and a growing
body of preclinical evidence in models of substance depen-
dence, suggest that selective DA D3 receptor antagonists may
be a point of therapeutic intervention for substance depen-
dence or abuse [for reviews see refs 1-6]. The observation that
selective DA D3 receptor antagonists regulate the motivation
to self-administer drugs and disrupt drug-associated cue-
induced craving has contributed in guiding state-of-the-art
medicinal chemistry research efforts to develop a highly
selective DA D3 receptor antagonist.
Chemistry
During the past decade, oneaspect of GSK research inCNS
drug discovery was directed toward the discovery of novel
chemical entities (NCEs) that selectively modulate the DA D3
receptor. Thesuccessful result of this work led to the discovery
of trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2yl)-
ethyl]cyclo-hexyl]-4-quinolinecarboxamide (SB-277011, 1)5,6
and to the more recent BAZ scaffolds 7-(1,3-dimethyl-1H-pyra-
zol-5-yl)-3-(3-{[4-methyl-5-(2-methyl-5-quinolinyl)-4H-1,2,4-
triazol-3-yl]thio}propyl)-2,3,4,5-tetrahydro-1H-3-benzazepine
(2),7 2-methyl-7-(3-{[4-methyl-5-(2-methyl-5-quinolinyl)-4H-
1,2,4-triazol-3-yl]thio}propyl)-6,7,8,9-tetrahydro-5H-[1,3]oxa-
zolo[4,5-h][3]benzazepine (3),8 and 8-(3-{[4-methyl-5-(2-meth-
yl-5-quinolinyl)-4H-1,2,4-triazol-3-yl]thio}propyl)-2-(trifluoro-
methyl)-7,8,9,10-tetrahydro-6H-[1,3]oxazolo[4,5-g][3]ben-
zazepine (4)9 (Figure 1).
We have recently reported7 that a series of 1,2,4-triazol-
3-yl-thiopropyl-tetrahydrobenzazepines, which have high in
vitro and in vivo selectivity, can significantly attenuate or
block the effects of drugs of abuse in a number of animal
models of addiction. The aim of the present work is related to
the pioneering and original substitution, for this specific
thiotriazole series, of the benzoazepine (BAZ) scaffold by a
aryl azabicyclo[3.1.0]hexane template that is endowed with
The major step forward from 1 to the last three BAZ
derivatives (2-4) was the introduction into the scaffold of
appropriate developability characteristics (i.e., acceptable P450
profile, low intrinsic clearance (Cli), and good bioavailability
(F%)) in preclinical species. Furthermore, in contrast to other
DA D3 receptor antagonists,5,6 derivative 2 showed a reduced
hERG liability in vitro and no prolongation of the QTc interval
in vivo.7 Given these positive preclinical results, our next goal
was to investigate the possibility to achieve a similar preclinical
profile using a different template. Accordingly, we planned to
*To whom correspondence should be addressed. Phone: þ39-045-
8218515. Fax: þ39-045-8218196. E-mail: Fabrizio.E.Micheli@gsk.com.
a Abbreviations: BAZ, benzoazepine; CPP, conditioned place prefer-
ence; ACh, acetylcholine ; hERG, human ether-a go-go Kþ channel; NCE,
novel chemical entity; PK, pharmacokinetic; P450, cytochrome P450; hCli,
human intrinsic clearance; MW, molecular weight; clogD, calculated logD;
PSA, polar surface area; F%, bioavailability; B/B, brain/blood; Clb, blood
clearance; Vd, distribution volume; SDM, site-directed mutagenesis;
FLIPR, fluorescent imaging plate reader; GPCR, G-protein coupled
receptor; TM, trans membrane; SPA, scintillation proximity assay; ECG,
electrocardiogram; DA, dopamine; NT, not tested.
r
pubs.acs.org/jmc
Published on Web 11/05/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 375
Figure 1. Structures of the previously reported GSK selective DA D3 receptor antagonists.
Scheme 1. General Synthetic Procedures for the Preparation of Compounds 5-105a
a (i) T3P in AcOEt; (ii) 4-methyl-3-thiosemicarbazide; (iii) NaOH; (iv) K2CO3/acetone; (v) K2CO3/DMF or CH3CN.
explore if, in this specific series of aryl thiotriazoles, the BAZ
scaffold could be replaced with an alternative basic moiety.
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.
Results and Discussion
There are several options to approach the potential replace-
ment of a certain portion of a molecule within a given scaffold:
it is possible either to use a database of commercial or in house
ring systems, to perform literature searches, to rely on de novo
design, or to go through a massive combinatorial task repla-
cing the given portion with all the fragments available in a
specific store. For this specific thiotriazole system, a mix of all
these techniques was used to replace the BAZ moiety, and a
number of different structures were identified. Among them,
the potential BAZ replacement that is reported in this manu-
script is represented by an aryl azabicyclo[3.1.0]hexane
moiety. The choice of this original scaffold was based on a
number of reasons including the attractive physicochemical
characteristics, predicted in silico (namely the polar surface
area and the calculated logD) and, despite the associated
structural complexity, the possibility to appropriately deco-
rate the pendant aryl ring of the azabicyclo[3.1.0]hexane due
to some in house synthetic experience.
In contrast to the previously described BAZ system,7 the
aryl azabicyclo[3.1.0]hexane contains two stereogenic cen-
ters. Accordingly, both the racemate and each single enan-
tiomer were submitted to test. Compounds were prepared
according to Scheme 1. Additional information can also be
found in refs 10,11.
The results of the exploration are reported in Tables 1-3.
The first compound of the series to be prepared and to be
considered the reference point of our exploration was the
racemic nonsubstituted phenyl azabicyclo[3.1.0]hexane 5 and
its pure enantiomers 6 and 7, respectively. This is usually an
important step in generating SAR to fully appreciate the role
of the substituents that will decorate a system, although the
unsubstituted systems might sometime behave differently
from the decorated ones. In this specific case, derivative 7
not only showed a similar potency at the DA D3 receptor
compared toderivative1 butalso had a 40-foldselectivity over
the DA D2 receptor. These results suggested that the working
hypothesis might be correct and constituted a good starting
point for further exploration. Interestingly enough, but not
unexpectedly, a difference in affinity at the DA D3 receptor
was also noticed in the two enantiomers. The docking7 of
derivative 2 in the DA D3 receptor model seemed to suggest
the need for a lipophilic area in the region where the BAZ sits
and the presence of some specific interactions with the hydro-
philic pyrazole substituent (close to S182 and S192). For that
reason, the meta/para position of the pendant aromatic ring
on the azabicyclo[3.1.0]hexane was appropriately explored,
By exploiting a screening cascade previously reported,7 all
the newly prepared compounds were assayed for their agonis-
tic and antagonistic properties using a functional GTPγS assay
expressing the human DA D3 receptor. To proceed with this
cascade, the NCEs had to fulfill the following key character-
istics: (1) at least 100-fold selectivity vs DA D2 and histamine
H1 receptors (functional assays), and (2) 100-fold selectivity vs
the hERG ion channel (dofetilide binding assay). Further-
more, to ensure that the selected templates were endowed
with appropriate pharmacokinetic (PK) and developability

376 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Micheli et al.
Table 1. Functional Activity at the Human DA D3 Receptor and Selectivity for Quinolinyl Derivativesa
entry
R
hD3-GTPγS fpKi
hD2-GTPγS fpKi
hH1-FLIPR pKb
hERG pIC50
PSAb
cLogDc
1
not applicable
not applicable
not applicable
not applicable
H (rac)
8.4
8.8
7.2
8.4
7.6
<6.3
8.3
7.9
7.4
6.8
8.8
7.7
8.7
8.4
<6.3
8.9
7.5
8.2
8.9
7.5
8.2
9.0
7.4
9.1
6.4
6.5
6.2
6.1
<5.6
6.3
7.3
7.2
NT
7.3
7.2
7.5
5.8
NT
NT
7.7
7.0
6.6
7.3
7.4
7.5
6.6
6.0
6.3
7.8
6.8
5.7
5.7
<5.0
5.7
5.7
5.3
6.1
6.2
5.8
6.0
6.6
6.6
6.6
6.9
6.3
6.6
6.2
6.2
6.2
5.6
6.7
5.8
5.7
6.8
69
65
98
73
47
47
47
47
47
47
47
47
47
47
47
47
71
56
56
56
47
47
47
47
3.8
5.3
5.0
6.3
3.4
3.4
3.4
5.2
5.2
5.2
5.4
5.4
5.4
4.5
4.5
4.5
3.1
3.5
3.5
3.5
4.3
4.3
4.3
4.5
2
3
<5.6
<6.1
6.8
4
5
6
H (se)
H (se)
<5.5
6.7
5.3
7
8
3,4-diCl (rac)
3,4-diCl (se)
3,4-diCl (se)
4-t-Bu (rac)
4-t-Bu (se)
4-t-Bu (se)
4-Br (rac)
4-Br (se)
9
6.4
5.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
6.5
5.7
6.1
5.4
<6.1
<6.1
5.2
4-Br (se)
4-CN (rac)
4-OMe (rac)
4-OMe (se)
4-OMe (se)
4-Cl (rac)
5.9
<6.4
5.9
<6.2
6.7
6.1
4-Cl (se)
4-Cl (se)
4-CF3 (rac)
<5.9
a fpKi = functional pKi obtained from the GTPγS functional assay. FLIPR = fluorescent imaging plate reader. SEM for D3 GTPγS, H1 FLIPR, and
2
HERG data sets is (0.1 and for the D2 GTPγS data is (0.2. (rac) = racemate. (se) = single enantiomer. b A . c ACD logD 7.4. version 11.
˚
and the results are reported in Table 1. The 3,4-dichloro
substitution (8-10) increased the lipophilicity to 5.2 without
leading to an increased potency at the desired target, while the
bulkier 4-t-butyl derivative (11-13, clogD = 5.4) increased
the potency at D3 and selectivity over the DA D2 receptor
(400-fold). This observation might result from the steric clash
with some residues in the DA D3 receptor of the substituents
in position 3 and 4 of the aryl ring with respect to the single
4-substitution, even if the binding pocket in the receptor
model appeared to be rather tolerant. A reduction of steric
hindrance and clogD was achieved with the bromo derivatives
(14-16, clogD = 4.5) and with the chloro derivatives (21-23,
clogD = 4.3) without affecting significantly the affinity at the
desired target. The introduction of the highly polar and
hydrophilic cyano derivative 17 led to a reduction in affinity
at the desired target with limited effect on hERG affinity.
The electron-donating properties of the methoxy derivatives
(18-20) did not seem to have a major influence on the affinity
in this series. The highest potency and selectivity at the desired
target (>1000 fold over DA D2 receptor and >100 over H1
and hERG targets) were achieved with the p-CF3 derivative
24. This derivative was also endowed with a promising in vitro
PK profile (inhibition on all P450 isoforms >1 μM and
relatively low hCli, namely 1.1 mL/min/g liver). In this specific
series, the higher affinity achieved at the DA D3 receptor was
linked to one specific isomer, which was characterized as the
enantiomer endowed with the (1S,5R) absolute stereochem-
istry. This attribution was achieved both through vibrational
circular dichroism (VCD) and X-ray techniques; further and
more specific details on this topic will be the subject of a
separate analytical communication. Once completed, this first
and very preliminary investigation of the left-hand side part of
the molecule was followed by an introductory exploration of
the right-hand side of the thiotriazole portion of the template.
Considering the overall activity shown by the substituents
reported in Table 1 on the aryl system, the trifluoromethyl
group was kept as the reference point, and the single
(1S,5R)-[3.1.0]hexane enantiomer was chosen for the explora-
tion; nonetheless, to confirm the validity of the working
hypothesis associated to the stereochemistry of the com-
pound, one of the derivatives was prepared as a racemate;
the single enantiomers were subsequently separated and were
both independently evaluated for their biological activity. The
results of this exploration are reported in Table 2. The
regioisomeric quinoline 25 showed an increase in potency
and a slightly reduced hERG activity; the introduction of
more basic/hydrophilic substituents (27-34) led to a general
slight reduction of activity both at the hERG and at the DA
D3 receptor, leading to a more balanced profile. Derivatives
27 and 28 showed low hERG affinity with a well-adjusted
profile which included good in vitro PK properties (inhibition
on all P450 isoforms was >10 μM for both derivatives and
their hCli was respectively 1.7 and 1.0 mL/min/g liver); there-
fore, both derivatives were further progressed to assess their in
vivo PK properties in preclinical species.12 After oral admini-
stration, they both showed moderate Clb in rats (18 and
13 mL/min/kg, respectively), identical half-life (T1/2 = 2.1 h),
similar distribution volumes (Vd = 2.5 and 2.2 L/kg, res-
pectively), and almost identical bioavailability (F% = 50
and 46%, respectively); the brain penetration values were also
comparable (B/B = 1.6 and 2, respectively), making them ideal
candidates for further progression.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 377
Table 2. Functional Activity at the Human DA D3 Receptor and Selectivity for 4-Trifluoromethyl Derivativesa
entry
R
hD3-GTPγS fpKi
hD2-GTPγS fpKi
hH1-FLIPR pKb
hERG pIC50
PSAb
cLogDc
25
26
27
28
29
30
31
32
33
34
35
36
2-methyl-6-quinolinyl (se)
phenyl (se)
9.6
9.2
9.0
9.0
8.8
9.3
9.1
9.6
9.2
9.3
9.0
7.3
6.2
6.8
7.3
7.4
6.9
7.0
7.3
7.1
7.1
7.4
7.2
7.4
7.5
7.5
6.3
6.2
5.3
5.4
5.5
6.7
6.1
5.7
6.1
6.0
6.1
6.2
47
34
47
60
60
47
47
47
60
60
60
60
4.5
4.1
3.1
1.4
2.1
3.9
2.7
2.1
2.6
2.1
2.1
2.1
2-methyl-3-pyridinyl (se)
4-pyridazinyl (se)
5-pyrimidinyl (se)
6.5
6.7
<6.0
6.4
6.2
3-methyl-2-furanyl (se)
6-methyl-3-pyridinyl (se)
2,4-dimethyl-1,3-thiazol-5-yl (se)
5-methyl-2-pyrazinyl (se)
4-methyl-1,3-oxazol-5-yl (rac)
4-methyl-1,3-oxazol-5-yl (se)
4-methyl-1,3-oxazol-5-yl (se)
6.6
6.3
6.9
7.0
<6.1
a fpKi = functional pKi obtained from the GTPγS functional assay. FLIPR = fluorescent imaging plate reader. SEM for D3 GTPγS, H1 FLIPR, and
2
HERG data sets is (0.1 and for the D2 GTPγS data is (0.2. b A . c ACD logD 7.4. version 11.
˚
Derivatives 32 and 34 also showed excellent in vitro proper-
ties considering both their affinity/selectivity values and their
PK profile; accordingly, they also underwent in vivo PK
evaluation in rats. The thiazolyl derivative 32 showed an
overall good PK profile after oral administration with a
between the basic nitrogen and Asp110 on transmembrane
helix 3 (TM3), the interaction with Ser192 (TM5) and Ser182
(second extracellular loop), and the potential π-stacking
interactions with Phe345 and His349 (TM6)). At the same
time, those appropriately identified substituents should be
able to disrupt the interactions of the molecule within the
hERG channel (namely the specific interactions with Tyr652
and Phe656 in the S6 helices and the ones in the pore turn
region of the channel model), particularly by reducing their
aromatic π interactions.
It is worth noting that the DA D3 receptor model can
explain the DA D3/D2 selectivity of these compounds because
of the hydrogen bond between the triazole ring and Thr368.
The corresponding residue in the DA D2 receptor is a
phenylalanine (Phe), which would be expected to cause a
severe steric clash with these ligands.
In the current exploration, in addition to the decorations of
the aryl group identified from the modeling work mentioned
above, a further selection of substituents with different phy-
sicochemical properties was also included to probe the SAR
for this series.
Among the different right-hand side heteroaromatic deri-
vatives reported in Table 2 and used in this exploration, we
would like to report here the results achieved with the oxazolyl
moiety present in derivative 35. These results are tabulated in
Table 3. The derivatives were synthesized and tested both as
racemates and single enantiomers to verify if the “favorite”
stereochemistry previously identified to achieve the higher
affinity on the DA D3 receptor (i.e., the (1S,5R) stereo-
chemistry) was specific only for that thiotriazole subseries
series or might have had a wider application in this specific
series of aryl [3.1.0] thiotriazoles.
moderate Clb (26 mL/min/kg), a half-life value (T1/2
=
2.0 h) comparable to that of previously analyzed derivatives,
a moderate distribution volume (Vd = 3.9 L/kg), a moderate
bioavailability (F% = 23%), and a good brain penetration
(B/B = 2.2). The oxazolyl derivative 34 showed a low Clb
(14 mL/min/kg), a comparable half-life (T1/2 = 3.2 h), a
similar distribution volume (Vd = 3.5 L/kg), and superior
bioavailability and brain penetration (F% = 85% and B/B =
3.6, respectively).
The (1S,5R) derivative 35, in agreement with the working
plan, was the product endowed with the highest affinity at the
DA D3 receptor and was therefore further characterized as
single enantiomer: its in vitro and in vivo PK profiles fully
matched the results already observed with the racemic deri-
vative 34. Considering their general profile and the good
selectivity window achieved, these results led to a further
characterization of all these products in the screening cascade
as described below.
At this point of the exploration, despite the identification of
a number of potentially interesting compounds to be fully
characterized along the screening cascade, a second SAR
expansion of the left-hand side was carried out. In this further
iteration, the newly identified heteroaromatic systems were
maintained on the right-hand side of the molecule, whereas a
computational strategy was applied as previously described.7
It was noticed that, in the previous BAZ template, the key
elements responsible for the activity of the molecule at the DA
D3 receptor were also responsible for the activity within the
hERG channel. At that time, the visualization within the DA
D3 receptor model and within the hERG channel model of the
amino acid residues interacting with specific regions of the
molecule allowed the rational design of the best substituents
able to maximize the window between these two affinities.
Briefly, both the DA D3 receptor (Figure 2A) and the hERG
channel model7 (Figure 2B) were used to dock the compounds
and to assess the substituents that maintain the specific
interactions in the DA D3 model (namely the salt bridge
Once again, it was decided to start the exploration using the
unsubstituted phenyl derivative 37 as reference compound,
and in agreement with the need of SAR generation, some of
the previously used substituents were also analyzed. In this
specific subseries, in agreement with what is reported in
Table 1, no major variation in the DA D3 affinity was noticed
among the 4-t-Bu (40), the 4-Br (42), and the 4-Cl (51)
substituents; the major difference was linked to lipophilicity
values and to the selectivity versus the H1 and DA D2
receptors of compound 51. A further reduction in the size of

378 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Micheli et al.
balanced profile, while the 2-F, 4-Me derivative 93 exhibited a
reduced affinity at the desired receptor; this might be related
to its lower lipophilicity as potentially confirmed by the results
of the 2F, 4-Cl derivative 96. The 4-F, 3-CF3 substitution (77)
led to the highest DA D3 affinity observed in this subseries,
possibly due to the substituent in position 3 potentially
interacting at best with the appropriate residues within the
receptor pocket, as also observed for derivative 63. Again, the
introduction of potentially electrodonating properties in the
methoxy derivative 99 led to a slight decrease in affinity in
agreement with what was observed for derivative 44. A
simultaneous substitution of both “meta” positions, as in
the 3,5- substituted derivative 89, showed positive results as
far as the affinity at the primary target was concerned, but the
selectivity at the DA D2 receptor was slightly reduced.
Among the compounds that were selected in agreement
with the receptor/channel models studies for their electron-
withdrawing ability and, therefore, for their potential ability
to negatively affect the π interactions within the hERG
channel, derivatives 51, 74, 77, 84, and 86 showed an overall
balanced affinity profile; from the in vitro PK point of view,
they showed the appropriate P450 and Cli parameters to be
further progressed in vivo and accordingly they were chosen
for further characterization. The best in vivo profiles obtained
after oral administration in rats were associated with deriva-
tives 51 and 74 that showed a moderate Clb (40 and 18 mL/
min/kg, respectively), a sufficiently long half-life (T1/2 = 1.3
and 3.7 h), a moderate distribution volume (Vd = 2.6 and
4.3 L/kg) associated with a good bioavailability (F% = 31%
and 47%), and brain penetration (B/B = 2.7 and 3.1,
respectively). Therefore, in addition to the compounds pre-
viously identified, these two derivatives were also selected for
further characterization.
To ensure that no potential gaps were left during the
chemical exploration of this new template, an automated
analysis of all the DA D3 fpKi and hERG pIC50 data was
performed using the SAR-Toolkit13 in addition to the “classi-
cal” computational techniques described so far.
The “R-group/D-score” analysis is available as part of the
SAR-Toolkit, a GSK proprietary suite of applications for
SAR data analysis, based on the Daylight Toolkit14 and
embedded within Spotfire DecisionSite.15 A highly versatile
fragmentation tool defines the core portion of the molecules
and the specific substituents (R-groups) within the set of
molecules of interest (e.g., RG-Core, RG-R1, and RG-R2).
Pairs of compounds which differ only in one R-group are then
automatically identified, and the activity of the compound
with an R-group chosen as reference is compared with that of
a molecule carrying areplacementR-group. Toassesswhether
the change is beneficial or detrimental for activity, a D-score is
calculated as the average difference in activity across all the
pairs of compounds carrying respectively the R-group of
interest and the reference R-group and differing only by that
R-group. All the compounds reported here constituted the
original data set. Compound 37 in Table 3 was used as a
reference structure. Using the right-hand side of the molecule
as core structure and making use of the pendant substituents
on the [3.1.0] scaffold as RG-R2, it appears quite clear from
Figure 3 that in this specific subseries more lipophilic substi-
tuents seem to boost the DA D3 activity. It is interesting to note
that bulky substituents in the ortho position of the aromatic
system (e.g., -CF3 65, -CH3 75) were less tolerated in this
specific subseries. This is probably due to a conformational
effect as the o-F derivative 74 (the fluorine atom being slightly
Figure 2. (A) Relevant interactions of derivative 35 indocking to the
DA D3 receptor model. Portions of the backbone ribbon has been
removed for clarity. (B) Interactions of compound 35 in the hERG
channel models. The backbone ribbon of the pore domain is included
but that of the S5 and S6 helices has been removed for clarity. For
details about the modeling techniques, please refer to ref 7.
the substituent in position 4 (F, 57) led to a reduced affinity at
the primary target. The slight reduction of DA D3 affinity
observed with the 4-OMe derivative 45 might be associated
with its electrodonating properties, as the same effect was not
observed with the 4-OCF3 derivative 67. The single substitu-
tion in position 3 of the aryl ring also seems to parallel the
results already achieved in the subseries reported in Table 1,
with the exception of the 3-CF3 derivative 63, where an
increase in the affinity at the primary target led also to a slight
increase in the affinity at the DA D2 receptor. The next step in
the analysis of the results of the exploration is related to the
double substitution of the phenyl system. This was done using
substituents that did not raise the clogD values to unaccep-
tably high values. The 3,4-bis-chloro substitution in derivative
70, in this specific subseries, did not suffer of the slight
decrease in affinity at the primary target previously reported
for the quinoline derivative 8; unfortunately, this substitution
did not achieve the desired selectivity over the DA D2
receptor. The 2-F, 4-CF3 derivative 74 showed an overall very

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 379
Table 3. Functional Activity at the Human DA D3 Receptor and Selectivity for 5-Oxazolyl Derivativesa
entry
R
hD3-GTPγS fpKi
hD2-GTPγS fpKi
hH1-FLIPR pKb
hERG pIC50
PSAb
cLogDc
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
phenyl (rac)
4-t-Bu (rac)
4-t-Bu (se)
7.9
9.1
8.5
9.5
8.7
9.4
6.4
8.3
8.7
7.0
8.1
9.0
6.6
8.9
9.4
6.3
8.3
9.1
7.9
8.2
8.1
6.3
8.3
8.3
6.4
9.3
9.3
7.8
7.5
8.7
9.0
7.3
8.5
9.0
7.6
8.8
6.9
8.9
7.0
8.9
9.6
7.1
8.8
6.9
8.4
9.1
7.2
8.9
9.2
9.3
6.7
9.2
9.1
7.6
7.1
<6.2
8.0
8.2
6.5
8.6
8.9
6.7
7.1
6.8
<5.6
6.6
6.3
7.9
8.1
6.0
<5.5
6.0
6.0
6.3
6.6
<5.5
6.4
7.2
8.0
6.9
7.2
6.5
7.8
6.6
7.7
7.4
6.7
6.1
5.6
6.2
7.1
6.1
7.4
6.1
6.5
7.1
6.3
7.9
<5.5
6.3
5.7
6.0
6.3
6.4
6.6
7.3
<5.7
<5.5
6.5
6.5
6.2
5.8
6.7
7.3
7.2
NT
7.1
6.1
5.6
6.5
7.3
5.5
7.2
7.0
5.8
6.2
6.4
6.0
6.4
6.3
6.1
5.9
5.7
5.6
5.8
5.4
5.2
6.1
6.4
6.1
5.8
6.5
5.9
5.7
6.5
5.7
5.7
6.0
6.0
5.5
5.1
5.6
5.2
6.7
6.6
6.5
6.0
6.3
6.0
6.0
6.3
6.1
6.4
5.7
5.6
6.3
5.9
5.4
5.0
6.0
5.5
5.4
6.4
6.2
6.2
6.0
5.7
5.7
5.2
6.0
4.4
6.3
6.0
6.4
5.9
60
60
60
60
60
60
60
69
69
69
69
69
69
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
69
69
69
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
69
1.0
3.0
3.0
3.0
2.2
2.2
2.2
1.1
1.1
1.1
1.0
1.0
1.0
1.9
1.9
1.9
1.9
1.9
1.9
1.3
1.3
1.3
1.0
1.0
1.0
1.8
1.8
1.8
2.4
2.4
2.4
2.4
2.8
2.8
2.8
2.9
2.9
2.9
2.6
2.5
2.5
2.5
2.4
2.4
2.4
2.8
2.8
2.8
2.3
2.3
2.3
1.8
1.8
1.8
2.0
2.0
2.0
2.7
2.7
2.7
1.9
<6.0
7.1
6.9
4-t-Bu (se)
4-Br (rac)
4-Br (se)
4-Br (se)
6.9
<5.9
6.9
6.6
4-OMe (rac)
4-OMe (se)
4-OMe (se)
<5.8
6.3
7.0
3-OMe (rac)
3-OMe (se)
3-OMe (se)
<6.4
6.5
6.8
4-Cl (rac)
4-Cl (se)
4-Cl (se)
3-Cl (rac)
<6.2
7.0
7.3
3-Cl (se)
3-Cl (se)
6.3
6.3
4-F (rac)
4-F (se)
6.4
6.5
4-F (se)
3-F (rac)
6.7
6.8
3-F (se)
3-F (se)
<6.2
7.5
7.5
3-CF3 (rac)
3-CF3 (se)
3-CF3 (se)
<6.4
<6.1
<6.4
6.8
2-CF3 (rac)
4-OCF3 (rac)
4-OCF3 (se)
4-OCF3 (se)
3,4-diCl (rac)
3,4-diCl (se)
3,4-diCl (se)
2-F, 4-CF3 (rac)
2-F, 4-CF3 (se)
2-F, 4-CF3 (se)
2-Me, 4-CF3 (rac)
3-CF3, 4-F (rac)
3-CF3, 4-F (se)
3-CF3, 4-F (se)
2-F, 5-CF3 (rac)
2-F, 5-CF3 (se)
2-F, 5-CF3 (se)
2-F, 3-CF3 (rac)
2-F, 3-CF3 (se)
2-F, 3-CF3 (se)
3-F, 4-CF3 (rac)
3-F, 4-CF3 (se)
3-F, 4-CF3 (se)
3-F, 5-CF3 (rac)
3-F, 5-CF3 (se)
3-F, 5-CF3 (se)
2-F, 4-Me (rac)
2-F, 4-Me (se)
2-F, 4-Me (se)
2-F, 4-Cl (rac)
2-F, 4-Cl (se)
2-F, 4-Cl (se)
3-CF3, 4-OMe (rac)
6.0
7.9
7.5
5.8
6.6
<6.2
6.2
<6.0
7.0
7.5
6.0
6.1
<6.1
6.8
6.9
6.6
7.2
6.2
7.1
<6.2
7.5
7.9
<6.4
<6.2
<6.1
6.2
<6.2
<6.1
6.5
6.8

380 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Table 3. Continued
Micheli et al.
entry
R
hD3-GTPγS fpKi
hD2-GTPγS fpKi
hH1-FLIPR pKb
hERG pIC50
PSAb
cLogDc
98
99
3-CF3, 4-OMe (se)
3-CF3, 4-OMe (se)
3-Cl, 4-OMe (rac)
3-Cl, 4-OMe (se)
3-Cl, 4-OMe (se)
3-OCF3 (rac)
6.7
8.9
8.3
8.5
6.7
8.8
7.3
9.0
<6.3
6.6
6.3
<5.6
7.4
6.9
5.9
5.9
5.4
5.7
6.1
6.2
6.0
6.0
69
69
69
69
69
69
69
69
1.9
1.9
1.9
1.9
1.9
2.3
2.3
2.3
100
101
102
103
104
105
6.4
<6.1
6.8
6.8
5.9
6.6
<5.5
6.5
3-OCF3 (se)
3-OCF3 (se)
<6.1
7.7
a fpKi = functional pKi obtained from the GTPγS functional assay. FLIPR = fluorescent imaging plate reader. SEM for D3 GTPγS, H1 FLIPR, and
2
HERG data sets is (0.1 and for the D2 GTPγS data is (0.2. b A . c ACD logD 7.4. version 11.
˚
less bulky than the hydrogen one), which is instead well
tolerated. These results were confirmed by the general trend
of the DA D3 activity vs Daylight’s ClogD, which is reported
inFigure 3B. Among the few outliers which can be observed in
this plot, derivatives 65 and 75 are endowed with RG-R2s
carrying a bulky substituent in ortho (their Hansch’s MR
values are respectively 5.02 and 5.65), while all the other
compounds have either a hydrogen or a fluorine in the ortho
positions (with their Hansch MR values equal to 1.03 and
0.92, respectively). Derivative 17 contains a p-cyano-phenyl in
RG-R2. Although the global polarity of the compound, as
expressed by tPSA, is not much higher than that for the other
molecules in the data set, this substituent is by far the most
polar RG-R2. Potentially, very polar substituents might not
be tolerated in this precise position in this specific series,
although this hypothesis must be further explored. Finally,
for derivative 9, the theory previously discussed about a
potential steric clash might be invoked; nonetheless, at the
current status of the exploration, it is not possible to exclude
any a priori hypothesis, including a potentially slightly diffe-
rent binding mode to the receptor within the subseries. As far
as derivative 7 is concerned, the lack of substituents on the aryl
ring, also in this case, might lead to a slightly different
behavior.
When the analysis was repeated with respect to the hERG
activity, it was evident that substituents on the right-hand side
which were very efficient in boosting the DA D3 activity (e.g.,
the furan and the 2-methyl-5-quinoline) were unfortunately
also good at increasing hERG. However, the RG-R2 plot
suggests that therewasslight roomfor adivergentSARforthe
two targets as reported in Figure 3C. The selection of the few
compounds along the screening cascade and the use of
receptor modeling techniques confirmed this possibility.
Further Characterization of Derivatives 27, 28, 32, 35, 51,
and 74. In agreement with the screening cascade, these
selected compounds underwent a further in vitro characte-
rization enlarging the selectivity panel using both in-house
testing and a selectivity screen available at Cerep as pre-
viously described.7 Because the compounds showed at least
100-fold selectivity over a wide range of receptors and
enzymes, they were submitted to a patch clamp hERG
electrophysiology assay7,16 to assess their overall activity
for this Kþ channel. The results of the experiments are
reported in Table 4.
remaining compounds in the expression of either nicotine or
cocaine-induced conditioned place preference7 (CPP).
Prior to these in vivo experiments, and for a better final
interpretation of the results, it was also decided to generate
affinity data in rat native tissues using [125I]-7-OH-PIPAT in
competition binding assays on brain homogenates from rat
nucleus accumbens and olfactory tubercles; results from
these experiments are reported in Table 5.
In the CPP paradigm, animals are given an injection of a
drug or vehicle and confined or “paired” to a specific
environment with distinct cues. This pairing of the animal
with specific cues after being given vehicle or drug is
repeated. Subsequently, on the test day, animals are not
given any treatment (drug-free state) and are allowed to
freely explore the CPP apparatus to determine if they prefer
an environment in which they previously received drug
compared to an environment in which they previously
received vehicle. If the animals approach and spend a
significantly greater amount of time in the drug-paired
environment, one can reasonably infer that the drug was
rewarding. Further details on this paradigm can be found in
the experimental part.
Separate studies were performed to investigate the effects
of the acute administration of derivatives 27, 28, 32, and 74
compared to vehicle on the expression of nicotine CPP in
male Sprague-Dawley rats. All the compounds were admi-
nistered ip (0.05, 0.1, 0.3, 1.0, 3.0 mg/kg) and the amount of
time spent in each chamber was determined using an auto-
mated timing system. The results are reported in Tables 6-9.
In addition, to further strengthen the available results6
indicating that antagonism at the DA D3 receptor is effica-
cious in attenuating the rewarding/reinforcing properties of
different drugs of abuse, two of these four derivatives
(namely 28 and 74), were also tested to investigate their acute
effects on the expression of cocaine CPP in male Sprague-
Dawley rats. Both compounds were given ip (0.05, 0.1, 0.3,
1.0, 3.0 mg/kg). The results of these experiments are reported
in Tables 10 and 11.
A one-way ANOVA revealed a significant main effect of
dose on the time spent in the nicotine or cocaine-paired
compartments, and the results clearly show that all the
compounds tested significantly reduced both nicotine-
(Table 6-9) and cocaine-induced CPP (Table 10-11) in a
dose-dependent manner.
On the basis of these new generated data, which consi-
dered the affinity at the DA D3 receptor, the overall in vitro
and in vivo PK properties of these derivatives and the in vivo
efficacy of DA D3 receptor antagonists in animal models
of addiction6 combined with our previous experience,7 it
was decided to temporarily remove derivatives 35 and 51
from further progression and to assess the efficacy of the
Additional experiments were performed to assess whether
or not these derivatives produced nonspecific behavioral
effects. Subsequently, the acute effects of these compounds
up to high exposure (i.e., 10 mg/kg ip) were determined using
spontaneous locomotor activity and motor coordination
using the accelerating rotarod. For locomotor activity
assessment, rats were treated with either saline or the test

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 381
Figure 3. (A) Pie chart for RG-R2 fragmentation. See text for a comprehensive explanation of the technique. (B) General trend of DA D3 fpKi
vs whole molecule lipophilicity expressed as ACD logD 7.4. Ver 11. Dots are color coded according to RG-R1: yellow, quinoline series
(Table 1); blue, oxazolo series (Table 3); red, other substituents (Table 2). (C) RG-R2 D-scores for hERG vs RG-R2 D-scores for D3. Labels
beneath the substituents are respectively D-score hERG, D-score D3. RG-R2 groups positioned above the unity line have on average a greater
effect on D3 than on hERG.
Table 4. Results from Patch Clamp hERG Electrophysiology Assay for
the Selected Derivativesa
Table 5. Profile of the Selected Derivatives in [125I]-7-OH-PIPAT
Competition Binding Assay on Brain Homogenates from Rat Brain
compd
27
28
32
35
51
74
compd
27
28
32
35
51
74
affinity at the hERG channel (μM) 0.39 0.47 0.35 0.20 0.11 0.46
a Results are expressed in μM values.
r DA D3 rat native tissue pKi 9.01 8.70 9.19 9.05 NT 8.38
For motor coordination assessment, rats were trained on
the accelerating rotarod (4-40 rpm over 270s; 7750, Ugo
Basile, Italy) twice daily for 3 consecutive days. On the test
day (day 4), rats were treated with vehicle, the four selected
compounds (1, 3, 10 mg/kg ip), or haloperidol (1 mg/kg ip) as
a positive control (n = 8 rats/group). Rats were then
repeatedly tested for their endurance performance on the
compounds (1, 3, 10 mg/kg ip). All rats were subsequently
placed in an AccuScan locomotor apparatus. Their locomo-
tor activity was measured for a 30 min period. An overall
ANOVA revealed that there was no significant effect of
treatment and no significant treatment ꢀ time interval
interaction (data not shown).

382 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Micheli et al.
Table 6. Effect of a Single ip Administration of Vehicle and 27
(0.05, 0.1, 0.3, 1, 3 mg/kg) on the Expression of the Conditioned Place
Preference Response to 0.6 mg/kg sc of (-)-Nicotine or Vehicle
(1 mL/kg sc) in Adult Male Sprague-Dawley Rats
Table 9. Effect of a Single ip Administration of Vehicle and 74
(0.05, 0.1, 0.3, 1, 3 mg/kg) on the Expression of the Conditioned
Place Preference Response to 0.6 mg/kg sc of (-)-Nicotine or Vehicle
(1 mL/kg sc) in Adult Male Sprague-Dawley Rats
time spent in compartments
(min) ( SEM
time spent in compartments
(min) ( SEM
treatment
pairings
drug given
on test day
treatment
pairings
drug given
on test day
paired
unpaired
paired
unpaired
vehicle/vehicle
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle
vehicle
7.51 ( 0.3
9.81 ( 0.3a
9.73 ( 0.4
9.87 ( 0.4
9.55 ( 0.4
7.91 ( 0.3b
7.77 ( 0.4b
7.49 ( 0.3
5.19 ( 0.3
5.27 ( 0.4
5.13 ( 0.3
5.45 ( 0.4
7.09 ( 0.3
7.23 ( 0.4
vehicle/vehicle
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle
vehicle
7.51 ( 0.3
9.39 ( 0.4a
8.68 ( 0.3
8.07 ( 0.3b
7.42 ( 0.3c
7.26 ( 0.2c
7.05 ( 0.2c
7.49 ( 0.3
5.61 ( 0.4
6.32 ( 0.3
6.93 ( 0.3
7.58 ( 0.3
7.84 ( 0.2
7.95 ( 0.2
27 (0.05 mg/kg)
27 (0.1 mg/kg)
27 (0.3 mg/kg)
27 (1.0 mg/kg)
27 (3.0 mg/kg)
74 (0.05 mg/kg)
74 (0.1 mg/kg)
74 (0.3 mg/kg)
74 (1.0 mg/kg)
74 (3.0 mg/kg)
a P < 0.0001 vs vehicle/vehicle/vehicle. b P < 0.0001 vs vehicle/
nicotine/vehicle.
a P < 0001 vs vehicle/vehicle/vehicle. b P < 001 vs vehicle/nicotine/
vehicle. c P < 0001 vs vehicle/nicotine/vehicle.
Table 7. Effect of a Single ip Administration of Vehicle and 28
(0.05, 0.1, 0.3, 1, 3 mg/kg) on the Expression of the Conditioned
Place Preference Response to 0.6 mg/kg sc of (-)-Nicotine or Vehicle
(1 mL/kg sc) in Adult Male Sprague-Dawley Rats
Table 10. Effect of a Single ip Administration of Vehicle and 28
(0.05, 0.1, 0.3, 1, 3 mg/kg) on the Expression of the Conditioned
Place Preference Response to 15 mg/kg ip of (-)-Cocaine or Vehicle
(1 mL/kg sc) in Adult Male Sprague-Dawley Rats
time spent in compartments
(min) ( SEM
time spent in compartments
(min) ( SEM
treatment
pairings
drug given
on test day
treatment
pairings
drug given
on test day
paired
unpaired
paired
unpaired
vehicle/vehicle
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle
vehicle
7.51 ( 0.4
9.87 ( 0.4a
9.59 ( 0.4
9.14 ( 0.4
8.49 ( 0.4b
7.65 ( 0.4c
7.10 ( 0.3c
7.49 ( 0.4
5.13 ( 0.4
5.41 ( 0.4
5.86 ( 0.4
6.51 ( 0.4
7.35 ( 0.4
7.90 ( 0.3
vehicle/vehicle
vehicle/cocaine
vehicle/cocaine
vehicle/cocaine
vehicle/cocaine
vehicle/cocaine
vehicle/cocaine
vehicle
vehicle
7.11 ( 0.4
10.83 ( 0.5a
9.79 ( 0.3b
9.36 ( 0.4c
8.70 ( 0.5d
7.89 ( 0.3d
7.36 ( 0.3d
7.90 ( 0.4
4.17 ( 0.5
5.21 ( 0.3
5.64 ( 0.4
6.30 ( 0.5
7.11 ( 0.3
7.64 ( 0.3
28 (0.05 mg/kg)
28 (0.1 mg/kg)
28 (0.3 mg/kg)
28 (1.0 mg/kg)
28 (3.0 mg/kg)
28 (0.05 mg/kg)
28 (0.1 mg/kg)
28 (0.3 mg/kg)
28 (1.0 mg/kg)
28 (3.0 mg/kg)
a P < 0.0001 vs vehicle/vehicle/vehicle. b P < 0.001 vs vehicle/
a P < 0001 vs vehicle/vehicle/vehicle. b P < 05 vs vehicle/cocaine/
vehicle. c P < 01 vs vehicle/nicotine/vehicle. d P < 0001 vs vehicle/
nicotine/vehicle.
nicotine/vehicle. c P < 0.0001 vs vehicle/nicotine/vehicle.
Table 8. Effect of a Single ip Administration of Vehicle and 32
(0.05, 0.1, 0.3, 1, 3 mg/kg) on the Expression of the Conditioned Place
Preference Response to 0.6 mg/kg sc of (-)-Nicotine or Vehicle
(1 mL/kg sc) in Adult Male Sprague-Dawley Rats
Table 11. Effect of a Single ip Administration of Vehicle and 74
(0.05, 0.1, 0.3, 1, 3 mg/kg) on the Expression of the Conditioned
Place Preference Response to 15 mg/kg ip of (-)-Cocaine or Vehicle
(1 mL/kg sc) in Adult Male Sprague-Dawley Rats
time spent in compartments
(min) ( SEM
time spent in compartments
(min) ( SEM
treatment
pairings
drug given
on test day
paired
unpaired
treatment
pairings
drug given
on test day
paired
unpaired
vehicle/vehicle
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle/nicotine
vehicle
vehicle
7.41 ( 0.4
9.82 ( 0.3a
9.58 ( 0.3
9.16 ( 0.2
8.23 ( 0.2b
7.54 ( 0.3c
7.16 ( 0.3c
7.49 ( 0.4
5.18 ( 0.3
5.42 ( 0.4
5.84 ( 0.4
6.77 ( 0.3
7.46 ( 0.4
7.84 ( 0.3
vehicle/vehicle
vehicle/cocaine
vehicle/cocaine
vehicle/cocaine
vehicle/cocaine
vehicle/cocaine
vehicle/cocaine
vehicle
vehicle
7.06 ( 0.4
10.81 ( 0.5a
10.44 ( 0.4
9.01 ( 0.4b
8.23 ( 0.3c
7.56 ( 0.3c
7.10 ( 0.3c
7.94 ( 0.4
4.19 ( 0.5
4.56 ( 0.4
5.89 ( 0.4
6.87 ( 0.3
7.34 ( 0.3
7.90 ( 0.3
32 (0.05 mg/kg)
32 (0.1 mg/kg)
32 (0.3 mg/kg)
32 (1.0 mg/kg)
32 (3.0 mg/kg)
74 (0.05 mg/kg)
74 (0.1 mg/kg)
74 (0.3 mg/kg)
74 (1.0 mg/kg)
74 (3.0 mg/kg)
a P < 0001 vs vehicle/vehicle/vehicle. b P < 001 vs vehicle/nicotine/
vehicle. c P < 0001 vs. vehicle/nicotine/vehicle.
a P < 0001 vs vehicle/vehicle/vehicle. b P < 001 vs vehicle/nicotine/
vehicle. c P < 0001 vs vehicle/nicotine/vehicle.
rotarod 30, 60, and 120 min after these treatments. Rotarod
latencies were measured with a 270 s cutoff time. Pretreat-
ment with haloperidol significantly decreased the time that
rats spent on the rotarod. In contrast, the selected derivatives
(27, 28, 32, 74) did not affect endurance performance at any
of the posttreatment times tested (data not shown).
A final important point to be assessed in this quest for new
and developable selective DA D3 receptor antagonist, as it
was done for the previously reported BAZ system,7 was
related to the demonstration of the lack of QTc prolongation
in vivo. It is well-known that an undesirable feature of some
drugs is their ability to delay cardiac repolarization, an effect
that can be measured as prolongation of the QT interval on
the surface electrocardiogram (ECG).17,18 Because of its
inverse relationship to heart rate, the QT interval is routinely
transformed (normalized) by means of various formulas into
a heart rate independent “corrected” value known as the QTc
interval. The QTc interval is intended to represent the QT
interval at a standardized heart rate of 60 bpm.
In agreement with our screening cascade, derivatives 27,
28, 32, and 74 were all evaluated for their ability to determine
QTc prolongation in vivo when assessed in the anesthetized
guinea pig model.19 All of the compounds were administered

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 383
intravenously in escalating doses from 0.5 to 15 mg/kg.
Despite the very high exposure reached at the top dose of
15 mg/kg (Cmax = 6237, 9577, 8322, and 8635 ng/mL for 27,
28, 32, and 74, respectively), the QTc interval was not
significantly increased in the guinea pig model by any of
the derivatives. Thus, there is a large window between the
efficacy and the potential side effects that was obtained in
preclinical species for these derivatives.
in 20 mM HEPES pH 7.4, 100 mM NaCl, 10 mM MgCl2,
60 μg/mL saponin, and 30 μM GDP is added. The third addition
was a 20% TAV addition of either buffer (agonist format) or
EC80 final assay concentration of agonist, Quinelorane, pre-
pared in assay buffer (antagonist format). The assay was started
by the addition of 29%TAV of GTPγ[35S] 0.38 nM final
(37 MBq/mL, 1160 Ci/mmol, Amersham). After all additions
assay plates were spun down for 1 min at 1000 rpm, assay plates
were counted on a Viewlux, 613/55 filter, for 5 min, between 2
and 6 h after the final addition.
Conclusions
The effect of the test drug over the basal generates EC50 value
by an iterative least-squares curve fitting program, expressed in
the table as pEC50 (i.e., -logEC50). The ratio between the
maximal effect of the test drug and the maximal effect of full
agonist, Quinelorane, generated the intrinsic activity (IA) value
(i.e., IA = 1 full agonist, IA < 1 partial agonist). fpKi values of
test drug were calculated from the IC50 generated by “antagonist
format” experiment using Cheng and Prusoff equation: fKi =
IC50/1 þ ([A]/EC50), where [A] is the concentration of the
agonist in the assay and EC50 is the agonist EC50 value obtained
in the same experiment. fpKi is defined as -logfKi.
Starting from the potent and selective DA D3 receptor
antagonists previously reported (1-4), a new series of 1,2,4-
triazol-yl azabicyclo[3.1.0]hexanes was identified. Rational
design and computational tools helped to design a number
of products endowed with high affinity and selectivity for the
DA D3 receptor. In addition, selected compounds of this
family series proved to have appropriate developability char-
acteristics (P450, Cli, F%) and good CNS penetration. As
described, a number of these compounds were also active in
the CPP model and were endowed with a large selectivity
window with respect to their affinity at the hERG channel,
leading to a total lack of QTc when assessed in vivo in the
anesthetized guinea pig model.
[
125I]-7-OH-PIPAT Competition Bbinding Assay on Brain
Homogenates from Rat Brain. Homogenates from frozen
nucleus accumbens and olfactory tubercles were prepared as
described by Burris et al. (1994).20 In saturation experiments,
increasing concentrations of [125I]-7-OH-PIPAT (15 pM-2 nM)
were incubated with 12 μg/well of homogenates for 45 min at
37 ꢀC in a final volume of 200 μL of 50 mM Tris-HCl (pH7.0),
50 mM NaCl, 100 μM Gpp(NH)p, and 0.02%BSA, i.e., condi-
tions which inhibit [125I]-7-OH-PIPAT binding to D2 and
5HT1A receptors.20 Nonspecific binding was determined by
the presence of 1 μM 1. In competition binding experiments,
increasing concentrations of 35 (17 pM-1 μM) were incubated
as above in the presence of 0.1 nM [125I]-7-OH-PIPAT.
Provided that in vivo preclinical evidence can be extrapo-
lated to human, selective DA D3 receptor antagonists belong-
ing to this new chemical series show high promise for the
treatment of drug addiction.
Experimental Section
Biological Test Methods. In Vitro Studies. [35S] GTPγS
Functional Binding Assay in Cell Membranes Expressing hD3
Receptors. In vitro functional studies were performed according
to the following [35S]-GTPγS protocol. Cells used in the study
are Chinese hamster ovary (CHO) cells.
Cell membranes were prepared as follows. Cell pellets were
resuspended in 10 volumes of 50 mM HEPES, 1 mM EDTA pH
7.4, using KOH. On the day, the following proteases were added
to the buffer just prior to giving the homogenization buffer.
10-6 M leupeptin (Sigma L2884) - 5000 ꢀ stock = 5 mg/mL
in buffer.
Reactions were stopped by filtration through GF/C 96-well
filter plates presoaked in 0.3% polyethylenimine using a cell
harvester. Filters were washed 3 times with 1 mL of ice-cold
50 mM Tris-HCl (pH7.7), and radioactivity was counted in a
microplate scintillation counter (Top Count, Perkin-Elmer).
Radioligand binding data were analyzed by nonlinear regres-
sion analysis using GraphPad Prism 4.0 (GraphPad Software,
CA). Determination of KD and Bmax of [125I]-7-OH-PIPAT from
saturation experiments was assessed by using one-site binding
(hyperbola) equation. Curve fitting from competition-binding
experiments was determined by using one site competition
equation after checking with F test (P < 0.05) that Hill slope
in the four parameter logistic equation was not statistically
different from 1.0. In this condition, IC50 values were converted
to Ki using the Cheng-Prusoff equation.33 Results are expressed
as mean pKi ( SEM.
25μg/mL bacitracin (Sigma B0125) - 1000 ꢀ stock = 25 mg/mL
in buffer.
1 mM PMSF - 1000 ꢀ stock = 17 mg/mL in 100% ethanol.
2 ꢀ 10-6 M pepstain A - 1000 ꢀ stock = 2 mM in 100%
DMSO.
The cells were homogenized by 2 ꢀ 15 s bursts in a 1 L glass
Waring blender in a class two biohazard cabinet. The resulting
suspension was spun at 500g for 20 min (Beckman T21 centri-
fuge: 1550 rpm). The supernatant was withdrawn with a 25 mL
pipet, aliquotted into prechilled centrifuge tubes, and spun
at 48000g to pellet membrane fragments (Beckman T1270:
23000 rpm for 30 min). The final 48000g pellet was resuspended
in homogenization buffer (4ꢀ the volume of the original cell
pellet). The 48000g pellet was resuspended by vortexing for 5 s
and homogenized in a dounce homogenizer 10-15 stokes.
The prep was distributed into appropriate sized aliquots
(200-1000 μL) in polypropylene tubes and store at -800 ꢀC.
Protein content in the membrane preparations was evaluated
with the Bradford protein assay.
The final top concentration of test drug was 3 μM in the
assay, and 11 points serial dilution curves 1:4 in 100% DMSO
were carried out using a Biomek FX. The test drug at 1% total
assay volume (TAV) was added to a solid, white, 384-well assay
plate. 50%TAV of precoupled (for 90 min at 4 ꢀC) membranes,
5 μg/well, and wheatgerm agglutinin polystyrene scintillation
proximity assay beads (RPNQ0260, Amersham), 0.25 mg/well,
[
125I]-7-OH-PIPAT ([125I](R)-trans-7-hydroxy-2-[N-propyl-
N-(30-iodo-20-propenyl)amino] tetralin, 81.4 TBq/mmol) was
purchased from PerkinElmer Life and Analytical Sciences.
Haloperidol and Gpp(NH)p (guanylyl-50-imidodiphosphate)
were obtained from Sigma Chemicals.
hERG-3H Dofetilide Binding Assay. hERG activity was mea-
3
sured using H dofetilide binding in a scintillation proximity
assay (SPA) format. The activity was measured with a Perkin-
Elmer Viewlux imager.
H1-FLIPR Assay. A functional response in CHO-hH1 cells
was measured using cytoplasmic calcium indicator, Fluo-4.
The change in cell fluorescence was measured in a FLIPR
(λex = 488 nm, λEM = 540 nm, Molecular Devices, UK).
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 recom-
binant human CYP450 microsomal protein (0.1 mg/mL; Cypex

384 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Micheli et al.
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) NaH-
CO3; 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 (Cli) Assay. Intrinsic clearance (CLi)
values were determined in rat and human liver microsomes.
Test compounds (0.5 μM) were incubated at 37 ꢀC for 30 min in
50 mM potassium phosphate buffer (pH 7.4) containing 0.5 mg
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 (CLi) 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 liver for all species. Values for CLi were expressed as
mL/min/g liver. The lower limit of quantification of clearance
was determined to be when <15% of the compound had been
metabolized by 30 min and this corresponded to a CLi value of
0.5 mL/min/g liver. The upper limit was 50 mL/min/g liver.
Patch Clamp hERG Electrophysiology Assay. On the days of
experimentation, a weighed amount of the test substance was
formulated in dimethyl sulfoxide (DMSO, lot no. U10781;
Sigma-Aldrich, UK) by shaking to give a stock concentration
of 10 mM of the test compound. The 10 mM stock solution was
serially diluted in DMSO to give further stock solutions of 1 and
0.1 mM. Aliquots of these stock solutions were added to bath
solution to achieve final perfusion concentrations of 0.1, 1, and
10 μM test compound. The corresponding vehicle concentration
in all test substance perfusion solutions was 0.1% DMSO. Test
substance stock formulations were freshly prepared on each day
of experimentation, stored at room temperature, and protected
from light.
between the patch electrodes (resistance range: 1.4-5.5 MΩ)
and individual cells. The membrane across the electrode tip was
then ruptured and the whole-cell patch-clamp configuration was
established. Once a stable patch had been achieved, recording
commenced in voltage-clamp mode, with the cell initially
clamped at -80 mV. Currents were evoked by stepping the
membrane potential to þ20 mV and then to -50 mV (tail
current). Test compound at 10, 1, and 0.1 μM was used to
produce (if any) inhibition of hERG tail current and therefore to
investigate the concentration-response relationship (n =
3 cells/concentration). The effect of the vehicle (0.1% DMSO)
was investigated in 3 cells. The effect of 100 nM E-4031 was
investigated in 2 of the vehicle treated cells to confirm the
sensitivity of the test system to an agent known to block hERG
current. All perfusion solutions were applied for approximately
10 min.
Anesthetized Guinea Pig Model for the Assessment of QT
Prolongation. Prior to the experiment, six male Hartley guinea
pigs (Charles River, France) weighing between 564 and 605 g on
the day of the test were anaesthetised with urethane (1-1.5 g/kg
ip), tracheotomized, artificially ventilated with a tidal volume of
approximately 1 mL/100 g at a rate of 54 cycles/min, and
surgically prepared for the experiment (i.e., insertion of cathe-
ters in appropriate blood vessels, see below, and placement of
surface electrodes for lead II electrocardiogram recording).
The animal’s temperature was kept constant between 36.8 and
38.0 ꢀC. Once physiological parameters were stabilized (approxi-
mately 30 min following surgery), each animal was given either the
vehicle (5% w/v dextrose in aqueous 0.9 w/v sodium chloride) or test
compound by the intravenous route (via the jugular vein) over
15 min/treatment.
The following parameters were recorded: arterial pressure (via
the carotid artery), heart rate, electrocardiography (i.e., RR, PQ,
QRS, and QT interval duration; the QT interval was corrected for
heart rate changes according to Fridericia, Bazett and Van de
Water’s formula). Parameters were recorded prior to dosing to
establish baseline measurements and continuously during the
infusion period but data were reported every 5 min during each
infusion period. In addition, blood samples (0.3 mL) were
collected via the femoral artery at the end of each infusion period
for toxicokinetic evaluations.
In Vivo Studies. All experiments were prereviewed and ap-
proved by a local animal care committee in accordance with the
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 Euro-
pean Directive 86/609/EEC on the care and welfare of labora-
tory animals.
Nicotine CPP. Male Sprague-Dawley rats (200 g at the start
of the pairings, Taconic Farms, Germantown, NY) were used in
all experiments. Animals were housed two per cage, and there
was no more than a 5% difference in body weight between the
cagemates. Animals were kept on a 12 h lights on/12 h lights off
schedule (lights on at 09.00 h). Food and water were freely
available. The conditioning and testing of all animals was
carried out between 11.00-18.00 h. All rats were naive and used
only once.
Reference Substance. E-4031 (batch no. MLE9446; Wako Pure
Chemical Industries Ltd.). Stock solutions of E-4031 (100 μM)
were prepared in reverse osmosis water, aliquoted, and stored at
approximately -20 ꢀC until use. On the day of use, the 100 μM
stock solution was added to bath solution to give a final perfusion
concentration of 100 nM.
An automated, two-chambered, Plexiglas CPP apparatus was
used as previously described,21-23 with modifications. Briefly, the
two pairing chambers of the apparatus were identical in dimen-
sions (25 cm ꢀ 14 cm ꢀ 36 cm) and were separated by removable
Plexiglas guillotine doors. The pairing chambers were composed
of distinct visual and tactile cues. The walls of one of the pairing
chamberswerewhite, withcage bedding onthe floor andthe walls
of the second chamber consisted of alternating white and black
boxes (1.2 cm ꢀ 1.8 cm) in a chessboard pattern and a Plexiglas
floor. The two pairing chambers were separated by a third,
neutral connecting tunnel, with one-half of the wall white and
the other half with black and white boxes.
Bath and Pipette Solutions. The composition of bath solution
was (mM): NaCl 137, KCl 4, CaCl2 1.8, MgCl2 1.0, D-glucose 10,
N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES)
10, pH 7.4 with 1 M NaOH. Pipette solution was prepared in
batches, aliquoted, and stored frozen until the day of use. The
composition of pipet solution was (mM): KCl 130, MgCl2 1.0,
ethylene glycol-bis(ss-aminoethyl ether)-N,N,N0,N0-tetraacetic
acid (EGTA) 5, MgATP 5, HEPES 10, pH 7.2, with 1 M KOH.
Study Design. Cells (passage number: 48) were transferred to
the recording chamber and continuously perfused (at approxi-
mately 1-2 mL/min) with bath solution at room temperature.
High resistance seals (seal resistances >1.5 GΩ) were formed

Article
Expression studies with the compounds reported in the tables
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 385
Table 12
or vehicle were divided into four phases: acclimation, handling,
conditioning, and testing. The animals were not exposed to the
chambers prior to the start of the pairings. During days 1-3,
animals were acclimated to the animal facility. During handling
(days 4-6), animals were transported to the laboratory and
handled for 5 min each. During conditioning (days 7-14),
animals were exposed to once-daily conditioning sessions. For
each conditioning session, animals (10 rats per group) were
injected with nicotine (0.6 mg/kg sc in a volume of 1 mL/kg) or
vehicle (1 mL/kg sc) and then immediately confined for 30 min in
an appropriate cue-specific chamber. During conditioning,
nicotine was always paired with 1 cue-specific environment,
and vehicle was paired with the other; nicotine or vehicle
exposure (and appropriate environmental pairing) alternated
from day to day. This was done over an 8-day period, i.e.,
animals were given four pairings with nicotine and there was a
24 h separation between exposure to vehicle and nicotine. The
animals in each group were randomly assigned to a 2 ꢀ 2
factorial design, with one factor being the pairing chamber
and the other factor being the order of conditioning. In the
counterbalanced procedure, the animals were randomly assi-
gned to one of the two pairing chambers so that half of the
subjects received the drug in one compartment (white walls with
bedding on the floor) and the other half in the other compart-
ment (alternating black and white boxes with a smooth chamber
floor). This procedure resulted in the animals receiving equal
exposure to the two compartments and due to random assign-
ment controlled for their side preference. Another group of 10
animals were paired with vehicle in both chambers of the
apparatus. On the test day (day 15), animals were randomly
divided into five groups of 10 and received the test compounds
or vehicle (10% 2-hydroxypropyl-β-cyclodextrin) in the home
cage 30 min before they were placed in the apparatus and
allowed free access to both chambers for 15 min. The amount
of time spent in each chamber was determined using an auto-
mated timing system.
analytical column
mobile phase
Zorbax SB-C18, 4.6 mm ꢀ 50 mm (1.8 μ)
amm acet, 5 mM þ 0.1% formic
acid/acetonitrile þ 0.1% formic acid
97/3 f 36/64 v/v in 3.5 min f 10/90 in 3.5 min
2.0 mL/min
gradient
flow rate
detection
MS
DAD, 210-350 nm
ESþ
Crudes were evaporated and purified by column chromatog-
raphy. The solids were taken up with dichloromethane and a
1.0 M HCl solution in diethylether was added; the solvent was
removed under reduced pressure to give product compounds as
hydrochloride salts.
Where no specific details are given, the retention times
reported refer to the following general analytical chromato-
graphic conditions of method A: column, X Terra MS C18
5 mm, 50 mm ꢀ 4.6 mm; mobile phase: A = NH4HCO3 soln 10n
mM, pH 10; B = CH3CN; gradient, 10% (B) for 1 min, from
10% (B) to 95% (B) in 12 min, 95% (B) for 3 min. Flow rate:
1 mL/min. UV wavelength range: 210-350 nm. Mass range:
100-900 amu. Ionization method: ESþ
The enantiomeric purity of each single enantiomer obtained
after preparative chromatography on chiral columns was always
verified on analytical column. For example, enatiomeric excess
has been measured on the separated enantiomers by means of
SFC analytical techniques. Typical conditions were derived from
ChiralpakAD-Hcolumn (25cm ꢀ 0.46 cm) with ethanol (þ0.1%
isopropylamine) 15% as modifier, flow rate 2.5 mL/min, P = 180
bar at 35 ꢀC with detection at 220 nm.
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%.
2-Methyl-5-[4-methyl-5-({3-[(1R,5S/1S,5R)-1-phenyl-3-aza-
bicyclo[3.1.0]hex-3-yl]propyl}thio)-4H-1,2,4-triazol-3-yl]quinoline
Hydrochloride (5). The compound was obtained as a white,
1
slightly hygroscopic, solid. H NMR (1H, DMSO-d6): δ: 10.4
(-)-Nicotine (þ)-bitartrate salt was used in all experiments
(Sigma-Aldrich Corporation, Saint Louis, MO) and was dis-
solved in sterile physiological saline and the pH adjusted to 7.4
with NaOH. The doses of nicotine were expressed as free base.
2-Hydroxypropyl-β-cyclodextrin was purchased from Tocris-
Cookson Chemical (St. Louis, MO). Data were analyzed with a
1-way ANOVA with a main factor of dose. Statistical signifi-
cance was set at a probability level of P < 0.05.
Cocaine CPP. The cocaine CPP procedure was identical to the
one described for nicotine except that (1) only four pairings were
used and (2) for each conditioning session, animals were injected
with (-)-cocaine HCl (15 mg/kg ip in a volume of 1 mL/kg) or
vehicle (1 mL/kg ip of deionized, distilled water). (-)-Cocaine
HCl was purchased from Sigma Chemicals (St. Louis, MO).
Chemical Procedures. General. Experimental. NMR spectra
were obtained on Varian INOVA spectrometers (300, 400, and
500 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). All mass spectrometric measure-
ments were performed using a Micromass Platform LCZ (Waters,
Manchester, UK) mass spectrometer operated in positive electro-
spray ionization mode. When LC/MS detection was performed,
analytical conditions were used as reported in Table 12.
(bs,1H), 8.3 (bs, 1H), 8.2 (d, 1H), 7.9 (t, 1H), 7.8 (d, 1H), 7.6 (bd,
1H), 7.4-7.3 (m, 5H), 4.0-3.5 (m/m, 2H), 3.7-3.45 (m/m, 2H),
3.5-3.3 (m, 7H), 2.73 (s, 3H), 2.3 (m, 3H), 1.60,1.1 (t,t 2H). MS:
m/z 456 [M þ H]þ.
Derivative 5 was separated to give the separated enantiomers
by semipreparative HPLC. Retention times given were obtained
using an analytical HPLC using a chiral column Chiralcel OD
5 μm, 250 mm ꢀ 4.6 mm; eluent A: n-hexane; B: 2-propanol,
gradient isocratic 25% B, flow rate 1 mL/min, detection UV at
200-400 nm. Enantiomer 6retention time = 39.2 min; enantiomer
7 retention time = 43.4 min.
5-[5-({3-[(1R,5S/1S,5R)-1-(3,4-Dichlorophenyl)-3-azabicyclo-
[3.1.0]hex-3-yl]propyl}thio)-4-methyl-4H-1,2,4-triazol-3-yl]-2-methyl-
quinoline Hydrochloride (8). The compound was obtained as a white,
slightly hygroscopic, solid (70% yield). 1H NMR (1H, DMSO-d6) δ:
10.46 (bs,1H), 8.58 (s, 1H), 7.4-7.2 (m, 5H), 4.04 (dd, 1H), 3.73 (m,
1H), 3.7 (s, 3H), 3.7-3.4 (m, 2H), 3.4-3.2 (m þ t, 4H), 2.39 (s, 3H),
2.17 (m, 3H), 1.64,1.1 (2t, 2H). MS: m/z 562 [M þ H]þ.
5-[5-({3-[(1R,5S/1S,5R)-1-(4-tert-Butylphenyl)-3-azabicyclo-
[3.1.0]hex-3-yl]propyl}thio)-4-methyl-4H-1,2,4-triazol-3-yl]-2-methyl-
quinoline Hydrochloride (11). The compound was obtained as a
1
white, slightly hygroscopic, solid. H NMR (1H, DMSO-d6): δ:
10.16 (bs,1H), 8.15 (dd, 2H), 7.89 (t, 1H), 7.76 (d, 1H), 7.49 (d, 1H),
7.36 (d, 2H), 7.23 (d, 2H), 4.05 (dd, 1H), 3.77 (dd, 1H), 3.58 (m, 2H),
3.44 (s, 3H), 2.7 (bm, 4H), 2.34 (s, 3H), 2.23 (t, 2H), 2.15 (t, 1H), 1.51
(t, 1H), 1.27 (s, 9H), 1.14 (m, 1H). MS: m/z 512 [M þ H]þ.
General Synthetic Procedures. Appropriate 4-methyl-5-aryl-
2,4-dihydro-3H-1,2,4-triazole-3-thiones, prepared by the aryl
acid in agreement with literature procedures, were suspended in
acetone and potassium carbonate and 1-bromo-3-chloropropane
was added. The so obtained 3-[(3-chloropropyl)thio]-4-methyl-
5-aryl-4H-1,2,4-triazoles were suspended/dissolved in dry acet-
onitrile or DMF and added (1S,5R/1R,5S) or (1S,5R) aryl
azabicyclo[3.1.0]hexanes in presence of potassium carbonate
according to Scheme 1.
Derivative 11 was separated to give the separated enantiomers by
chiral chromatography. Retention times given were obtained using
an analytical supercritical fluid chromatography (Gilson) using a
chiral column Chiralpak AD-H, 25 cm ꢀ 0.46 cm, eluent CO2
containing 20% (ethanol þ 0.1% 2-propanol), flow rate 2.5 mL/
min, P 194 bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 12
retention time = 7.0 min; enantiomer 13 retention time = 7.8 min.

386 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Micheli et al.
5-[5-({3-[(1R,5S/1S,5R)-1-(4-Bromophenyl)-3-azabicyclo[3.1.0]-
hex-3-yl]propyl}thio)-4-methyl-4H-1,2,4-triazol-3-yl]-2-methyl-
quinoline Hydrochloride (14). The compound was obtained as a
white, slightly hygroscopic, solid. 1H NMR (1H, DMSO-d6) δ:
10.28 (bs,1H), 8.16 (dd, 2H), 7.89 (dd, 1H), 7.76 (d, 1H), 7.55 (d,
2H), 7.49 (d, 1H), 7.28 (d, 2H), 4.06 (bm, 1H), 3.77 (bm, 1H), 3.6
(bm, 2H), 3.44 (s, 3H), 3.5-3.2 (bm, 4H), 2.71 (s, 3H), 2.23 (m,
3H), 1.58/1.14 (t/m, 2H). MS: m/z 534 [M þ H]þ.
2-Methyl-6-{4-methyl-5-[(3-{(1S,5R)-1-[4-(trifluoromethyl)-
phenyl]-3-azabicyclo[3.1.0]hex-3-yl}propyl)thio]-4H-1,2,4-tri-
azol-3-yl}quinoline Hydrochloride (25). The compound was
obtained as a white, slightly hygroscopic, solid. 1H NMR
(DMSO-d6) δ: 10.41 (bs, HCl), 8.67 (bs, 1H), 8.47 (s, 1H), 8.2
(s, 2H), 7.72 (m, 1H), 7.68 (m, 2H), 7.49 (m, 2H), 4.07 (m, 1H),
3.74 (dd, 1H), 3.72 (s, 3H), 3.64 (dd, 1H), 3.51 (m, 1H), 3.3 (m,
4H), 2.81 (s, 3H), 2.28 (m, 1H), 2.19 (m, 2H), 1.72 (t, 1H), 1.19 (t,
1H). MS: m/z 524 [M þ H]þ. Retention time = 10.17 min
(method A).
Derivative 14 was separated to give the separated enantiomers.
Retention times given were obtained using an analytical super-
critical fluid chromatography (Gilson) using a chiral column
Chiralcel OJ-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 10%
(ethanol þ 0.1% isopropylamine), flow rate 2.5 mL/min, P 196
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 15 retention
time = 56.8 min; enantiomer 16. Retention time = 62.5 min. The
absolute configuration of enantiomer 15 was assigned using
comparative VCD and comparative OR analyses of the corres-
ponding free base to be 5-[5-({3-[(1R,5S)-1-(4-bromophenyl)-3-
azabicyclo[3.1.0]hex-3-yl]propyl}thio)-4-methyl-4H-1,2,4-triazol-
3-yl]-2-methylquinoline. The absolute configuration of enantiomer
16 was assigned as described above to be 5-[5-({3-[(1S,5R)-1-(4-
bromophenyl)-3-azabicyclo[3.1.0]hex-3-yl]propyl}thio)-4-methyl-
4H-1,2,4-triazol-3-yl]-2-methylquinoline.
(1S,5R)-3-{3-[(4-Methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio]-
propyl}-1-[4-(trifluoromethyl)phenyl]-3-azabicyclo[3.1.0]hexane
Hydrochloride (26). The compound was obtained as a white,
slightly hygroscopic, solid. 1H NMR (DMSO-d6) δ: 10.3 (bs,),
7.7 (m, 4H), 7.57 (m, 4H), 7.49 (m, 1H), 4.11 (d, 1H), 3.76 (d,
1H), 3.66 (m, 1H), 3.61 (s, 3H), 3.52 (m, 1H), 3.30 (m, 4H), 2.29
(m, 1H), 2.17 (m, 2H), 1.63 (m, 1H), 1.22 (m, 1H). MS: m/z 459
[M þ H]þ.
(1S,5R)-3-(3-{[4-Methyl-5-(2-methyl-3-pyridinyl)-4H-1,2,4-
triazol-3-yl]thio}propyl)-1-[4-(trifluoromethyl)phenyl]-3-azabicyclo-
[3.1.0]hexane Hydrochloride (27). The compound was obtained as a
whitish, slightly hygroscopic, solid. 1H NMR (DMSO-d6) δ: 10.84
(bs, HCl), 8.77 (dd, 1H), 8.19 (bd, 1H), 7.71 (d, 2H), 7.66 (m, 1H),
7.51 (d, 2H), 4.08 (dd, 1H), ca. 3.8 (s, 3H), 3.8-3.3 (m, 7H), 2.54 (s,
3H), 2.30 (m, 1H), 2.22 (m, 2H), 1.83 (m, 1H), 1.20 (m, 1H). NMR
(19F, DMSO): δ -60.8. MS: m/z 474 [M þ H]þ. Retention time =
9.35 min (method A).
Enantiomer 15: specific optical rotation of the corresponding
free base [R]D = þ47ꢀ (CHCl3, T = 20 ꢀC, c = 0.066 g/mL).
Enantiomer 16: specific optical rotation of the corresponding
free base [R]D = -42ꢀ (CHCl3, T = 20 ꢀC, c = 0.065 g/mL).
4-[(1R,5S/1S,5R)-3-(3-{[4-Methyl-5-(2-methylquinolin-5-yl)-
4H-1,2,4-triazol-3-yl]thio}propyl)-3-azabicyclo[3.1.0]hex-1-yl]-
benzonitrile Hydrochloride (17). The compound was obtained as
a white, slightly hygroscopic, solid. 1H NMR (1H, DMSO-d6) δ:
10.45 (bs,1H), 8.26 (bd, 1H), 8.17 (d, 1H), 7.93 (t, 1H), 7.8 (d/d,
3H), 7.5 (d, 1H), 7.46 (d, 2H), 4.09 (d, 1H), 3.76 (d, 1H), 3.67 (t,
1H), 3.6-3.2 (bm, 5H), 3.43 (s, 3H), 2.73 (s, 3H), 2.34 (m, 1H),
2.25 (quint., 2H),1.71/1.22 (dt, 2H). MS: m/z 481[M þ H]þ.
5-[5-({3-[(1R,5S/1S,5R)-1-(4-Methoxyphenyl)-3-azabicyclo-
[3.1.0]hex-3-yl]propyl}thio)-4-methyl-4H-1,2,4-triazol-3-yl]-2-
methylquinoline Hydrochloride (18). The compound was obtained
as a white, slightly hygroscopic, solid. 1H NMR (1H, DMSO-d6)
δ: 10.57 (bs,1H), 8.28 (bs, 1H), 8.2 (d, 1H), 7.94 (t, 1H), 7.82
(d, 1H), 7.56 (d, 1H), 7.25 (d, 2H), 6.91 (d, 2H), 4.01 (dd, 1H), 3.7
(m, 1H), 3.74 (s, 3H), 3.6-3.2 (m, 6H), 3.42 (s, 3H), 2.75 (s, 3H),
2.24 (quint, 2H), 2.08(quint, 1H), 1.62/1.05 (t/t, 2H). MS:m/z 486
[M þ H]þ.
(1S,5R)-3-(3-{[4-Methyl-5-(4-pyridazinyl)-4H-1,2,4-triazol-3-yl]-
thio}propyl)-1-[4-(trifluoromethyl)phenyl]-3-azabicyclo[3.1.0]hexane
Hydrochloride (28). The compound was obtained as a white, slightly
hygroscopic, solid. 1H NMR (DMSO-d6) δ: 10.24 (bs, HCl), 9.56
(m, 1H), 9.39 (m, 1H), 8.01 (s, 1H), 7.64 (m, 2H), 7.44 (m, 2H), 4.08
(m, 1H), 3.68 (s, 3H), 3.58 (bm, 1H), 3.7-3.3 (m, 6H), 2.26 (m, 1H),
2.13 (m, 2H), 1.58 (t, 1H), 1.14 (t, 1H). MS: m/z 461 [M þ H]þ.
Retention time = 8.84 min (method A).
(1S,5R)-3-(3-{[4-Methyl-5-(5-pyrimidinyl)-4H-1,2,4-triazol-3-yl]-
thio}propyl)-1-[4-(trifluoromethyl)phenyl]-3-azabicyclo[3.1.0]hexane
Hhydrochloride (29). The compound was obtained as a off-white,
slightly hygroscopic, solid. MS: m/z 461 [M þ H]þ. Retention
time = 8.92 min (method A).
(1S,5R)-3-(3-{[4-Methyl-5-(3-methyl-2-furanyl)-4H-1,2,4-tri-
azol-3-yl]thio}propyl)-1-[4-(trifluoromethyl)phenyl]-3-azabicyclo-
[3.1.0]hexane Hydrochloride (30). The compound was obtained
as a white, slightly hygroscopic, solid. 1H NMR (DMSO-d6) δ:
10.56 (bs, HCl), 7.85 (d, 1H), 7.71 (d, 2H), 7.51 (d, 2H), 6.64 (d,
1H), 4.08 (dd, 1H), 3.75 (dd, 1H), 3.71 (s, 3H), 3.7-3.3 (m, 4H),
3.27 (t, 2H), 2.31 (m, 1H), 2.28 (s, 3H), 2.18 (m, 2H), 1.74 (t, 1H),
1.21 (t, 1H). MS: m/z 463 [M þ H]þ. Retention time = 10.72 min
(method A).
(1S,5R)-3-(3-{[4-Methyl-5-(6-methyl-3-pyridinyl)-4H-1,2,4-
triazol-3-yl]thio}propyl)-1-[4-(trifluoromethyl)phenyl]-3-azabicyclo-
[3.1.0]hexane Hydrochloride (31). The compound was obtained as
a white, slightly hygroscopic, solid. MS: m/z 474 [M þ H]þ.
Retention time = 9.47 min (method A).
(1S,5R)-3-(3-{[5-(2,4-Dimethyl-1,3-thiazol-5-yl)-4-methyl-4H-
1,2,4-triazol-3-yl]thio}propyl)-1-[4-(trifluoromethyl)phenyl]-3-azabi-
cyclo[3.1.0]hexane Hhydrochloride (32). The compound was ob-
tained as a white, slightly hygroscopic, solid. 1H NMR (DMSO-d6)
δ: 10.61 (bs, 1H), 7.69 (d, 2H), 7.49 (d, 2H), 4.06 (d, 1H), 3.72 (t,
1H), 3.63 (d, 1H), 3.52 (t, 1H), 3.48 (s, 3H), 3.34 (t, 2H), 3.27 (t, 2H),
2.68 (s, 3H), 2.33 (m, 3H), 2.28 (m, 1H), 2.18 (m, 2H), 1.73 (t, 1H),
1.18 (t, 1H). MS: m/z 494 [M þ H]þ. Retention time = 9.79 min
(method A).
(1S,5R)-3-(3-{[4-Methyl-5-(5-methyl-2-pyrazinyl)-4H-1,2,4-tri-
azol-3-yl]thio}propyl)-1-[4-(trifluoromethyl)phenyl]-3-azabicyclo-
[3.1.0]hexane Hydrochloride (33). The compound was obtained
as a white, slightly hygroscopic, solid. 1H NMR (DMSO-d6) δ:
10.41 (bs, HCl), 9.11 (bs, 1H), 8.63 (bs, 1H), 7.66 (d, 2H), 7.44 (d,
2H), 4.02 (dd, 1H), 3.83 (s, 3H), 3.68 (d, 1H), 3.6-3.2 (m, 6H),
2.54 (s, 3H), 2.25 (m, 1H), 2.14 (m, 2H), 1.65 (m, 1H), 1.14
Derivative 18 was separated to give the separated enantiomers.
Retention times given were obtained using an analytical super-
critical fluid chromatography (Gilson) using a chiral column
Chiralpak AD-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 20%
(ethanol þ 0.1% isopropyl alcohol), flow rate 2.5 mL/min, P 194
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 19 retention
time = 39.2 min; enantiomer 20 retention time = 43.4 min.
5-[5-({3-[(1S,5R/1R,5S)-1-(4-Chlorophenyl)-3-azabicyclo[3.1.0]-
hex-3-yl]propyl}thio)-4-methyl-4H-1,2,4-triazol-3-yl]-2-methylqui-
noline Hydrochloride (21). The compound was obtained as a
1
white, slightly hygroscopic, solid. H NMR (CD3OD) δ: 8.95
(d,1H), 8.39 (d, 1H), 8.28 (t, 1H), 8.13 (d, 1H), 7.96 (d, 1H), 7.37
(m, 4H), 4.17 (d, 1H), 3.93 (d, 1H), 3.71 (m, 2H), 3.62 (s, 3H), 3.5
(2 m, 4H), 3.04 (s, 3H), 2.37 (m, 2H), 2.27 (m, 1H), 1.55 (m, 1H),
1.31 (m, 1H). MS: m/z 490 [M þ H]þ.
Derivative 21 was separated to give the separated enantiomers.
Retention times given were obtained using an analytical super-
critical fluid chromatography (Berger) using a chiral column
Chiralpak AD-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 25%
(ethanol þ 0.1% isopropylamine), flow rate 2.5 mL/min, P 180
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 22. Retention
time = 24.3 min; enantiomer 23 retention time = 26.5 min.
2-Methyl-5-{4-methyl-5-[(3-{(1S,5R)-1-[4-(trifluoromethyl)-
phenyl]-3-azabicyclo[3.1.0]hex-3-yl}propyl)thio]-4H-1,2,4-tri-
azol-3-yl}quinoline Hydrochloride (24). MS: m/z 490 [M þ H]þ.
Retention time = 10.12 min (method A).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 387
(m, 1H). MS: m/z 475 [M þ H]þ. Retention time = 10.15 min
(method A).
retention time = 22.2 min. Specific optical rotation of the
corresponding free base: [R]D = -51ꢀ (CHCl3, T = 20 ꢀC,
c = 0.00913 g/mL); enantiomer 43 retention time = 30.8 min.
Specific optical rotation of the corresponding free base: enan-
tiomer 2, specific optical rotation of the corresponding free base
[R]D = þ27ꢀ (CHCl3, T = 20 ꢀC, c = 0.0113 g/mL).
(1R,5S/1S,5R)-3-(3-{[4-methyl-5-(4-methyl-1,3-oxazol-5-yl)-
4H-1,2,4-triazol-3-yl]thio}propyl)-1-[4-(trifluoromethyl)phenyl]-
3-azabicyclo[3.1.0]hexane Hydrochloride (34). The compound
1
was obtained as a white, slightly hygroscopic, solid. H NMR
(DMSO-d6) δ: 10.16 (bs, 1H), 8.58 (s, 1H), 7.72 (d, 2H), 7.51 (d,
2H), 4.1 (dd, 1H), 3.78 (dd, 1H), 3.70 (s, 3H), 3.66 (m, 2H), 3.29
(t, 2H), 2.5 (bm, 2H), 2.39 (s, 3H), 2.33 (quint, 2H), 2.19 (m, 1H),
1.62/1.23 (t/t, 2H). MS: m/z 464 [M þ H]þ.
(1R,5S/1S,5R)-1-(4-Methoxyphenyl)-3-(3-{[4-methyl-5-(4-methyl-
1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-azabicyclo-
[3.1.0]hexane Hydrochloride (44). The title compound was ob-
tained as a white slightly hygroscopic solid. 1H NMR (DMSO-
d6) δ: 10.18 (bs, 1H), 8.58 (s, 1H), 7.24 (d, 2H), 6.91 (d, 2H), 3.97
(dd, 1H), 3.74 (s, 3H), 3.7 (s, 3H), 3.7 (m, 1H), 3.6-3.2 (m, 4H),
3.27 (t, 2H), 2.39 (s, 3H), 2.15 (quint, 2H), 2.07 (quint, 1H), 1.49/
1.05 (2t, 2H). MS: m/z 426 [M þ H]þ.
(1S,5R)-3-(3-{[4-Methyl-5-(4-methyl-1,3-oxazol-5-yl)-4H-
1,2,4-triazol-3-yl]thio}propyl)-1-[4-(trifluoromethyl)phenyl]-3-
azabicyclo[3.1.0]hexane Hydrochloride (35). The compound
was obtained as a off white solid, slightly hygroscopic, solid.
1H NMR (DMSO-d6) δ: 10.16 (bs, 1H), 8.58 (s, 1H), 7.72 (d,
2H), 7.51 (d, 2H), 4.1 (dd, 1H), 3.78 (dd, 1H), 3.70 (s, 3H), 3.66
(m, 2H), 3.29 (t, 2H), 2.5 (bm, 2H), 2.39 (s, 3H), 2.33 (quint,
2H), 2.19 (m, 1H), 1.62/1.23 (t/t, 2H). MS: m/z 464 [M þ H]þ.
(1R,5S)-3-(3-{[4-Methyl-5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-
triazol-3-yl]thio}propyl)-1-[4-(trifluoromethyl)phenyl]-3-azabi-
Compound 44 was separated to give the separated enantiomers.
Retention times given were obtained using an analytical super-
critical fluid chromatography (Gilson) using a chiral column
Chiralpak AS-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 8%
(ethanol þ 0.1% isopropylamine), flow rate 2.5 mL/min, P 190
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 45 retention
time = 28.7 min; enantiomer 46 retention time = 36.4 min.
(1S,5R/1R,5S)-3-(3-{[4-Methyl-5-(4-methyl-1,3-oxazol-5-yl)-
4H-1,2,4-triazol-3-yl]thio}propyl)-1-[3-(methyloxy)phenyl]-3-azabi-
cyclo[3.1.0]hexane Hydrochloride (47). The title compound was
obtained as a white, slightly hygroscopic solid. 1H NMR (DMSO-
d6) δ: 10.16 (bs,1H), 8.58 (d, 1H), 7.26 (dd, 1H), 6.85 (m, 3H), 4.03
(dd, 1H), 3.77 (s, 3H), 3.72 (dd, 1H), 3.7 (s, 3H), 3.6-3.3 (bm, 4H),
3.28 (t/m, 2H), 2.39 (s, 3H), 2.18 (m, 3H), 1.53-1.1 (t/m, 2H). MS:
m/z 426 [M þ H]þ.
1
cyclo[3.1.0]hexane Hydrochloride (36). H NMR (DMSO-d6)
δ: 10.16 (bs, 1H), 8.58 (s, 1H), 7.72 (d, 2H), 7.51 (d, 2H), 4.1
(dd, 1H), 3.78 (dd, 1H), 3.70 (s, 3H), 3.66 (m, 2H), 3.29 (t, 2H),
2.5 (bm, 2H), 2.39 (s, 3H), 2.33 (quint, 2H), 2.19 (m, 1H), 1.62/
1.23 (t/t, 2H). MS: m/z 464 [M þ H]þ.
(1R,5S/1S,5R)-3-(3-{[4-Methyl-5-(4-methyl-1,3-oxazol-5-yl)-
4H-1,2,4-triazol-3-yl]thio}propyl)-1-phenyl-3-azabicyclo[3.1.0]-
hexane Hydrochloride (37). The compound was obtained as a
white, slightly hygroscopic, solid. 1H NMR (DMSO-d6) δ: 10.46
(bs,1H), 8.58 (s, 1H), 7.4-7.2 (m, 5H), 4.04 (dd, 1H), 3.73 (m,
1H), 3.7 (s, 3H), 3.7-3.4 (m, 2H), 3.4-3.2 (m þ t, 4H), 2.39 (s,
3H), 2.17 (m, 3H), 1.64,1.1 (2t, 2H). MS: m/z 396 [M þ H]þ.
(1R,5S/1S,5R)-1-(4-tert-Butylphenyl)-3-(3-{[4-methyl-5-(4-
methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-
azabicyclo[3.1.0]hexane Hydrochloride (38). The compound
was obtained as a white, slightly hygroscopic, solid. 1H NMR
(CD3OD) δ: 8.4 (s, 1H), 7.42 (d, 2H), 7.28 (d, 2H), 4.11 (d,
1H), 3.88 (d, 1H), 3.8 (s, 3H), 3.65 (m, 2H), 3.43 (t, 2H), 3.39
(t, 2H), 2.47 (s, 3H), 2.29 (m, 2H), 2.21 (m, 1H), 1.44 (m, 1H),
1.33 (s, 9H), 1.3 (m, 1H). MS: m/z 452 [M þ H]þ.
Compound 47 was separated to give the separated enantiomers.
Retention times given were obtained using an analytical super-
critical fluid chromatography (Berger) using a chiral column
Chiralcel OJ-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 13%
(2-propanol þ 0.1% isopropylamine), flow rate 2.5 mL/min, P 180
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 48 retention
time = 24.2 min; enantiomer 49 retention time = 26.8 min.
(1S,5R/1R,5S)-1-(4-Chlorophenyl)-3-(3-{[4-methyl-5-(4-methyl-
1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-azabicyclo-
[3.1.0]hexane Hydrochloride (50). The title compound was ob-
tained as a white, slightly hygroscopic solid. 1H NMR (DMSO-
d6) δ: δ 9.93(bs,1H), 8.58 (s, 1H), 7.42 (d, 2H), 7.33 (d, 2H), 4.04
(dd, 1H), 3.75 (dd, 1H), 3.7 (s, 3H), 3.5 (m, 2H), 3.3 (bm, 4H),
2.39 (s, 3H), 2.2 (m, 1H), 2.15 (m, 2H), 1.47-1.14 (2t, 2H). MS:
m/z 431 [M þ H]þ.
(1R,5S)-1-(4-tert-Butylphenyl)-3-(3-{[4-methyl-5-(4-methyl-
1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-azabicyclo-
[3.1.0]hexane Hydrochloride (39). The compound was obtained
as a white, slightly hygroscopic, solid. 1H NMR (CD3OD) δ: 8.4
(s, 1H), 7.42 (d, 2H), 7.28 (d, 2H), 4.11 (d, 1H), 3.88 (d, 1H), 3.8
(s, 3H), 3.65 (m, 2H), 3.43 (t, 2H), 3.39 (t, 2H), 2.47 (s, 3H), 2.29
(m, 2H), 2.21 (m, 1H), 1.44 (m, 1H), 1.33 (s, 9H), 1.3 (m, 1H).
MS: m/z 452 [M þ H]þ.
Compound 50 was separated to give the separated enantio-
mers. Retention times given were obtained using an analytical
supercritical fluid chromatography (Gilson) using a chiral col-
umn Chiralpak AS-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing
15% (ethanol þ 0.1% isopropylamine), flow rate 2.5 mL/min,
P 190 bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 51
retention time = 7.8 min. Specific optical rotation of the
corresponding free base: [R]D = -25ꢀ (CHCl3, T = 20 ꢀC,
c = 0.0066 g/mL). Enantiomer 52 retention time = 9.7 min.
Specific optical rotation of the corresponding free base: [R]D =
þ29ꢀ (CHCl3, T = 20 ꢀC, c = 0.0068 g/mL)
(1S,5R)-1-(4-tert-Butylphenyl)-3-(3-{[4-methyl-5-(4-methyl-1,3-
oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-azabicyclo-
[3.1.0]hexane Hydrochloride (40). The compound was obtained as
a white, slightly hygroscopic, solid. 1H NMR (CD3OD) δ: 8.4 (s,
1H), 7.42 (d, 2H), 7.28 (d, 2H), 4.11 (d, 1H), 3.88 (d, 1H), 3.8 (s,
3H), 3.65 (m, 2H), 3.43 (t, 2H), 3.39 (t, 2H), 2.47 (s, 3H), 2.29 (m,
2H), 2.21 (m, 1H), 1.44(m, 1H), 1.33 (s, 9H), 1.3(m, 1H). MS: m/z
452 [M þ H]þ.
(1S,5R/1R,5S)-1-(3-Chlorophenyl)-5-methyl-3-(3-{[4-methyl-
5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-
azabicyclo[3.1.0]hexane Hydrochloride (53). The title compound
(1R,5S/1S,5R)-1-(4-Bromophenyl)-3-(3-{[4-methyl-5-(4-methyl-
1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-azabicyclo-
[3.1.0]hexane Hydrochloride (41). The compound was obtained
as a white, slightly hygroscopic, solid. 1H NMR (DMSO-d6) δ:
10.29 (bs,1H), 8.58 (s, 1H), 7.55 (dd, 2H), 7.27 (dd, 2H), 4.03 (dd,
1H), 3.73 (dd, 1H), 3.7 (s, 3H), 3.55 (m, 2H), 3.5-3.2/3.28 (m þ t,
4H), 2.39 (s, 3H), 2.19 (m, 3H), 1.59/1.12 (2t, 2H). MS: m/z 474
[M þ H]þ.
1
was obtained as a white slightly hygroscopic solid. H NMR
(DMSO-d6) δ: 9.88 (bs,1H), 8.58 (s, 1H), 7.43 (d, 1H), 7.4-7.2
(m, 3H), 4.06 (dd, 1H), 3.75 (dd, 1H), 3.7 (s, 3H), 3.62-3.54 (t/m,
2H), 3.5-3.3 (bm, 4H), 2.39 (s, 3H), 2.25 (m, 1H), 2.15 (m, 2H),
1.46-1.19 (2 m, 2H). MS: m/z 431 [M þ H]þ.
Compound 53 was separated to give the separated enantiomers.
Retention times given were obtained using an analytical super-
critical fluid chromatography (Berger) using a chiral column
Chiralpak AD-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 15%
(ethanol þ 0.1% isopropylamine), flow rate 2.5 mL/min, P 180
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 54 retention
time = 29.6 min; enantiomer 55 retention time = 32.0 min.
Compound 41 was separated to give the separated enantio-
mers. Retention times given were obtained using an analytical
supercritical fluid chromatography (Gilson) using a chiral col-
umn Chiralpak AS-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing
10% (ethanol þ 0.1% isopropyilamine), flow rate 2.5 mL/min,
P 199 bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 42

388 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Micheli et al.
(1S,5R/1R,5S)-1-(4-Fluorophenyl)-3-(3-{[4-methyl-5-(4-methyl-
1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-azabicyclo-
[3.1.0]hexane Hydrochloride (56). The title compound was ob-
tained as a white slightly hygroscopic solid. 1H NMR(DMSO-d6)
δ: 10.06 (bs,1H), 8.58 (s, 1H), 7.36 (dd, 2H), 7.19 (t, 2H), 4.02
(dd, 1H), 3.74 (dd, 1H), 3.7 (s, 3H), 3.55 (m, 2H), 3.5-3.2 (bm,
4H), 2.39 (s, 3H), 2.15 (m, 3H), 1.49-1.1 (2t, 2H). MS: m/z 414
[M þ H]þ.
4.04/3.74 (2dd, 2H), 3.7 (s, 3H), 3.6-3.2 (m, 4H), 3.28 (t, 2H), 2.39
(s, 3H), 2.26 (quint, 1H), 2.15 (quint, 2H), 1.53/1.2 (2t, 2H). MS:
m/z 464 [M þ H]þ.
Compound 69 was separated to give the separated enantiomers.
Retention times given were obtained using an analytical super-
critical fluid chromatography (Gilson) using a chiral column
Chiralpak AS-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 8%
(ethanol þ 0.1% isopropylamine), flow rate 2.5 mL/min, P 190
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 70 retention
time = 38.0 min; enantiomer 71 retention time = 40.8 min.
(1R,5S/1S,5R)-1-[2-Fluoro-4-(trifluoromethyl)phenyl]-3-(3-{[4-
methyl-5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}-
propyl)-3-azabicyclo[3.1.0]hexane Hydrochloride (72). The title
compound was obtained as a white slightly hygroscopic solid.
1H NMR (DMSO-d6) δ: 10.28 (bs,1H), 8.58 (s, 1H), 7.73 (d,
1H), 7.6 (m, 2H), 4/ 3.57 (d/m, 2H), 3.79 (d, 1H), 3.69 (s, 3H),
3.5-3.2 (vbm, 1H), 3.27 (t, 2H), 2.5 (m, 2H), 2.4 (m, 1H), 2.38 (s,
3H), 2.14 (quint, 2H), 1.62/1.16 (2t, 2H). MS: m/z 481 [M þ H]þ.
Compound 72 was separated to give the separated. Retention
times given were obtained using an analytical HPLC using a
chiral column Chiralpak AD-H 5 μm, 250 mm ꢀ 4.6 mm, eluent
A, n-hexane; B, 2-propanol, gradient isocratic 15% B, flow rate
0.8 mL/min, detection UV at 200-400 nm. Enantiomer 73
retention time = 15.4 min; enantiomer 74 retention time =
16.3 min.
Compound 56 was separated to give the separated. Retention
times given were obtained using an analytical supercritical fluid
chromatography (Gilson) using a chiral column Chiralpak
AS-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 6% (ethanol
þ 0.1% isopropylamine), flow rate 2.5 mL/min, P 190 bar,
T 35 ꢀC, detection UV at 220 nm. Enantiomer 57 retention
time = 26.2 min; enantiomer 58 retention time = 32.4 min
(1S,5R/1R,5S)-1-(3-Fluorophenyl)-3-(3-{[4-methyl-5-(4-methyl-
1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-azabicyclo-
[3.1.0]hexane Hydrochloride (59). The title compound was ob-
tained as a white slightly hygroscopic solid. 1H NMR(DMSO-d6)
δ: 10.21 (bs,1H), 8.58 (d, 1H), 7.4 (m, 1H), 7.2-7.0 (m, 3H), 4.03
(dd, 1H), 3.75 (dd, 1H), 3.7 (s, 3H), 3.61 (t/m, 1H), 3.52 (m, 1H),
3.3 (m, 2H), 3.28 (t, 2H), 2.38 (s, 3H), 2.25 (m, 1H), 2.16 (m, 2H),
1.57-1.17 (t/m, 2H). MS: m/z 414 [M þ H]þ.
Compound 59 was separated to give the separated. Retention
times given were obtained using an analytical supercritical fluid
chromatography (Gilson) using a chiral column Chiralpak
AS-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 6% (ethanol
þ 0.1% isopropylamine), flow rate 2.5 mL/min, P 190 bar,
T 35 ꢀC, detection UV at 220 nm. Enantiomer 60 retention
time = 24.6 min; enantiomer 61 retention time = 26.0 min
(1R,5S/1S,5R)-3-(3-{[4-Methyl-5-(4-methyl-1,3-oxazol-5-yl)-
4H-1,2,4-triazol-3-yl]thio}propyl)-1-[3-(trifluoromethyl)phenyl]-
3-azabicyclo[3.1.0]hexane Hydrochloride (62). The title compound
was obtained as a white slightly hygroscopic solid. 1H NMR
(DMSO-d6) δ: 10.5 (bs,1H), 8.58 (s, 1H), 7.7-7.5 (m, 4H), 4.09
(m, 1H), 3.8-3.2 (m, 8H), 3.29 (t, 2H), 2.39 (s, 3H), 2.3 (m, 1H),
2.18 (m, 2H), 1.68 (t, 1H), 1.21 (t, 1H). MS: m/z 464 [M þ H]þ.
Compound 62 was separated to give the separated. Retention
times given were obtained using an analytical supercritical fluid
chromatography (Gilson) using a chiral column Chiralpak AD-H,
25 cm ꢀ 0.46 cm, eluent CO2 containing 10% (ethanol þ 0.1% 2-
propanol), flow rate 22 mL/min, P 190 bar, T 36 ꢀC, detection UV
at 220 nm. Enantiomer 63 retention time = 17.6 min; enantiomer
64 retention time = 18.4 min
(1S,5R/1R,5S)-3-(3-{[4-Methyl-5-(4-methyl-1,3-oxazol-5-yl)-
4H-1,2,4-triazol-3-yl]thio}propyl)-1-[2-methyl-4-(trifluoromethyl)-
phenyl]-3-azabicyclo[3.1.0]hexane Hydrochloride (75). The title
compound was obtained as a white slightly hygroscopic solid.
1H NMR (DMSO-d6) δ: 10.25 (bs,1H), 8.58 (s, 1H), 7.6 (m, 3H),
3.97-3.7 (dd/m, 2H), 3.79/3.4 (dd/m, 2H), 3.69 (s, 3H), 3.27 (t,
2H), 2.5 (m, 2H), 2.48 (s, 3H), 2.38 (s, 3H), 2.2 (m, 1H), 2.13
(quint, 2H), 1.61-1.01 (2t, 2H). MS: m/z 478 [M þ H]þ.
(1R,5S/1S,5R)-1-[4-Fluoro-3-(trifluoromethyl)phenyl]-3-(3-{[4-
methyl-5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}-
propyl)-3-azabicyclo[3.1.0]hexane Hydrochloride (76). The title
compound was obtained as a white slightly hygroscopic solid.
1H NMR (DMSO-d6) δ: 10.2 (bs, 1H), 8.58 (s, 1H), 7.75 (dm,
1H), 7.72 (m, 1H), 7.53 (t, 1H), 4.06 (dd, 1H), 3.74 (dd, 1H), 3.7
(s, 3H), 3.6 (m, 2H), 3.4 (m, 2H), 3.28 (t, 2H), 2.39 (s, 3H), 2.26
(m, 1H), 2.18 (m, 2H), 1.54 (t, 1H), 1.22 (dd, 1H). MS: m/z 481
[M þ H]þ.
Compound 76 was separated to give the separated. Retention
times given were obtained using an analytical supercritical fluid
chromatography (Gilson) using a chiral column Chiralpak AS-H,
25 cm ꢀ 0.46 cm, eluent CO2 containing 8% (ethanol þ 0.1%
isopropylamine), flow rate 2.5 mL/min, P 180 bar, T 35 ꢀC,
detection UV at 220 nm. Enantiomer 77 retention time = 25.9
min; enantiomer 78 retention time = 28.3 min.
(1R,5S/1S,5R)-3-(3-{[4-Methyl-5-(4-methyl-1,3-oxazol-5-yl)-
4H-1,2,4-triazol-3-yl]thio}propyl)-1-[2-(trifluoromethyl)phenyl]-
3-azabicyclo[3.1.0]hexane Hydrochloride (65). The title com-
1
pound was obtained as a white slightly hygroscopic solid. H
NMR (DMSO-d6) δ: 10.48 (bs,1H), 8.55 (s, 1H), 7.9-7.6 (m,
4H), 3.9-3.1 (bm, 8H), 3.68 (s, 3H), 2.36 (s, 3H), 2.13 (m, 2H),
1.66 (m, 1H), 1.2 (m, 1H), 1.1 (m, 1H). MS: m/z 464 [M þ H]þ.
(1S,5R/1R,5S)-3-(3-{[4-Methyl-5-(4-methyl-1,3-oxazol-5-yl)-
4H-1,2,4-triazol-3-yl]thio}propyl)-1-{4-[(trifluoromethyl)oxy]-
phenyl}-3-azabicyclo[3.1.0]hexane Hydrochloride (66). The title
compound was obtained as a white slightly hygroscopic solid. 1H
NMR (DMSO-d6) δ: 10.33 (bs,1H), 8.58 (s, 1H), 7.43 (d, 2H),
7.36 (d, 2H), 4.04 (dd, 1H), 3.73 (dd, 1H), 3.7 (s, 3H), 3.6-3.2
(bm, 6H), 2.39 (s, 3H), 2.2 (m, 3H), 1.61-1.16 (2t, 2H). MS: m/z
480 [M þ H]þ.
(1R,5S/1S,5R)-1-[2-Fluoro-5-(trifluoromethyl)phenyl]-3-(3-{[4-
methyl-5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}-
propyl)-3-azabicyclo[3.1.0]hexane Hydrochloride (79). The title
compound was obtained as a white slightly hygroscopic solid.
1H NMR (CD3OD) δ: 8.41 (s,1H), 7.8 (m, 1H), 7.74 (m, 1H),
7.39 (t, 1H), 4.13 (d, 1H), 3.95 (d, 1H), 3.81 (s, 3H), 3.73 (bd,
1H), 3.54 (d, 1H), 3.48 (m, 2H), 3.41 (m, 2H), 2.48 (s, 3H), 2.39
(m, 1H), 2.28 (q, 2H), 1.58 (m, 1H), 1.35 (m, 1H). MS: m/z 482
[M þ H]þ.
Compound 79 was separated to give the separated. Retention
times given were obtained using an analytical supercritical fluid
chromatography (Berger) using a chiralcolumnChiralpakAD-H,
25 cm ꢀ 0.46 cm, eluent CO2 containing 7% (ethanol þ 0.1%
isopropylamine), flow rate 2.5 mL/min, P 180 bar, T 35 ꢀC,
detection UV at 220 nm. Enantiomer 80 retention time = 21.2
min; enantiomer 81 retention time = 22.7 min.
Compound 66 was separated to give the separated enantiomers.
Retention times given were obtained using chiral column Chiracel
OD, 25 cm ꢀ 0.46 cm, eluent A: n-hexane; B: 2-propanol, gradient
isocratic 10% B v/v, flow rate 1 mL/min, detection UV at 220 nm.
Enantiomer 67 retention time = 28.3 min; enantiomer 68 reten-
tion time = 50.6 min.
(1R,5S/1S,5R)-1-(3,4-Dichlorophenyl)-3-(3-{[4-methyl-5-(4-
methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-azabi-
cyclo[3.1.0]hexane Hydrochloride (69). The title compound was
obtained as a white slightly hygroscopic solid. 1H NMR (DMSO-
d6) δ: 10.11 (vbs, 1H), 8.58 (s, 1H), 7.6 (d þ d, 2H), 6.29 (dd, 1H),
(1R,5S/1S,5R)-1-[2-Fluoro-3-(trifluoromethyl)phenyl]-3-(3-{[4-
methyl-5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}-
propyl)-azabicyclo[3.1.0]hexane Hydrochloride (82). The title
compound was obtained as a white slightly hygroscopic solid.
1H NMR (CD3OD) δ: 8.46 (s,1H), 7.75-7.65 (m, 2H), 7.38 (t,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 389
1H), 4.1 (d, 1H), 3.93 (d, 1H), 3.78 (s, 3H), 3.71 (d, 1H), 3.54 (d,
1H), 3.48 (t, 2H), 3.38 (t, 2H), 2.45 (s, 3H), 2.36 (m, 1H), 2.25 (m,
2H), 1.54 (m, 1H), 1.34 (m, 1H). MS: m/z 481 [M þ H]þ.
Compound 82 was separated to give the separated. Retention
times given were obtained using an analytical supercritical fluid
chromatography (Gilson) using a chiral column Chiralpak AD-H,
250 mm ꢀ 4.6 mm, eluent n-hexane/ethanol 88/12 (isocratic), flow
rate 1 mL/min, P 200-400 bar, T 36 ꢀC, detection UV at 200-
400 nm. Enantiomer 83 retention time = 20.4 min; enantiomer 84
retention time = 23.0 min.
(1R,5S/1S,5R)-1-[4-(Methyloxy)-5-(trifluoromethyl)phenyl]-
3-(3-{[4-methyl-5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]-
thio}propyl)-azabicyclo[3.1.0]hexane Hydrochloride (97). The title
compound was obtained as a white slightly hygroscopic solid. 1H
NMR (CD3OD) δ: 8.37 (s,1H), 7.57 (m, 2H), 7.17 (d, 1H) 3.9 (m,
4H), 3.77 (s, 3H), 3.74 (m, 1H), 3.65-3.30 (m, 6H), 2.44 (s, 3H),
2.21 (m, 2H), 2.13 (m, 1H), 1.43 (t, 1H), 1.24 (m, 1H). MS: m/z 494
[M þ H]þ.
Compound 97 was separated to give the separated. Retention
times given were obtained using an analytical supercritical fluid
chromatography (Gilson) using a chiral column Chiralpak AD-
H, 250 mm ꢀ 4.6 mm, eluent n-hexane/ethanol 70/30 (isocratic),
flow rate 0.8 mL/min, detection UV at 200-400 nm. Enantiomer
98 retention time = 15.5 min; enantiomer 99 retention time =
17.5 min.
(1R,5S/1S,5R)-1-[3-Fluoro-4-(trifluoromethyl)phenyl]-5-methyl-
3-(3-{[4-methyl-5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]-
thio}propyl)-3-azabicyclo[3.1.0]hexane Hydrochloride (85). The title
compound was obtained as a white slightly hygroscopic solid. 1H
NMR (CD3OD) δ: 8.4 (s, 1H), 7.55 (t, 1H), 7.37 (d, 1H), 7.32 (d,
1H), 4.2 (d, 1H), 3.91 (d, 1H), 3.81 (s, 3H), 3.76 (d, 1H), 3.67 (d,
1H), 3.51 (t, 2H), 3.43 (t, 2H), 2.47 (s, 3H), 2.41 (m, 1H), 2.31 (m,
2H), 1.61 (t, 1H), 1.45 (t, 1H). MS: m/z 496 [M þ H]þ.
(1R,5S/1S,5R)-1-[3-Chloro-4-(methyloxy)phenyl]-3-(2-{[4-methyl-
5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-3-
azabicyclo[3.1.0]hexane Hydrochloride (100). The title com-
1
pound was obtained as a white slightly hygroscopic solid. H
Compound 85 was separated to give the separated. Retention
times given were obtained using an analytical supercritical fluid
chromatography (Berger)using achiralcolumnChiralpakAD-H,
25 cm ꢀ 0.46 cm, eluent CO2 containing 10% (ethanol þ 0.1%
isopropylamine), flow rate 2.5 mL/min, P 180 bar, T 35 ꢀC,
detection UV at 220 nm. Enantiomer 86 retention time = 27.1
min; enantiomer 87 retention time = 31.0 min.
NMR (CD3OD) δ: 8.4 (s,1H), 7.35 (m, 1H), 7.1 (m, 2H), 4.01
(m,1H), 3.89 (m,4H), 3.8 (s, 3H), 3.6-3.3 (m, 6H), 2.47 (s, 3H),
2.23 (m, 2H), 2.19 (m, 1H), 1.4 (m, 1H), 1.2 (m, 1H). MS: m/z
460 [M þ H]þ.
Compound 100 was separated to give the separated enantio-
mers. Retention times given were obtained using an analytical
supercritical fluid chromatography (Berger) using a chiral column
Chiralpak AD-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 25%
(ethanol þ 0.1% isopropyilamine), flow rate 2.5 mL/min, P 180
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 101 retention
time = 13.7 min; enantiomer 102 retention time = 16.2 min.
(1R,5S/1S,5R)-1-[3-(2-{[4-Methyl-5-(4-methyl-1,3-oxazol-5-yl)-
4H-1,2,4-triazol-3-yl]thio}propyl)-1-{3-[(trifluoromethyl)oxy]-
phenyl}-3-azabicyclo[3.1.0]hexane Hydrochloride (103). The
title compound was obtained as a white slightly hygroscopic
solid. 1H NMR (CD3OD) δ: 8.41 (s, 1H), 7.49 (t, 1H), 7.3 (s,
1H), 7.37 (d, 1H), 7.24 (m,1H), 4.17 (d,1H), 3.9 (d, 1H), 3.8 (s,
3H), 3.69 (d, 2H), 3.51 (t, 2H), 3.42 (t, 2H), 2.47 (s, 3H), 2.3 (m,
3H), 1.57 (dd, 1H), 1.36 (t, 1H). MS: m/z 480 [M þ H]þ.
Compound 103 was separated to give the separated enantio-
mers. Retention times given were obtained using an analytical
supercritical fluid chromatography (Berger) using a chiral column
Chiralpak AD-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 12%
(ethanol þ 0.1% isopropyilamine), flow rate 2.5 mL/min, P 180
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 104 retention
time = 10.7 min; enantiomer 105 retention time = 12.0 min.
Computational Modeling. Construction of the hERG Models.
The closed state of the hERG channel was modeled based on its
homology with the bacterial Kcsa structure from Streptococcus
lividans, solved in 1998 by McKinnon et al.24 Alignment of the
pore region and the S6 helix is obvious from the key GYG motif
(GFG in hERG) and the key hinge glycine in S6. The alignment
of the S5 helices of hERG and Kcsa, however, was not obvious.
This was based finally on the observation of a highly conserved
glutamate at the extracellular side of S5, which is found in most
potassium channels. An NMR structure of the S5-SS1 loop
peptide has been published,25 which suggests that this region is
largely helical. This was added to the model as a helix-turn-
β-strand motif. The tetrameric ensemble was fully minimized
using the CHARMm program,26 with an initial 500 steps of
Steepest Descent, followed by 5000 steps of ABNR, and a
constant dielectric of 5. Helical H-bonding distance constraints
were used to maintain the overall fold of the bundle.
(1R,5S/1S,5R)-1-[3-Fluoro-5-(trifluoromethyl)phenyl]-3-(3-{[4-
methyl-5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}-
propyl)-azabicyclo[3.1.0]hexane Hydrochloride (88). The title
compound was obtained as a white slightly hygroscopic solid.
1H NMR (CD3OD) δ: 8.46 (s,1H), 7.54 (bs, 1H), 7.47 (bd, 1H),
7.41 (bd, 1H), 4.19 (d, 1H), 3.09 (d, 1H), 3.87 (s, 3H), 3.71 (m,
2H), 3.51 (t, 2H), 3.46 (t, 2H), 2.49 (s, 3H), 2.33 (m, 3H), 1.67 (m,
1H), 1.39 (m, 1H). MS: m/z 482 [M þ H]þ.
Compound 88 was separated to give the separated enantiomers.
Retention times given were obtained using an analytical super-
critical fluid chromatography (Gilson) using a chiral column
Chiralpak AD-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 10%
(ethanol þ 0.1% isopropyilamine), flow rate 2.5 mL/min, P 180
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 89 retention
time = 12.6 min; enantiomer 90 retention time = 14.7 min.
(1R,5S/1S,5R)-1-(2-Fluoro-4-methylphenyl)-3-(3-{[4-methyl-
5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-
3-azabicyclo[3.1.0]hexane Hydrochloride (91). The title com-
1
pound was obtained as a white slightly hygroscopic solid. H
NMR (CD3OD) δ: 8.39 (s, 1H), 7.26 (t, 1H), 7.01 (m, 2H), 3.93
(m,1H), 3.77 (m, 4H), 3.61 (m, 1H), 3.41-3.38 (m, 5H), 2.47 (s,
3H), 2.36 (s, 3H), 2.23 (m, 2H), 2.19 (m, 1H), 1.45 (t, 1H), 1.21 (t,
1H). MS: m/z 428 [M þ H]þ.
Compound 91 was separated to give the separated enantiomers.
Retention times given were obtained using an analytical super-
critical fluid chromatography (Berger) using a chiral column
Chiralpak AD-H, 25 cm ꢀ 0.46 cm, eluent CO2 containing 25%
(ethanol þ 0.1% isopropyilamine), flow rate 2.5 mL/min, P 180
bar, T 35 ꢀC, detection UV at 220 nm. Enantiomer 92 retention
time = 13.7 min; enantiomer 93 retention time = 16.2 min.
(1R,5S/1S,5R)-1-[4-(4-Chloro-2-fluorophenyl]-3-(3-{[4-methyl-
5-(4-methyl-1,3-oxazol-5-yl)-4H-1,2,4-triazol-3-yl]thio}propyl)-
azabicyclo[3.1.0]hexane Hydrochloride (94). The title com-
1
pound was obtained as a white slightly hygroscopic solid. H
NMR (CD3OD) δ: 8.27 (s, 1H), 7.3 (t, 1H), 7.1 (m, 2H), 3.95 (d,
1H), 3.8 (d, 1H), 3.67 (s, 3H), 3.56 (dd, 1H), 3.4-3.2 (m, 5H),
2.34 (s, 3H), 2.15 (m, 3H), 1.4 (t, 1H), 1.18 (t, 1H). MS: m/z 448
[M þ H]þ.
An open state model was also built using the rat Kv1.2
structure as a template.27 The same S5 glutamate, pore GYG,
and S6 glycine residues were used to generate the alignment. Use
was made of the S5-SS1 loop model constructed for the closed
state structure and the same CHARMm conditions were used
for final refinement.
Compound 94 was separated to give the separated enantio-
mers. Retention times given were obtained using an analytical
HPLC using a chiral column Chiralpak AD-H 5 μm, 250 mm ꢀ
21 mm; eluent A, n-hexane; B, ethanol þ 0.1% isopropylamine,
gradient isocratic 20% B, flow rate 6 mL/min, detection UV at
225 nm. Enantiomer 95 retention time = 18.7 min; enantiomer
96 retention time = 20.6 min.
Construction of the Dopamine D3 Receptor Models. The model
of the dopamine D3 receptor was built using our “in house”

390 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Micheli et al.
program GPCR_Builder.28 This is an automated 7TM receptor
homology modeling program which incorporates a database of
all known human and rodent Family A receptor sequences and a
prealigned set of transmembrane domains for these. The models
are based on the TM domain taken from the crystal structure of
bovine rhodopsin.29 A special set of optimized 3D templates are
used to add the extracellular loops. An internal rotamer library
is then applied to all residue side chains except those conserved
residues known to be involved in an important functional role
(e.g., the asparagines in TM1 and TM7, the aspartate on TM2,
and the DRY motif on TM3). The program automatically
generates a CHARMm script, which can be used for model
optimization and ligand docking. Typically, the same set of
conditions as those described for the hERG models above were
used during the minimization stage. A strict alignment between
rhodopsin and dopamine D3 gives rise to a very short loop
length (2 residues) between the disulfide bonded cysteine in the
second extracellular loop and the top of TM5. During the
optimization some unfolding of the extracellular side of TM5
is therefore allowed by relaxing the helical distance constraints
in this region. This is in keeping with the reported SCAM results
carried out by Javitch et al. on the D2 receptor.30
Automated Docking of Ligands in the Receptor and Channel
Models. Partial atomic charges fitted to the electrostatic poten-
tial derived from semiempirical wave functions (MNDO) were
calculated with MOPAC.31 All calculations were performed in
vacuum.
Automatic docking experiments were performed with Monte
Carlo sampling followed by energy minimization (MCEM)
using MacroModel routines and AMBER* force field. Subse-
quently poses were ranked according to their interaction energy
defined as the difference between the energy of the complex
(Ecomplex) and the energy of the ligand (Eligand) and that of the
protein (Eprotein):
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for their consistency with with SAR data generated within the
same structural class.
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Acknowledgment. We thank all the colleagues who helped
in generating the data reported in this manuscript, Drs.
Caterina Mazzoni, Chiara Savoia, and Maria Pilla, and Enzo
Valerio. We also thank in particular Drs. Anne Andorn and
Barbara Bertani for the fruitful discussion during the pre-
paration of this manuscript.
[
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