H. Aissaoui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5729–5733
5733
shown that almorexant (or ACT-078573), a dual OX1R/OX2R antag-
onist that crosses the blood–brain barrier decreases alertness and
increases non-REM and REM sleep in a dose-dependent manner
when administered at the beginning of the dark active phase in
rats.5
markers of both non-REM and REM sleep in rats following oral
administration (at 300 mg/kg).
Acknowledgments
Tested under the same conditions (compound given orally at
the beginning of the dark active phase), compound 47 was the only
representative of this series that showed signs of activity on sleep
and wake stages. It was tested at 100 and 300 mg/kg po and
induced an apparent dose-dependent decrease in home cage activ-
ity (P < 0.0001, one-way ANOVA; Fig. 1A). The effect was significant
at 300 mg/kg (P < 0.0001, Bonferroni post hoc analysis) when com-
pared to vehicle control. We also observed a decrease in time spent
in active wake over the 12 h night period which was not significant
((P > 0.05, one-way ANOVA; Fig. 1B). Time spent in non-REM and
REM sleep were both increased in a dose-dependent manner over
the 12 h night period following administration (P = 0.0318 for non-
REM sleep and P = 0.0263 for REM sleep, one-way ANOVA). The
effect was significant at 300 mg/kg (P < 0.05, Bonferroni post hoc
analysis, Fig. 1C and D) when compared to vehicle control. Body
temperature was not significantly affected at any dose (P > 0.05,
one-way ANOVA).
BBB penetration was evaluated at two different time points
(1 and 3 h) following oral administration of 100 mg/kg. None of
those compounds (47, 48, and 50) showed substantial total brain
penetration. Concentration in brain was very low, in the range of
10–20 ng/g. Compound 47 showed the highest brain concentration
observed for this class of compounds, reaching 66 ng/g 3 h follow-
ing administration.
These total brain concentrations were sufficient to induce elec-
trophysiological signs of non-REM and REM sleep when compound
47 was administered at three times higher dosage (300 mg/kg po,
Fig. 1). Time spent in non-REM and REM sleep increased in physi-
ological proportion: over the 12 h night period, non-REM sleep rep-
resented 86% of the total sleep time for vehicle-treated animals and
83% for animal treated with compound 47 (at 300 mg/kg) and REM
sleep represented 14% of the total sleep time for the vehicle group
and 17% for the compound 47 group. These pharmacological effects
were achieved at total brain concentrations which are likely to be
lower than those needed for almorexant to elicit similar effects on
non-REM and REM sleep.5 Characteristic physicochemical parame-
ters (such as protein binding in plasma and brain or partitioning
into lipid and aqueous compartments) are likely to contribute to
differences in free extracellular concentrations reached in the brain
for compounds of different classes.
The authors thank K. Menyhart and C. Müller for carrying out
the FLIPR assays, S. Delahaye, A. Weigel and C. Gnerre for perform-
ing the pharmacokinetic experiments, J. Hazemann, J. Gabillet, V.
Egger, S. Paput, P. Rebmann, U. Fusco, D. Trachsel, D. Lehman, R.
Haener, C. Fischer for expert technical support, T. Weller, C.
Hubschwerlen, and M. Clozel for constantly supporting the project
and Prof. H. Ramuz for stimulating discussions.
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
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6. FLIPR assay. Chinese hamster ovary (CHO) cells expressing the human orexin
receptors (hOX1R or hOX2R) were seeded into 96-well plates and incubated at
37 °C in 5% CO2 with the cytoplasmic fluorescent calcium indicator fluo-3 AM
(Molecular Probes). After washing the cells, intercellular Ca2+ mobilization was
monitored as
a change in cell fluorescence intensity by FLIPR (Molecular
Devices). Differing concentrations of orexin antagonists were added to the
plates prior to addition of orexin A. For each antagonist, IC50 (the concentration
of compound needed to inhibit 50% of the agonistic response) is calculated.
7. Heffernan, G. D. U.S. Patent 6,562,979, 2003.
8. In vivo experiments were done on male Wistar rats. Rats are maintained on a 12-h
light/12-h dark cycle.For pharmacokinetic studies, the formulation used for iv
dosing was
a 5%/95% (v/v) mixture of DMSO and 30/70 hydroxypropyl-b-
cyclodextrine: sterile water and for po dosing was 100% PEG-400. Serial blood
samples were collected into containers with Na-EDTA as anti-coagulant at various
time-points and blood centrifuged to yield plasma. These studies used three
animals (po) and one or two animals (iv).For BBB penetration studies, we
measured the concentration of the OX receptor antagonist in plasma, CSF and
brain sampled 1 and 3 h following oral administration. Plasma and brain are
collected from the same animal at the same time ( 5 min). Blood is sampled from
the vena cava caudalis intocontainers with EDTA as anticoagulant and centrifuged
to yield plasma. CSF is sampled from the cisterna magna with careful attention to
avoid blood contamination. Brain is sampled after cardiac perfusion of 10 mL NaCl
0.9% and homogenized into 1 volume of cold phosphate buffer (pH 7.4).
For pharmacodynamic sleep studies, animals were implanted with miniature
radiotelemetric implants (Data Sciences International) under general anesthesia.
Those implants consist of two pair of differential leads; one pair for cranial
placement to record the electroencephalogram (EEG) and one pair placed in either
side of the muscular neck to record the electromyogram (EMG). This technology
allows stress-free acquisition of vigilance and sleep stages, spontaneous activity
and body temperature from freely moving rats in their home cage environment.
Compounds were administrated orally at the beginning of the night dark cycle and
formulated in100% PEG-400. Ineach experiment, we used groups of7 or 8 rats, ina
crossover design, with at least 4-day washout periods separating consecutive
administrations.
Despite such differences, our investigations with these N-gly-
cine-sulfonamides confirm the unique characteristics of dual orex-
in receptor antagonists. By transiently blocking the effects of
endogenous orexin-peptides, those antagonists mimic the physio-
logical state of sleep occurring when orexinergic neurons stop
firing.4
In conclusion, we have described a novel series of potent dual
orexin antagonists based on a N-glycine sulfonamide scaffold.
SAR studies based on screening hit 1 have been outlined. A combi-
nation of a 2-chloro-5-dimethylamino-phenyl or a 6-chloro-3-
methyl-benzisothiazole moiety in the anilinic region with polar
heterocyclic systems in the tertiary amide region led to the most
potent dual orexin receptor antagonists found so far in the litera-
ture. Generally, the series exhibited good oral bioavailability but
was associated with low BBB penetration. We further demon-
strated the ability of compound 47 to increase electrophysiological