Cell experiment:

Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL Penicillin, 100 μg/mL Streptomycin and 10 % heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells / well into 384-well black clear bottom sterile plates. The seeded plates are incubated overnight at 37℃ in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1 :1), diluted in HBSS containing 0.1 % bovine serum albumin (BSA), NaHCO3: 0.375g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM. Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1 % bovine serum albumin (BSA), NaHCO3: 0.375g/L and 20 mM HEPES. On the day of the assay, 50 μL of staining buffer (HBSS containing 1 % FCS, 20 mM HEPES, NaHCO3: 0.375g/L, 5 mM probenecid and 3 μM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37℃ in 5% CO2 followed by equilibration at RT for 30 min before measurement. Within the Fluorescent Imaging Plate Reader, antagonists are added to the plate in a volume of 10 μL/well, incubated for 120 min and finally 10 μL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by an approximate EC70 (for example 5 nM) of orexin-A with vehicle in place of antagonist. The IC50 value is determined[1].

Nemorexant (ACT-541468) is a potent orexin receptor antagonist extracted from patent WO2015083094A1, compound example 7, has IC50s of 2 nM and 3 nM for Ox1 receptor and Ox2 receptor, respectively.

Nemorexant (Compound example 7) is a orexin receptor antagonist with IC50s of 2 nM and 3 nM for Ox1 receptor and Ox2 receptor, respectively[1].

[1]. BOSS, Christoph, et al. USE OF BENZIMIDAZOLE-PROLINE DERIVATIVES. WO2015083094A1.