Discovery process and pharmacological characterization of a novel dual orexin 1 and orexin 2 receptor antagonist useful for treatment of sleep disorders

Article abstract of DOI:10.1016/j.bmcl.2011.06.086

The hypothalamic peptides orexin-A and orexin-B are potent agonists of two G-protein coupled receptors, namely the OX₁ and the OX₂ receptor. These receptors are widely distributed, though differentially, in the rat brain. In particul

Full text of DOI:10.1016/j.bmcl.2011.06.086

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5562–5567
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery process and pharmacological characterization of a novel dual orexin
1 and orexin 2 receptor antagonist useful for treatment of sleep disorders
Romano Di Fabio a, , Annalisa Pellacani a, Stefania Faedo a, Adelheid Roth a, Laura Piccoli a, Philip Gerrard a,
⇑
Rod A. Porter b, Christopher N. Johnson b, Kevin Thewlis b, Daniele Donati a, Luigi Stasi a, Simone Spada a,
Geoffrey Stemp b, David Nash b, Clive Branch b, Leanda Kindon c, Mario Massagrande a, Alessandro Poffe a,
Simone Braggio a, Elisabetta Chiarparin a, Carla Marchioro a, Emiliangelo Ratti a, Mauro Corsi a
a GlaxoSmithKline Medicine Research Centre, Via A. Fleming, 4, 37135 Verona, Italy
b GlaxoSmithKline New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
c GlaxoSmithKline Medicine Research Centre, Particles Generation Control and Engineering, Product Development, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The hypothalamic peptides orexin-A and orexin-B are potent agonists of two G-protein coupled recep-
tors, namely the OX1 and the OX2 receptor. These receptors are widely distributed, though differentially,
in the rat brain. In particular, the OX1 receptor is highly expressed throughout the hypothalamus, whilst
the OX2 receptor is mainly located in the ventral posterior nucleus. A large body of compelling evidence,
both pre-clinical and clinical, suggests that the orexin system is profoundly implicated in sleep disorders.
In particular, modulation of the orexin receptors activation by appropriate antagonists was proven to be
an efficacious strategy for the treatment of insomnia in man. A novel, drug-like bis-amido piperidine
derivative was identified as potent dual OX1 and OX2 receptor antagonists, highly effective in a pre-clin-
ical model of sleep.
Received 20 April 2011
Revised 15 June 2011
Accepted 18 June 2011
Available online 30 June 2011
Keywords:
Orexin receptor antagonist
G-Protein coupled receptor (GPCR)
Central nervous system (CNS)
Sleep disorders
Ó 2011 Elsevier Ltd. All rights reserved.
Hypnotic agent
Conformational analysis
Structure–activity relationship (SAR)
The neuropeptides orexin-A (OX-A) and orexin-B (OX-B), also
called hypocretin 1 and hypocretin 2, respectively, were originally
isolated in the rat hypothalamus and derive from the proteolytic
cleavage of prepro-orexin,1 a common 133 aminoacids (AA) poly-
peptide precursor. Orexins were originally identified as natural li-
gands of the former orphan G-protein coupled receptor (GPCR)
HFGAN72, now known as the orexin-1 receptor (OX1).1,2 Neuro-
anatomical studies revealed that orexin containing neurons origi-
nate exclusively in the lateral and posterior hypothalamus1–3
projecting mainly in the brain stem nuclei (e.g., locus coeruleus
and raphe nuclei), the hypothalamic nuclei (e.g., the arcuate nu-
cleus and the tuberomammillary nucleus)3–5 and throughout the
cerebral cortex, hence suggesting a major role in the regulation
of sleep and arousal states. The projection of orexin neurons
throughout the entire neuroaxis, with the exception of cerebellum,
is paralleled by the distribution of the orexin receptors.6–8 In par-
ticular, the action of OX-A and OX-B is mediated via two class A
GPCRs, namely OX1 and OX2 receptors, which are 64% homologous
and, like the peptides themselves, are highly conserved across
mammalian species. OX-A binds similarly to OX1 and OX2 recep-
tors, whereas OX-B recognises preferentially the OX2 receptor.
In addition, these receptors have been associated with different
signal transduction pathways, which may account for differential
biological functions.3 A great deal of evidence suggests that the
orexin system is critical in the regulation of arousal and sleep. In
particular, it has been observed that: (a) intracerebroventricular
(icv) injection of OX-A in the rat caused increases in locomotor
activity, rearing and grooming behavior, findings which were
supported by the evidence that OX-A was able to increase locus
coeruleus (LC) firing;9,10 (b) electroencephalogram (EEG) and
electromiogram (EMG) recordings in the rat reveal that icv OX-A
administered at the beginning of the normal sleep period increased
time spent awake and decreased both rapid eye movement (REM)
sleep and slow wave (deep) sleep;11 (c) mice devoid of the orexin
precursor prepro-orexin and the OX1/OX2 dual KO mouse exhibit a
behavioral phenotype similar to that of narcolepsy;12 (d) an OX2
receptor mutation, producing a non-functional receptor, was asso-
ciated with narcolepsy in dog;13 in addition, narcolepsy in man
was associated with reduced orexin levels in the cerebrospinal
fluid (CSF);14 (f) the dual OX1 and OX2 receptor antagonist almo-
rexant15,16 as well as, more recently, MK-4305,17 another dual
⇑
Corresponding author. Tel.: +39 045 8218879; fax: +39 045 8218196.
E-mail address: romano.difabio@aptuit.com (R. Di Fabio).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.086

R. Di Fabio et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5562–5567
5563
Table 1
OX1 and OX2 receptor antagonist, have shown clinical efficacy in
In vitro functional activity (pKi) at h-OX1 and h-OX2 receptors
primary insomnia studies. All these findings, confirm that a dual
orexin receptor antagonist has the potential to treat sleep disor-
ders; in addition, exploiting a different biochemical pathway than
both benzodiazepins and non-benzodiazepine hypnotic agents, the
orexin antagonists were expected to be devoid of typical side ef-
fects associated to zolpidem, the current gold standard drug for
insomnia. As part of a wide drug discovery program aimed at iden-
tifying drug-like dual OX1 and OX2 receptor antagonists, an HTS
was performed in house in a chinese hamster ovary (CHO) cell line
transfected with the recombinant human OX1 receptor, using a
FLIPR calcium based functional assay protocol. Selectivity screen-
ing was performed against CHO cell line transfected with the re-
combinant human OX2 receptor. From this screening activity the
series of compounds of type A, shown in Figure 1, were identified
and selected for further investigation based upon the encouraging
observed target activity.
In particular, racemic compounds 2 and 3, shown in Table 1,
exhibited signs of in vitro potency at both the OX1 and OX2 recep-
tors (compound 2, pKi = 6.9 and 6.3, compound 3, pKi = 6.4 and 6.2
at the OX1 and OX2 receptor, respectively). Hence, the general
investigative program highlighted in Table 1, was initiated to elab-
orate the key structural features for maximizing the in vitro activ-
ity at both receptors and optimize pharmacokinetic properties,
namely the investigation of the SAR of the disubstituted amide bot-
tom region and the role of the substitution of the right-hand side
(RHS) part of the chemical series selected.
As shown in Table 1, an initial key observation was the positive
effect of the presence of an additional aromatic moiety in the bot-
tom region of the molecule, that is, introducing a 2-biaryl or a
1-naphyl moiety, and concomitant substitution at the para position
by a fluorine atom of the RHS phenyl ring. In particular, compound
5 showed good target activity at both receptors (pKi = 8.3 and 7.7 at
the OX1 and OX2 receptor, respectively), while 6 featured good OX1
activity and was moderately selective over OX2 (pKi = 8.2 and 6.9 at
the OX1 and OX2 receptor, respectively). From an additional itera-
tion focused on the exploration of the effect of substituents of the
RHS phenyl ring, the 2,4-difluoro substituted derivative 7 was
identified as the most potent dual orexin receptor antagonist
belonging to this sub-series (pKi = 9.8 and 9.1 at the OX1 and OX2
receptor, respectively).18 However, this class of compounds was
characterized by high in vitro clearance (Cli) in both human and
rat liver microsomes; in particular, compound 5 exhibited a Cli
>50 ml/min/g of total liver. The high metabolic instability was
hypothesized to be due either to the high lipophilicity and/or the
presence of the ether functionality in the RHS, potentially respon-
X
N
2
R
1
O
R
Entry
R1
R2
X
OX1 pKia
OX2 pKia
n
1b
—
—
—
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
NHCO
NHCO
NHCO
N(CH3)CO
CONH
CH2CO
NHCONH
9.5
6.9
6.4
5.6
8.3
8.2
9.8
8.1
9.9
7.4
6.3
7.3
8.0
9.0
9.4
6.3
6.2
<5.5
7.7
6.9
9.1
7.7
9.6
7.5
5.9
7.5
7.7
8.3
20
4
4
4
4
4
4
4
8
8
4
4
4
6
2b
3-OCH3-C6H4
3-CN-C6H4
3,4-Cl-C6H3
2-C6H5–C6H4
1-C10H7
2-C6H5–C6H4
2-C6H5–C6H4
2-C6H5–C6H4
2-C6H5–C6H4
2-C6H5–C6H4
2-C6H5–C6H4
2-C6H5–C6H4
2-C6H5–C6H4
3,4-Cl
3,4-Cl
3,4-Cl
4-F
4-F
2,4-F
H
4-F
4-F
4-F
H
3b
4b
5b
6c
7c
8c
9ac
9bd
10c
11c
12c
13c
H
H
a
b
c
pKi values are the mean of n experiments each performed in duplicate.
Racemate.
(S)-Enantiomer.
(R)-Enantiomer.
d
The introduction of a fluorine atom in the para position of the
RHS aromatic ring further increased the in vitro affinity (compound
9a, Table 1, pKi = 9.9 and 9.6 at the OX1 and OX2 receptor, respec-
tively) along with a partial reduction of the intrinsic clearance
(Cli = 19 ml/min/g and 23 ml/min/g in rat and human liver micro-
somes, respectively). Notably, the corresponding (R)-enantiomer
9b was significantly less active (pKi = 7.4 and 7.5 at the OX1 and
OX2 receptor, respectively). A substantial drop of in vitro affinity
was observed for the N-methyl derivative 10, the reverse amide
11 and the ketone derivative 12, giving an initial insight into the
required spatial orientation of the aromatic moiety present in the
RHS region of the molecule to maximize the in vitro affinity. Con-
versely, the urea derivative 13, demonstrated good in vitro affinity,
particularly at the OX1 receptor (pKi = 9.0 and 8.3 at the OX1 and
OX2 receptor, respectively).
Following these initial results, a wide exploration of the bottom
region of the molecule was undertaken with the specific objective
of replacing the lipophilic biaryl moiety by more hydrophilic het-
erocycles. A series of five-membered and six-membered ring
substituted heterocycles was introduced, replacing the central aryl
ring (namely, pyrazoles, triazoles, thiazoles, isoxazoles, pyrazines);
then, an attempt to replace the ‘pendant’ terminal phenyl ring
mainly by pyridines was performed.19 This exploration led to the
identification of the 2-phenyl-5-methyl thiazole ‘motif’ as the pre-
ferred piperidine amide substituent offering best balance among
in vitro activity, OX1 versus OX2 receptor selectivity ratio and
reduction of the general lipophilicity. Having optimized the bottom
region of the molecule, the optimization of the secondary amide
aryl moiety present in the RHS region of the compounds was
undertaken managing to replace the amide phenyl moiety with a
focus set of heterocycles.19 The 4-carboxyamidebenzofuran
group20 was selected as an instrumental moiety to maximize the
in vitro activity at both the OX1 and OX2 receptor. N-[((2S)-1-{[5-
(4-Fluorophenyl)-2-methyl-1,3-thiazol-4-yl]carbonyl}-2-piperidi-
sible for an easy oxidation at the
a position to the oxygen atom. La-
ter on, in vitro metabolite identification studies confirmed the
metabolic liability of the aryl ether moiety. Hence, to stabilize this
class of molecules, while maintaining high in vitro activity at both
the OX1 and OX2 receptors, attention was drawn on modifying the
ether linkage. Among the functional groups investigated, the sec-
ondary amide 8 (Table 1, pKi = 8.1 and 7.7 at the OX1 and OX2
receptor, respectively) showed very encouraging in vitro potency.
H
N
R'
N
O
N
O
O
N
O
O
1
(SB-649868)19 was
S
nyl)methyl]-1-benzofuran-4-carboxamide
R
identified as one the most in vitro potent dual OX1 and OX2 recep-
tor antagonist known at that time (pKi = 9.4 and 9.5 at the OX1 and
OX2 receptor, respectively), featuring signs of reduction of the in Cli
both in the rat and human liver microsomes (Cli = 8.7 mL/min/g
and 15.1 mL/min/g, respectively). This data, when corrected with
F
A
1 (SB-648868)
Figure 1. Chemical series A identified by HTS and chemical structure of compound
1 (SB-649868).

5564
R. Di Fabio et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5562–5567
the fraction unbound,21 suggested a low to moderate in vivo clear-
ance, therefore accelerating its in vivo pharmacokinetic character-
ization. Compound 1 was smoothly prepared as shown in Scheme
1, by two sequential amidation reactions from the known chiral
diamine template 15.22
From an in depth analysis of its 1H NMR spectrum, it was ob-
served that the chemical shifts of the protons present in
a position
to the disubstituted amide nitrogen were associated with unex-
pected values, suggesting the presence in solution of a mixture of
the E and Z rotamers, as depicted Figure 2, originating from the
slow rotation around the disubstituted amide N–C@O bond. The
conformation of those rotamers was postulated by rotating frame
Overhauser 2D ROESY experiments on the equilibrium mixture.
This data suggested that in both rotamers the six membered piper-
idine core adopts a twisted-chair conformation, as shown in Figure
2, while the exocyclic CH2NHCOR group occupies an axial position.
In the Z conformation the presence of weak ROE effects between
Figure 2. Low energy conformations predicted for compound 1 using systematic
conformational search using parameterized force field for gas phase organic
molecules. These structures are consistent with the ROEs constraints derived from
NMR measurements.
the aromatic rings suggests a
p-stacking interaction, while in the
E conformation the methyl group present in the thiazole ring
shows several spatial correlations with the RHS aromatic ring, call-
ing for a close proximity of these moieties.
Then, 1H NMR exchange experiments were carried out to mea-
sure thermodynamics and kinetics of the interconversion process.
The E rotamer was found to be the thermodynamically most stable
in all solvents, and the interconversion half-life values of the two
rotamers, at T = 25 °C, were less than 1 s. The D D
G#ZE and GE#Z activa-
tion energy values for rotation of the amide bond in DMSO-d6 were
18.0 0.2 kcal/mol and 17.8 0.2 kcal/mol, respectively.23
The solid-state structure of compound 1, determined by X-ray
crystallography, as depicted in Figure 3, shown that the most stable
E conformation in solution, was the only one present in the crystal-
line state. Notably, the energy stabilization deriving from postu-
lated weak
p-stacking in the Z conformation does not seem to
drive the crystal packing while the preferred orientation of the
C@O of the disubstituted amide looks responsible for the confor-
mation adopted by compound 1.
In summary, the information generated by NMR experiments
suggested the preferential presence in solution of a ‘U-shaped’ E
conformer in rapid equilibrium with the Z conformer, whereas
the former one was the only conformer observed in the crystalline
Figure 3. X-ray crystal structure of compound 1.
state by X-ray analysis. Recently, this finding was confirmed inde-
pendently by Merck’s scientists investigating a different class of
dual orexin receptor antagonists,24 giving specific insights on the
spatial pharmacophore requirements to design novel series of
compounds.
CO2H
N
S
Compound 1 was selected for further biological and pharmaco-
kinetic characterization based on the high in vitro potency at both
orexin receptors and the reduced Cli observed. As reported in
Table 2, it exhibited pKi values of 9.50 and 9.40 on human OX1
and OX2 receptors, respectively. Slightly higher functional poten-
cies were found for the rat receptors (pKi of 9.93 and 10.11 on
OX1 and OX2 receptors, respectively). This similarity was not wholly
unexpected as it is well established that sequence homologies of
the orexin peptides and receptors are highly conserved across
NHBoc
F
NH2
N
16
N
b
N
a
N
O
O
NHBoc
N
H
S
S
F
F
15
18
17
c
H
N
N
O
O
Table 2
N
O
Functional potency and receptor binding affinity values (fpKi) obtained using
recombinant cell lines expressing human and rat OX1 and OX2 receptors
S
fpKi SEMa
n
fpKi SEMa
n
F
h-OX1
h-OX2
r-OX1
r-OX2
9.50 0.02
9.40 0.05
9.93 0.08
10.11 0.11
38
23
6
9.12 0.12b
3
3
—
—
1
8.89 0.08b
a) i. 16, (COCl)2, DMF c.a., CH2Cl2, room temp, 6 h; ii. 15, CH2Cl2, TEA,
room temp, overnight; b) TFA, CH2Cl2, room temp, 3 h; c) i. 1-benzofurane-
4-carboxylic acid, (COCl)2, DMF c.a., CH2Cl2, room temp, 5 h; ii. 18, TEA,
room temp, overnight.
—
—
6
a
fpKi values are the mean of n experiments each performed in duplicate.
Displacement binding study using [3H]almorexant as radioligand.
b
Scheme 1. Synthesis of compound 1.

R. Di Fabio et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5562–5567
5565
mammalian species.3 In addition, no agonism was observed with
compound 1 when tested per se up to the concentration of 1 M.
Table 3
Sleep promoting effects of compound 1 administered at 3, 10 and 30 mg/kg, po in rat
during active phase (starting time CT18)
l
Compound 1 displaced, in CHO cells overexpressing both the
human recombinant OX1 and OX2 receptors, [3H]almorexant25 in
a concentration dependent manner. The calculated receptor affin-
ity values as reported in Table 2, were pKi = 9.12 and 8.89 for the
human OX1 and OX2 receptor, respectively, confirming a similar
potency for compound 1 at both receptor subtypes. When tested
Vehicle
3 mg/kg
10 mg/kg
30 mg/kg
Awake
218 11.32 194 10.8
117 4.6**
11.6 1.62**
165.2 5.4**
26.6 7.7**
17.7 2.2**
87 6.7**
NREM latency
NREM sleep
REM latency
REM sleep
46.1 10.0
78.1 11.3
146.3 54.2
3.22 0.73
19.0 6.2**
99.3 10.9
30.1 17.7**
6.5 1.0
8.3 1.4**
186.6 8.0**
13.8 2.2**
24.1 12.1**
in the CEREP selectivity screen, compound compound 1, at 1 lM
Values are expressed as time post-dosing in minutes as mean SEM (n = 8).
p <0.01 for each dose versus corresponding vehicle as determined by one-way
Anova followed by Dunnett’s test.
concentration, produced less than 50% inhibition of binding in
the CEREP test battery, which included GPCRs, ion channels and
enzymes, resulting in >1000 time selectivity for the orexin target
receptors.
**
Pharmacokinetic studies in the male CD rat, performed at 1 mg/
kg, iv and 3 mg/kg, po,26 demonstrated an excellent pharmacoki-
netic profile for a hypnotic agent featuring moderate clearance in
plasma (Clp = 24 mL/min/kg), short half-life of (<0.6 h) and a low
volume of distribution (Vss = 1.1 l/kg), coupled with excellent oral
(EEG) and electrooculogram (EOG). Simultaneous recording of
EEG and EOG allows the accurate derivation of the key sleep param-
eters, namely: awake, non-REM (NREM) sleep and REM sleep. These
effects were assessed at 3, 10 and 30 mg/kg, po dose, over a 5 h per-
iod in the active phase of the rat, starting the recording phase at CT
18 (light off at CT 12) of the rat light–dark cycle. CT18 was specifi-
cally chosen to give a maximum window to assess the hypnotic ef-
fects of the compound. In these experimental conditions, as shown
both in Table 3 and Figure 5, vehicle treated rats showed a latency
time to sleep (time required for the first sleep episode of a duration
of 1 min) of 46 10 min and a total sleep time (sum of NREM and
REM sleep time) of 81 11 min, recorded over a 5 h period. In addi-
tion, compound 1 produced a dose related reduction in sleep la-
tency, with a maximal effect at 30 mg/kg (8.3 min 1.4, as shown
in Fig. 6). The reduction of latency to sleep was statistically signifi-
cant at all doses tested (p <0.01 for all doses).
bioavailability (F = 85%) and good exposure in plasma (Cmax
=
333 ng/mL). A brain to blood ratio (B/B) of 0.1:1 was observed
1 h after iv administration, a value in line with the expected parti-
tion between the two compartments based on the lower tissue
binding observed in vitro in brain tissues (fraction unbound/
brain = 5.28%) with respect to plasma proteins (fraction unbound/
plasma = 1.34%).27 These data suggest that compound 1 is brain
penetrant and is not substrate for brain efflux transporters (e.g.,
P-glycoprotein).
The ability of compound 1 to antagonise in vivo OX receptors
has been determined in the OX-A evoked grooming model in the
rat. Intracerebroventricular (icv) injection of Ox-A (3 lg/5 ll) in-
The hypnotic effect observed was marked and demonstrates
that a dual OX1 and OX2 receptor antagonist is able to facilitate
sleep induction in the rat. In addition, compound 1 produced a
robust dose dependent increase in total sleep time over the 5 h
testing period, effect statistically significant at both 10 mg/kg
and 30 mg/kg doses (81 11 min for vehicle; 182 4.6 min for
10 mg/kg, p <0.01; 213 6.7 p <0.01 for 30). A more detailed
analysis of the sleep patterns showed that compound 1 induced
a robust dose dependent effect in terms of significant increase of
both NREM and REM sleep time (Table 3).
duced a robust grooming response in the male CD rat. OX1 receptor
antagonists are able to attenuate this behaviour and the model has
been used as a pharmacodynamic (PD) readout for the investiga-
tion of in vivo effects mediated via the OX1 receptor.28,29
Data are expressed as time spent in grooming and numbers of
bouts (mean SEM, n = 8) over 1 h of observation. Statistically sig-
nificant differences compared to vehicle treated animals are indi-
cated as ⁄⁄p <0.01 and statistically significant reversal of OX-A
induced grooming is indicated as ##p <0.01 (ANOVA followed by
Dunnett’s t test).
Finally, to determine if compound 1 caused impairment in mo-
tor coordination when administered orally at 1, 3 and 10 mg/kg or
in combination with ethanol, motor performance in male CD rats
was investigated using an accelerated rotarod apparatus. In order
to differentiate results obtained, zolpidem was included in the
study as positive control. Results obtained (Fig. 6) showed that zol-
pidem reduced ability of rats to remain on rotarod. As expected,
when administered in combination with ethanol the motor impair-
ing effects of zolpidem were potentiated. Conversely, compound 1,
The compound effect was assessed over 1 h on rat grooming as
time and frequency (bouts), starting immediately after OX-A injec-
tion. Compound 1, administered orally 3 h before OX-A injection at
doses of 1, 3 and 10 mg/kg, caused a dose-dependent reduction of
OX-A induced grooming as measured by total time spent grooming
and number of grooming bouts (p <0.01 at 3 and 10 mg/kg po, Fig. 4).
Following these positive results, the hypnotic profile of com-
pound 1 was assessed in the Circadian Time (CT) model in male
CD rats,30 by using telemetric recording of electroencephalogram
25
**
800
**
20
600
400
15
10
**
**
**
**
200
0
5
0
Veh
Veh
Veh
1
3
10
Veh
1
3
10
Compound 1 (mg/kg p.o.)
Compound 1 (mg/kg p.o.)
Figure 4. Effects of compound 1 (1, 3 and 10 mg/kg po) on time spent in grooming (a) and frequency (b) in the OX-A evoked grooming model in the rat.

5566
R. Di Fabio et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5562–5567
Figure 5. Effects of compound 1 (3, 10 and 30 mg/kg po) on sleep latency and total sleep time in the rat CT18 model.
Compound1/Veh
Compound1/EtOH
Zolpidem/Veh
120
100
80
120
100
80
Zolpidem/EtOH
60
* *
60
* *
* *
40
40
20
0
20
0
Veh
1
3
10
Veh
1
3
10
mg/kg p.o.
mg/kg p.o.
Figure 6. Assessment of potential for induction of locomotor deficits by compound 1 and zolpidem and potentiation by ethanol (1 g/kg, ip) using an accelerated rotarod
apparatus.
when given alone or combined with ethanol, failed to cause motor
coordination impairment in the rotarod model in the rat at all dose
tested.
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Data are expressed as mean SEM (n = 7–9). ⁄p <0.01 refer to
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In conclusion, a large body of evidence suggests that the orexin
system plays a key role in the control of arousal and sleep/wake
behavior and that dual OX1 and OX2 receptor antagonists may pro-
vide innovative hypnotics of truly novel mechanism of action.31,32
Compound 1 was identified as a in vitro potent and selective dual
OX1 and OX2 receptor antagonist, able to revert in the rat the
grooming behavior induced by icv administration of OX-A. Phar-
macokinetic analysis and in vivo activity in a pre-clinical sleep
model showed an appropriate profile of compound 1 as a hypnotic
agent, without inducing animal motor impairment at the active
dose. A comparative characterization of compound 1 with respect
to almorexant in animal models of insomnia has recently been
published,33 confirming the excellent in vivo efficacy observed in
house. Based on these characteristics, compound 1 was selected
as a drug candidate and successfully progressed to clinical phase
studies.
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Acknowledgment
Professor William Clegg and Dr. Sophie Dale (University of
Newcastle) are thanked for the GSK funded X-ray crystallographic
analysis of compound 1.

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