Discovery, synthesis, selectivity modulation and DMPK characterization of 5-azaspiro[2.4]heptanes as potent orexin receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2013.02.093

Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor antagonist class has been discovered. SAR and Pharmacokinetic optimization of this series

Full text of DOI:10.1016/j.bmcl.2013.02.093

Accepted Manuscript
Discovery, synthesis, selectivity modulation and DMPK characterization of 5-
azaspiro[2.4]heptanes as potent orexin receptor antagonists
Luigi Piero Stasi, Roberto Artusi, Clara Bovino, Benedetta Buzzi, Luca
Canciani, Gianfranco Caselli, Fabrizio Colace, Paolo Garofalo, Silvia
Giambuzzi, Patrice Larger, Ornella Letari, Stefano Mandelli, Lorenzo Perugini,
Sabrina Pucci, Matteo Salvi, PierLuigi Toro
PII:
S0960-894X(13)00280-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.02.093
BMCL 20211
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
29 January 2013
17 February 2013
20 February 2013
Please cite this article as: Stasi, L.P., Artusi, R., Bovino, C., Buzzi, B., Canciani, L., Caselli, G., Colace, F., Garofalo,
P., Giambuzzi, S., Larger, P., Letari, O., Mandelli, S., Perugini, L., Pucci, S., Salvi, M., Toro, P., Discovery,
synthesis, selectivity modulation and DMPK characterization of 5-azaspiro[2.4]heptanes as potent orexin receptor
antagonists, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.02.093
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery, synthesis, selectivity modulation and DMPK characterization of 5-
azaspiro[2.4]heptanes as potent orexin receptor antagonists.
Luigi Piero Stasia,∗ Roberto Artusia, Clara Bovinoa, Benedetta Buzzia, Luca Cancianic, Gianfranco Casellib,
Fabrizio Colacea, Paolo Garofalob, Silvia Giambuzzic, Patrice Largerc, Ornella Letarib, Stefano Mandellia,
Lorenzo Peruginia, Sabrina Puccia, Matteo Salvia, PierLuigi Toroa
aRottapharm Madaus, Medicinal Chemistry Department, Monza via Valosa di Sopra, 9 20900, Italy
bRottapharm Madaus, Pharmacology & Toxicology Department, Monza via Valosa di Sopra, 9 20900, Italy
cRottapharm Madaus, Translational Sciences & Pharmacokinetics Department, Monza via Valosa di Sopra, 9 20900, Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine
series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor
antagonist class has been discovered. SAR and Pharmacokinetic optimization of this
series is herein disclosed. Lead compound 15 exhibits potent activity against orexin 1
and orexin 2 receptors along with low cytochrome P450 inhibition potential, good
brain penetration and oral bioavailability in rats.
Keywords:
Keyword_1 Orexin antagonists
Keyword_2 DORA
Keyword_3 7TM
Keyword_4 DMPK
Keyword_5 TDI
2013 Elsevier Ltd. All rights reserved.
Orexins, also known as hypocretins, are hypothalamic
neuropeptides secreted by a discrete number of neurons in the
lateral and posterior hypothalamus. The orexin system is highly
conserved across species and it is based on two hypothalamic
peptides, orexin A (OXA) and orexin B (OXB), arising from a
common 130 amino acids precursor protein named prepro-orexin
peptide. Discovered in 1998 by independent research groups1, the
orexin peptides were found to bind at two previously identified
orphan G-protein coupled receptors, which following this
discovery were classified as orexin 1 (OX1) and orexin 2 (OX2)
receptors. These receptors are widely distributed, though
differentially, in the brain and periphery. In the central nervous
system (CNS) OX1 receptors are found mainly in the locus
coeruleus, frontal cortex, ventral tegmental area and lateral
hypothalamus while OX2 receptors are mainly located into
system (CNS) orexin receptors are distributed in the gut, in
adipose brown tissue and in pancreatic islets5. As shown in many
studies, OXA binds with similar affinity to OX1 and OX2,
whereas OXB displays higher affinity vs. OX2. Neuronal OX1
and OX2 display a common signal transduction mechanism based
on the activation of Gq proteins followed by the modulation of
several ion channel activities to enhance synaptic transmission.
Considerable evidences have been accumulated in literature
regarding the utility of antagonizing the orexin receptors.
Selective blockade of central OX1 receptors has been reported to
modulate addiction and craving to rewarding drugs such as
alcohol, cocaine and morphine6. The modulation of binge eating
episodes, induced by highly palatable food, has been recently
observed in rats by using a centrally acting OX1 antagonist7.
Selective OX1 receptor antagonists (SORA) also have been
shown to be potentially useful in managing other disorders like
tuberomammillary nucleus2,3,4
. Outside the central nervous
———
∗ Corresponding author: Tel.:+39 0397390630; Fax: +39 0397390673; e-mail: luigi.stasi@rottapharm.com.

diabetes8 or post-menopausal syndrome9. However no SORA
reached the clinical stage so far to validate the aforementioned
hypothesis, most probably because of the poor drug likeness of
the tools identified so far (among which the widely used SB-
334867 and GSK105986510, Figure 1). OXA has a central pro-
arousal effect in several species11 and for this reason dual OX1
and OX2 receptor antagonists (DORA) have been intensively
Corey’s reagent in 41% yield; reduction of this lactam by
LiAlH4 in THF at reflux gave the amino-alcohol derivative (iii)
in 74% yield. Pyrrolidine (iv) was then prepared by
hydrogenation of (iii) over Pd/C. Protection of the cyclic amine
moiety with boc anhydride gave the key alcohol intermediate (v)
in 82% yield. Intermediate (v) was then used in two alternative
synthetic pathways (Scheme 2) leading to advanced
intermediates (x), which were the immediate precursors of the
compounds to screen against orexin receptors. In particular, the
oxidation of alcohol (v) was accomplished by using
TEMPO/BAIB procedure19 in 82% yield followed by a reductive
amination with the desired amino heterocycle and Na(OAc)3BH,
usually in DCE at room temperature to get intermediates (vii).
studied to identify novel hypnotic agents. Almorexant12,13
,
Suvorexant14 and SB-64986815 DORAs (Figure 1) achieved
clinical proof of concept, being able to enhance sleep duration
and reduce latency to fall asleep in patients. Remarkably, these
clinical endpoints achievement were obtained without the side
effects profile typical of benzodiazepines and Z-drugs (the
current clinical gold standards in insomnia) such as motor
impairments, next day hangover, potentiation of alcohol effects
and memory impairment. Recently, the use of DORA has been
associated to the potential treatment of Alzheimer disease, by
modulating the level of amyloid αβ42 filaments into the brain16.
This discovery is paving the way to the use of the DORA class in
the poorly treated neurodegenerative CNS disorders, where the
unmet clinical need is extremely high.
b
a
O
O
N
N
N
O
O
OH
(iii)
(ii)
(i)
c
Cl
d
O
O
O
N
N
N
H
N
N
N
H
O
OH
OH
O
O
N
O
N
N
N
(v)
(iv)
CF3
Scheme
1.
Synthesis
of
(S)-tert-butyl
6-(hydroxymethyl)-5-
Almorexant (DORA)
Suvorexant (DORA)
azaspiro[2.4]heptane-5-carboxylate, key intermediate (v). Reagents and
conditions: a) DMF, NaH, trimethylsulphoxonium iodide, r.t., 41% yield. b)
THF, LiAlH4, reflux, 74% yield c) H2, Pd/c (10%), EtOAc/AcOH, r.t., quant.
d) H2O, NaOH, (Boc)2O, 82% yield.
O
N
H
N
N
HN
HN
N
O
Br
Alternatively, when reductive aminations on aldehyde (vi) were
problematic, due to the poor nucleophylicity of the amino
heterocycles employed, alcohol (v) was transformed into its
phtalimido derivative (viii) by Mitsunobu reaction in 97% yield;
hydrazinolysis of (viii) gave amino derivative (ix) in 88% yield.
Intermediate (ix) was then reacted with the desired halogen
heterocycles by using a SnAr reaction to get the heteroaryl
intermediates (vii). These were transformed in the advanced
intermediates (x) by removing the Boc group with TFA in DCM)
(Scheme 2).
O
O
N
F
N
GSK1059865 (SORA)
SB-334867 (SORA)
Figure 1. Representative examples of SORA and DORA antagonists.
We have been recently engaged17 in the orexin antagonists
arena and we have identified a novel 6-azaspiro[2.5]octan-6-yl
SORA drug-like series. We then decided to expand the
exploration of this series by contracting the core six membered
piperidine ring into a five membered pyrrolidine ring while
keeping the distinctive cyclopropane spiro-ring system at the
same relative position with respect to the nitrogen carrying the
amide bond (Figure 2). We in fact believed that the distinctive
cyclopropane spiro-ring system was an important feature for
modulating the selectivity among the orexin receptors. We also
decided to keep the same absolute configuration of the chiral
center (2-S) in the novel series. No obvious clues were reported
in literature regarding the impact of this transformation into the
SAR against the OX1 and OX2 receptors.
H
N
H
N
N
N
RHS
N
N
S
O
Cl
N
S
N
O
Cl
2nd ring
LHS
1st ring
LHS
Figure 2. Planned exploration of our SORA series.
Scheme 2. Synthesis of compounds 2, 4, 6 and 7-32. Reagents and
conditions: a) DCM, BAIB, TEMPO, 81% yield. b) Ph3P, Phtalimide, THF,
DEAD, r.t., 97% yield. c) d) EtOH, NH2NH2(H2O), r.t., 88% yield. e) DMF,
The new 6-azaspiro[2.5]octane
synthesized (Scheme 1) starting from the known18 (3R,7aS)-6-
Methylene-3-phenyl-tetrahydro-pyrrolo[1,2-c]oxazol-5-one (i)
which was converted to the cyclopropane intermediate (ii) by
scaffold core could be

K2CO3, Hal-Het, MW 100°C. f) DCM, TFA, r.t.. g) N-methyl morpholine, 2-
chloro-4,6-dimethoxy-1,3,5-triazine, 1,4-dioxane, r.t. to 60°C.
penetration capability for drugs it is often used in preliminary
screens to understand the CNS potential of new drugs.
Final compounds were obtained by reacting intermediates (x)
with the desired carboxylic acids or acyl chlorides by using
standard coupling procedures (Scheme 2). Compounds were then
screened in a functional cell-based assay (CHO or HEK-293 cells
stably expressing human recombinant OX1 or OX2 receptors,
respectively) based on OXA or OXB stimulated intracellular
calcium mobilization using a combination of calcium-sensitive
dyes and a fluorescent imaging plate reader (FLIPR)20.
Cmpd
2
Clpa
F%b
Vdssc
B/Pd
CyP450se
11.0
40
6.21
0.03
All<53%
6
33.1
NT
0.48
0.2
All<34%
Table 2. (a) rat Clp given in ml/min/Kg, 1mg/kg iv dose (b) rat oral
bioavailability, 1mg/Kg po dose (c) Vdss given in L/Kg (d) rat brain to
plasma ratio obtained after 1mg/kg iv dose administration (e) CyP450s
GentestTM microplated – based fluorimetric assay with recombinant human
P450 enzymes against CYP450 3A4, 2D6, 1A2, 2C19 and 2C9 isoforms
(CYP3A4 two substrates were used, DBF = Dibenzyl fluorescein and BFC =
7-benzyloxy-4-trifluoromethyl coumarin): data reported as % inhibition @
1µM compound’s concentration. NT= not tested.
With chemistry set-up, the first round of exploration was aimed
at comparing the effect of spiro-pyrrolidine instead of spiro-
piperidine ring by leaving intact the other two points of
variations, namely the left hand side (LHS) amides and the right
hand side (RHS) heterocycles. RHSs and LHSs were initially
chosen among those used in the exploration of the spiro-
piperidine scaffold17. First data showed this modification had
some influence on OX1 activity with respect to the former series:
compounds 2 and 4 were found slightly more active against OX1
receptor with respect to compounds 1 and 3, whereas compounds
6 reported a 1 log unit drop of potency with respect to its
corresponding spiro-piperidine 5. For compounds 1-6 a wide
selectivity against the OX2 receptor was achieved (Table 1).
Compound 2 showed a very low B/P ratio (in the range of blood
content of brain tissue) suggesting the preclusion of the brain
compartment for this product. Also compound 6 was submitted to
rat DMPK (iv and B/P studies only) showing moderate in vivo
plasmatic clearance and significant brain penetration, thus
showing that in this series it is possible to achieve good
metabolic stability, oral bioavailability and both a peripheral or a
central distribution pattern (Table 2). Based on these
encouraging results we decided to enlarge the exploration of the
spiro-pyrrolidine series initially by expanding the RHSs used so
far; in this round we also decided to introduce the known thiazol-
phenyl group as LHS because its usage in the preparation of
orexin antagonists was widely described. In addition to that, it
proved very easy to prepare (Table 3).
a
a
Compound
1
n
LHS
RHS
OX1
OX2
1
7.6
<5.0
<5.0
<5.0
2
3
0
1
8.2
7.5
a
a
Compound
7
n
RHS
OX1
OX2
1
7.9
<5.0
7.1
4
5
0
1
8.1
8.2
<5.0
<5.0
8
0
1
0
0
0
0
0
8.3
7.7
8.4
7.3
7.5
6.8
6.1
9
<5.0
7.7
7.6
8.6
6.8
7.0
10
11
12
13
14
6
0
7.1
<5.0
Table 1. (a) pKb values in Ca2+ FLIPR functional assays are means from at
least two independent experiments.
Spiro-pyrrolidine 2 emerged from this first iteration as an
interesting OX1 selective hit and for this reason it was further
progressed in rat DMPK studies and cytochrome P450s inhibition
(CyP450) characterization (Table 2). In rats compound 2 showed
low in vivo plasmatic clearance (Clp) and good oral
bioavailability; its ability to cross the blood brain barrier was
investigated by sampling the concentration of the compound in
brain homogenate one hour after its iv administration and by
comparing it against its plasma level at the same time point.
Although this procedure gives a static picture of the brain

Table 3. (a) pKb values in Ca2+ FLIPR functional assays are means from at
least two independent experiments.
ring, a known structural alert for mechanism-based inactivation
of Cytochrome P450 Enzymes21(Table 5). From this iterations
we discovered that polarity was well tolerated in both the LHS
aromatic rings (see ad e.g. in Table 5 cmpds 15 vs. 19), that the
methyl in position 2 of the first LHS pyridine ring is important
for OX activity (Table 5, cmpds 15 vs. 21) and that the second
LHS pyrimidine ring could be well replaced with a benzyloxy
moiety (Table 5, cmpds 15 vs. 22). We also observed that the
introduction of polarity in the position 2 of first LHS pyridine
was generally not tolerated (see ad e.g. in Table 5 cmpds 15 vs.
20 & 23). Interestingly the introduction of a CN group into the
first LHS ring was well tolerated (Table 5, entry 26).
To our surprise spiro-pyrrolidine compounds 8 and 10-14 (Table
3) emerged from this new array showing a dual OX1/OX2
antagonist activity. Clearly, OX2 activity was found to be
peculiar to the spiro-pyrrolidine series because spiro-piperidine
compounds 7 and 9 were showing a selective OX1 activity
pattern even when decorated with the thiazolo phenyl LHS
(Table 3). However, the spiro-pyrrolidine moiety was not
sufficient per se to gain OX2 activity which, we discovered, was
indeed linked to the nature of both the amide LHS and the
pyridine RHS. For the LHS, shifting from a phenyl-pyrimidine to
a thiazolo-phenyl system was key to get OX2 activity on the new
series (compared to e.g. compounds 8 vs. 2, Table 3 and 2
respectively). The 5-CF3-pyridine moiety RHS was also
discovered to be important to increase OX2 activity on the new
series (compared to e.g. compounds 10 vs. 8, Table 3). Very
interestingly a couple of examples prepared in the pyrimidine
RHS sub-series (compounds 12 and 14) showed an OX2 preferred
antagonist pattern. In particular the 4,6-dimethyl substitution
pattern (compound 12) brought a 40-fold increase in the OX2
potency when compared with its 6-methyl derivative (compound
14). This result suggested that the 4-position of the pyrimidine
RHS could be further explored in order to enhance OX2 activity
in this sub-series.
Cmpd
10
LHS
OX1
OX2
ClogPb
Cyp450sc
a
a
8.4
7.7
5.29
All<20%
15
8.4
7.5
3.09
All<28%
In view of their potent OX1/OX2 antagonism profile,
compounds 10 and 12 were submitted to DMPK characterization:
in vivo rat PK, CyP450s inhibition and CYP 3A4 Time
Dependent Inhibition21 (TDI) (Table 4).
16
17
18
19
8.1
8.6
6.7
8.9
7.2
8.0
5.0
8.3
4.43
5.25
3.70
3.96
All<22%
All<20%
All<39%
All<85%
CyP450se
3A4 IC50
All<20%
>100μM
All<16%
7.9μM
Cmpd Clpa F%b
Vdssc
3.0
B/Pd
0.6
3A4 TDIg
Yes
f
10
12
55.2
41.4
20
NT
0.7
0.1
Yes
Table 4. (a) rat plasmatic clearance given in ml/min/Kg, 1mg/kg iv dose (b)
rat oral bioavailability, 1mg/Kg po dose (c) Vdss: Volume of distribution at
steady state, given in L/Kg (d) CyP450s GentestTM microplated – based
fluorimetric assay with recombinant human P450 enzymes against CYP450
3A4, 2D6, 1A2, 2C19 and 2C9 isoforms (CYP3A4 two substrates were used,
DBF = Dibenzyl fluorescein and BFC = 7-benzyloxy-4-trifluoromethyl
coumarin): data reported as % inhibition @ 1µM compound’s concentration.
NT= not tested. (f) 3A4 BFC (g) Time Dependent Inhibition of CYP3A4
enzyme with BFC as substrate was examined by comparing IC50 value from
experiment with pre-incubation (30 min) to the IC50 value from experiment
without pre-incubation; a substantial decrease in IC50 after pre-incubation
(IC50 fold shift >1.5) is characteristic of compound showing TDI.
20
21
7.0
7.7
6.9
5.0
5.01
2.59
All<58%
All<47%
22
8.6
6.7
8.6
8.7
8.6
7.1
8.0
6.6
5.50
4.63
3.87
4.19
All<71%
All<41%
All<89%
NT
Both compounds showed a higher plasmatic clearance with
respect to the lead compound 2 (Table 2), although the
moderate-high metabolism in this case did not compromise the
oral absorption of compound 10. The measured brain-plasma
ratio of compound 10 was much higher with respect to
compounds 12 and especially 2 (respectively in Table 4 & Table
2) thus showing how the brain penetration of this series was
deeply influenced from chemical variations on both the RHS and
LHS points of variation. Further, compound 10, although having
a very low CyP450s inhibition profile, still carried on some
concerns because it showed to be a time dependent inhibitor
(TDI) of the 3A4 enzyme. Following these results, we decided to
focus the exploration of the newly identified spiro-pyrrolodine
series in order to achieve DORA endowed with improved
physico-chemical features and possibly with no TDI liability. For
this reason we decided to modulate the polarity of the LHS to
achieve less lipophilic compounds while removing the thiazole
23
24
25

LHS (entry 29, table 6) . Further, the increase in polarity was not
well tolerated (entry 27, Table 6). Interestingly, compound 28
was identified as an OX2 selective antagonist (>100 fold
26
8.0
8.1
3.25
All<32%
separation between OX2 and OX1 activities) which is
remarkable achievement considering that the starting point of this
exploration was an OX1 selective series.
Eventually, compounds 1522 and 1723 (Table 5), in light of their
good in vitro features, were further characterized into DMPK
assays (in vivo rat PK and CYP 3A4 TDI) as shown in Table 7.
a
Table 5. (a) pKb values in Ca2+ FLIPR functional assays are means from at
least two independent experiments. (b) ClogP was measured by using
ChemBioDrawUltra 12.0.2 (c) CyP450s GentestTM microplated–based
fluorimetric assay with recombinant human P450 enzymes: data reported as
% inhibition @ 3µM compound’s concentration against CYP450 3A4, 2D6,
1A2, 2C19 and 2C9 isoforms (CYP3A4 two substrates were used, DBF =
Dibenzyl fluorescein and BFC = 7-benzyloxy-4-trifluoromethyl coumarin).
NT= not tested.
e
Cmpd
15
Clpa F%b
Vdssc
B/Pd
CYP3A4 IC50
3A4 TDIf
47.5
61.3
33
11
2.0
0.5
Yes
>100μM
Importantly, from this exploration emerged that, by keeping a
CF3-pyridine as the RHS, OX2 activity could be maintained
through a broader range of different LHS amides than it was
previously noted in the initial exploration, where all active
compounds were OX1 selective (even when the amide LHS was a
phenyl-pyrimidine moiety, which at the beginning of the
exploration was giving exclusively OX1 selective compounds,
compare ad e.g. entry 2, 4 and 6 vs. 16 and 25, Table 2 and 5,
respectively). We thus decided to further explore the RHS
pyridine pattern of substitution, particularly considering the
excellent gain in activity against OX2 receptor observed with this
strategy. For this reason a small library of 4-substituted pyridine
compounds was prepared (Table 6).
17
4.4
0.4
No
64 μM
Table 7. (a) rat Clp given in ml/min/Kg, 1mg/kg iv dose (b) rat oral
bioavailability, 1mg/Kg po dose (c) Vdss given in L/Kg (d) rat brain to
plasma ratio obtained after 1mg/kg iv dose administration (e) CyP450s
GentestTM microplated – based fluorimetric assay with recombinant human
P450 enzymes: data reported as IC50 on 3A4 BFC (f) Time Dependent
Inhibition of CYP3A4 enzyme with BFC as substrate was examined by
comparing IC50 value from experiment with pre-incubation (30 min) to the
IC50 value from experiment without pre-incubation; a substantial decrease in
IC50 after pre-incubation (IC50 fold shift >1.5) is characteristic of compound
showing TDI.
Pyridine derivative 15, a compound with greatly reduced (2-logs
fold) lipophilicity with respect to lead molecule 10, showed in
rats a moderate-high plasma clearance, decent brain penetration
and acceptable oral bioavailability together with a very low
CyP450s inhibitory profile; this compound however still showed
TDI on CYP 3A4 enzyme (Table 7). This issue was eventually
overcome with compound 17, which showed slightly higher in
vivo rat Clp with respect to 15, low CyP450s inhibitory profile
but no TDI on 3A4 enzyme (Table 7). This data set was
suggesting that the replacement of the first LHS thiazole ring
(compound 10 vs. compound 17) was sufficient to rule out the
TDI effect on the 3A4 enzyme. However, when a pyrimidine ring
was installed instead of the phenyl moiety on the second LHS
ring TDI was again measurable (compound 15 vs. compound 17);
therefore, in this molecular context, the pyrimidine ring
apparently contributed to the observed TDI behavior of this
compound.
Cyp450sb
a
a
Cmpd
27
LHS
RHS
OX1
NI
OX2
NI
ClogPc
All<17%
1.72
All<39%
3.09
28
6.0
8.2
7.8
7.3
8.1
8.1
8.5
8.6
7.8
7.1
All<55%
5.29
29
All<69%
5.79
In conclusion, starting from an OX1 selective series, we
identified new potent dual OX1/OX2 antagonists. Compound 15
showed good in vitro and in vivo DMPK profile. Further, the
CYP3A4 TDI issue was eliminated in this new series by
replacing the LHS thiazole ring, used to decorate the spiro-
pyrrolidine scaffold, with a pyridine moiety.
30
All<72%
4.19
31
All<57%
4.43
32
Acknowledgments
Table 6. (a) pKb values in Ca2+ FLIPR functional assays are means from at
least two independent experiments. (b) CyP450s GentestTM microplated –
based fluorimetric assay with recombinant human P450 enzymes against
CYP450 3A4, 2D6, 1A2, 2C19 and 2C9 isoforms (CYP3A4 two substrates
were used, DBF = Dibenzyl fluorescein and BFC = 7-benzyloxy-4-
trifluoromethyl coumarin): data reported as % inhibition @ 1µM compound’s
concentration. (c) ClogP was measured by using ChemBioDrawUltra 12.0.2
The authors thank Cristiano Falcioni for in vivo rat DMPK
work, Dr. Mariella Artusi and Mr. Roberto Beretta for pre-
formulation work and the members of the Rottapharm Madaus
R&D Analytical Chemistry group for their assistance in the
structural confirmation of the compounds described in this
manuscript.
References and notes
From this exploration emerged that very good OX2 activity
was achievable by using 4-CF3-pyridine RHSs (e.g. compounds
29 and 30, Table 6). OX1 activity was however generally lower
with respect to corresponding 3-CF3-pyridine derivatives
(compared to e.g. compounds 15 vs. 28, Table 5 and 6
respectively) except when thiazole-phenyl was used as amide
1. (a) Sakurai, T.; Amemiya, A.; Ishii, M.; Matsuzaki, I.; Chemelli,
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22. Compound 15 analytical data (C24H23F3N6O, Molecular Weight =
468.5): 1HNMR (400MHz, CDCl3) δ ppm 8.6-8.85 (m, 1H), 8.36-
8.58 (m, 1H), 8.19-8.33(m, 1H), 7.47-7.50 (m, 1H), 7.27-7.34 (m,
2H), 7.08-7.19 (m, 2H), 6.01-6.50 (m, 1H), 4.73-4.87(m, 1H),
3.64-3.87 (m, 2H), 3.35-3.48 (m, 1H) 3.10-3.13 (m, 1H) 2.56-2.67
(m, 3H) 2.37-2.53 (m, 1H), 1.60-1.66 (m, 1H), 0.45-0.77 (m, 4H);
HPLC-MS [MH]+ = 469.
23. Compound 17 analytical data (C26H25F3N4O, Molecular Weight =
466.5): 1H NMR (400MHz, CDCl3) δ ppm = 8.97-8.55 (m, 2 H),
8.46-8.09 (m, 2 H), 7.60 -7.30 (m, 2 H), 7.25-6.92 (m, 2 H), 6.55
(d, J = 8.8 Hz, 1 H), 4.84 (d, J = 4.9 Hz, 1 H), 4.04 - 2.73 (m, 4 H),
2.29 (dd, J = 8.3, 12.7 Hz, 1 H), 1.63 (d, J = 8.8 Hz, 1 H), 1.03 -
0.03 (m, 4 H); HPLC-MS [MH]+ = 468.

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Discovery, synthesis, selectivity modulation
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azaspiro[2.4]heptanes as potent orexin
receptor antagonists
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Luigi Piero Stasi, Roberto Artusi, Clara Bovino, Benedetta Buzzi, Luca Canciani, Gianfranco Caselli,
Fabrizio Colace, Paolo Garofalo, Patrice Larger, Ornella Letari, Stefano Mandelli, Lorenzo Perugini,
Sabrina Pucci, Matteo Salvi, PierLuigi Toro