OX₂ Receptor

Recently, the X-ray crystal structure of the human OX2R bound to a dual orexin receptor antagonist suvorexant was solved at 2.5 Å. By use of this X-ray structure, the binding mode of Orexin 2 Receptor Agonist /26 with OX2R was examined by molecular-docking calculations (see Supporting Information). The resulting binding mode of Orexin 2 Receptor Agonist /26 (Figure 3) suggested that the dimethylcarbamoyl group on Orexin 2 Receptor Agonist /26 was located deep in the ligand-binding site of OX2R, and its carbonyl group formed a hydrogen bond with N324 (transmebrane helix 5, TM5). Compound 26 also used a nitrogen atom of the ethylenediamine moiety to form an additional hydrogen bond with the main-chain carbonyl group of C210 (TM3). The biphenyl group of 26 was located in the hydrophobic pocket consisting of V138 (TM3), F227 (TM5), Y317 (TM6), I320 (TM6), and V353 (TM7) to make hydrophobic interactions. The sulfonylamide group of 26 was in proximity to T111 (TM2), H350 (TM7), and Y354 (TM7) of OX2R (see Supporting Information’ Figure 2). Interestingly, 26 directed two oxygens of sulfonylamide group to hydroxyl groups of T111 (TM2) and Y354 (TM7) and imidazole ring of H350 (TM7), respectively. The sulfonylamide group of 26 may form hydrogen bonds in the structural changes associated with receptor activation. This may explain that the carboxyamide derivative 29 was inactive because the direction of oxygen was different between carboxyamide and sulfonylamide groups. T111 (TM2) of OX2R is mutated to Ser in OX1R. This mutation might be related to high selectivity for OX2R of compounds in Table 2. The benzene groups in the middle and terminal portions of 26 also formed hydrophobic interactions with V114 (TM2), W120 (ECL1), and I130 (TM3) and with Y343 (TM7) and F346 (TM7). The neighborhood of the B-ring of compound 26 was given in Supporting Information’s Figure 3. This also may explain that compounds 23, 26, and 28 with a substituent group at the 3-position possessed potent activity because we could see large space in the direction of 3-position to accommodate the substituent group. Figure 4 compares the binding modes of suvorexant and 26 with OX2R. As reported in the literature, suvorexant adopted a π-stacked horseshoe-like conformation to direct its seven-membered ring to TM5 and TM6 of OX2R. This binding mode was believed to be responsible for the OX2R antagonist activity of suvorexant, as the seven-membered ring seems to inhibit inward movements of TM5 and TM6 relative to the rest of the TM bundle, which may be a general trigger for GPCR activation. In contrast, 26 binds to OX2R with an extended conformation, and we detected some spatial allowance between 26 and TM5 and TM6 of OX2R. Therefore, we could consider that the binding of 26 to OX2R may induce the inward movements of TM5 and TM6, which would allow 26 to have OX2R agonist activity.[1]

---

In vitro activity: Orexin 2 Receptor Agonist is a potent and selective agonist of the Orexin2 receptor; it is selective only for OX2R (the EC50 ratio between OX1R and OX2R is 70). The neuropeptide family known as orexins controls sleep and wakefulness by binding to orexin receptors 1 (OX1R) and 2 (OX2R), two G-protein-coupled receptors. Orexin receptor agonists, particularly OX2R agonists, may prove beneficial for the mechanistic treatment of narcolepsy/cataplexy, according to genetic and pharmacologic evidence. We hereby announce the identification of 4-methoxy-N,N-dimethyl-3-[N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl)sulfamoyl] as a potent (EC50 on OX2R is 0.023 μM) and OX2R-selective (OX1R/OX2R EC50 ratio is 70) agonist. -(1,1-biphenyl)-3-carboxamide 26/Orexin 2 Receptor Agonist .

Orexin receptors 1 (OX1R) and 2 (OX2R) are two G-protein-coupled receptors that are the targets of the orexin family of neuropeptides, which controls sleep and wakefulness. Mechanistic therapy of the sleep disorder narcolepsy/cataplexy may benefit from the use of orexin receptor agonists, particularly OX2R agonist, according to genetic and pharmacologic evidence. The discovery of 4′-methoxy-N,N-dimethyl-3′-[N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl)sulfamoyl] is reported herein as a potent (EC50 on OX2R is 0.023 μM) and OX2R-selective (OX1R/OX2R EC50 ratio is 70) agonist. -(Biphenyl-1,1′) 26-carboxamide -3.

---

In Table 2, the most potent compound was 3-methyl derivative 26/Orexin 2 Receptor Agonist. Meanwhile, the most selective compound for OX2R was 3-methoxy derivative 23. However, compounds 11 and 22–28 in Table 2 were barely soluble in water, which led to difficulty in confirming in vivo activity, although every compound showed satisfactory activities and potencies in vitro. Therefore, we introduced the 2-dimethylamino group on the B-ring to give more polar derivative 30 (Figure 5) and then converted 30 into dihydrochloride salt 31 (analog of Orexin 2 Receptor Agonist), which could be dissolved in saline at 1.3 M. To evaluate the pharmacological effect of 31 in vivo, we administered it intracerebroventricularly in the light phase and observed the effect on sleep/wake states by recording EEG/EMG in C57BL/6J mice. 31 promoted wakefulness in a dose-dependent manner (Figure 6). 260 nmol of 31 increased wake time to 53 min, to a similar degree reported with 3.0 nmol of orexin A (58 min). In contrast, 31 did not increase wakefulness in OX1R/OX2R double knockout (DKO) mice, demonstrating that the effect of 31 requires orexin receptors as expected. The intraperitorial injection of 31 afforded similar effects [1].

In vitro binding assay [1]
Evaluations of binding affinity to human OX1R or OX2R were carried out according to the literature. Cell membrane homogenates of human OX1R expressing CHO-K1 cells or human OX2R expressing HEK-293 cells (20 µg protein) were incubated for 60 min at 22 °C with 0.1 nM [125I]orexin-A in the absence or presence of the test compound in a buffer containing 25 mM Hepes/NaOH (pH 7.4), 0.5 mM EDTA, 2.5 mM CaCl2 and 2.5 mM MgCl2 (OX1R) or 25 mM Hepes/NaOH (pH 7.4), 2.5 mM CaCl2, 1 mM MgCl2 and 0.5% BSA (OX2R). Nonspecific binding was determined in the presence of 1 µM SB-334867 (OX1R) or orexin-B (OX2R). Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with an ice-cold buffer containing 50 mM Tris-HCl and 150 mM NaCl (OX1R) or 50 mM Tris-HCl (OX2R) using a 96-sample cell harvester. The filters were dried then counted for radioactivity in a scintillation counter using a scintillation cocktail. The results were expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound was orexin-A (OX1R) or orexin-B (OX2R), which was tested in each experiment at several concentrations to obtain a competition curve.

---

Modeling of binding mode of 26/Orexin 2 Receptor Agonist  with OX2R [1]
All calculations were performed using the Schrödinger suite 2013-2. The 2D structure of 26/Orexin 2 Receptor Agonist  was first converted into 3D structures using the LigPrep2.7 program. The protonation state of 26 was predicted using Epik2.5 program4 . Then, the conformational search was carried out using the ConfGen2.5 program5 , and the resulting conformers were used in the following docking calculations. The X-ray structure of the OX2R complexed with suvorexant (PDB ID: 4S0V) was used as a receptor for docking6 . The structure was prepared using the Protein Preparation Wizard in Maestro9.5. The missing atoms for side chains were compensated for using Prime3.3 program 7 . Finally, the structure was minimized using force-field OPLS 2005. We removed H2O molecules before docking calculations, except for HOH4021 and HOH4025 located in the ligand-binding site of OX2R. As HOH4021 and HOH4025 forms water-mediated hydrogen bond networks between side chains of N324 and R328 of OX2R and between H350 of OX2R and suvorexant, respectively, these water molecules seem to be important for structural stability of ligand-binding site or ligand-recognition. The docking calculation of 26 against OX2R was performed using the IFD 2006 protocol as implemented in Schrödinger suite 2013-28 . Box center for the “Receptor Grid Generation” protocol were set to a centroid of suvorexant binding to OX2R. Initial Glide docking was performed using van der Waal radius scaling of 0.8 for both protein and ligand, and the maximum number of poses per conformer was set to 2. Prime was used to refine residues within 5.0 Å of ligand poses. Re-docking was performed in Glide using standard settings. The generated poses were ranked according to IFDScore to select the top 10% docked complexes. The free energies of these complexes (Gcomplexs) were then estimated by the molecular mechanics generalized Born surface area (MM-GBSA) method using Prime program, and we finally selected a docked complex with the lowest Gcomplex as the interaction model. In order to test our procedure, we applied it for the suvorexant-OX2R complex. The result is shown in Figure 1. The resulting model, i.e., the top-ranked pose, reproduced all interactions between suvorexant and OX2R and the water-mediated hydrogen bonds between suvorexant and OX2R through HOH4025 observed in the crystal structure. The positional and conformational root mean square deviations (RMSDs) were 1.30 and 0.99 Å, respectively. These results suggested that our procedure is appropriate for producing reliable interaction model between 26 and OX2R.

---

Orexin 2 Receptor Agonist is an effective and specific Orexin2 receptor agonist that has an EC50 of 0.023 μM on OX2R. It is selective only for OX2R (EC50 ratio between OX1R and OX2R is 70). Orexin receptors 1 (OX1R) and 2 (OX2R) are two G-protein-coupled receptors that are the targets of the orexin family of neuropeptides, which controls sleep and wakefulness.

Cell lines and cell culture [1]
After 48 h post transfection using FuGENE 6 according to the manufacture’s recommendations, retrovirus fluid was harvested. For the generation of receptor expressing cells, CHO (Chinese Hamster Ovary) K1 cells were infected with a retroviral supernatant. After 24 h post infection, infected cells were selected with 10 µg/mL of puromycin. To generate stable indicator cell lines, constitutively receptor expressing CHO-K1 cell lines were transfected with NFAT-response luciferase reporter plasmid (pNFAT-TA-Luc) together with pSV2neo which carried antibiotic resistance gene, by using FuGENE 6. After 24 h, the transfected cells were selected with 1 mg/mL G418. G418 resistance cells were seeded in Dulbecco’s Modified Eagle Medium containing 5% fetal bovine serum, 1 x non-essential amino acids, 100 U/mL penicillin, and 100 µg/mL streptomycin, 10 µg/mL of puromycin, 1 mg/mL G418 and isolated a single clone. Cell lines co-transfected with human OX1R or OX2R cDNA and an NFAT-response luciferase reporter were generated. These cell lines were maintained in above medium.

---

Calcium mobilization assay [1]
CHO-K1 cells stably expressing human OX1R (CHOOX1R) or OX2R (CHOOX2R) were seeded in a 96-well-plate (10,000 cells/well) and incubated with 5% FBS/DMEM at 37 °C for 48 h. Then, cells were loaded with 5µM fluorescent calcium indicator Fura 2-AM in Hanks balanced salt solution (HBSS: GIBCO) including 20 mM HEPES, 2.5 mM Probenecid, 5% CremophorEL, and 0.1% BSA at 37 °C for 1 h. The cells were washed once and added with 75 µL of HBSS buffer. Then, cells were treated with 25 µL of various concentrations of test compounds or orexin A (OXA: diluted in PBS/0.1% BSA). The increase of the intracellular Ca2+ concentration after the application of test compounds or OXA was moni- S19 tored by FDSS 3000 system. The EC50 value of each compounds was calculated by Graph Pad Prism 5J (MDF)

---

Luciferase assay [1]
CHOOX1R or CHOOX2R cells were seeded (10,000 cells/well) in a 96-well-plate and grown to confluence for 24 h. Cells were treated with indicated test compounds or OXA. OXA (diluted in PBS/0.1%BSA) was used positive control. Six hours later, the medium was removed by suction. The cell monolayer was lysed in 50 µL of Steay-Glo luciferase reagent and luciferase activity was determined by ARVO x5. The Steady-Glo reagent was diluted 10-fold in 25 mM Tris (pH 7.5)/10% Glycerol/1% triton X-100 as Working solution.

In vivo experiments [1]
Experimental mice were individually housed and kept on a 12 h: 12 h light: dark schedule at an ambient temperature of 23 ± 1 °C and under specific pathogen-free conditions. 8- to 12-wk-old male C57BL/6J mice were anesthetized with isoflurane (4%) and remained anesthetized with isoflurane (2%), and then were surgically installed with a guide cannula into left lateral ventricle for intracerebroventricular (ICV) administration and were simultaneously implanted with electroencephalograph/electromyography (EEG/EMG) electrodes. Two EEG electrodes were implanted over the right hemisphere coordinates, and two EMG electrodes were implanted over both sides of the trapezius muscle. All mice were recovered for at least 1 week and were habituated with the recording tether for 1 week before experiments. After habituation, baselines of EEG/EMG were recorded for 1 day, and at ZT6 on the next day, ICV administrations were performed under transient anesthesia with isoflurane through a cannula by using a syringe pump and an oil-filled syringe. An injection volume of 5.0 µL was delivered over a 10 min period to each mouse. Mice were immediately returned to home cages and EEG/EMG recorded. After saline administration, we administrated 32, 130, 260 nmol of 31 (water soluble analog of Orexin 2 Receptor Agonist ) on every other day. EEG/EMG signals were analyzed by using custom semi-automated software. We assessed the wake time for 2 h after ICV administration. All values are expressed as means ± SEM. In wild-type mice, data were analyzed with a repeated one-way ANOVA, and then individual groups were compared with a Bonferroni test. Differences were considered significant when P <0.05, P <0.001. In OX1R/OX2R DKO mice, data were analyzed with a paired t-test.

Barely soluble in water [1]

[1]. Design and Synthesis of Non-Peptide, Selective Orexin Receptor 2 Agonists. J Med Chem. 2015 Oct 22;58(20):7931-7.

Researchers paid attention to the sulfonamide group of hit compound 1 and synthesized more than 1000 compounds. Those compounds were tested against a CHO cell line expressing the human orexin receptor using the NFAT-luciferase assay and the calcium influx assay. We found the first lead compound 5, then focused on the transformation of the A-ring and the B-ring in 5. As a result, the first, potent, and selective agonist 26/Orexin 2 Receptor Agonist for OX2R was discovered. The conversion from the sulfonamide group in 26/Orexin 2 Receptor Agonist to the carbonylamide group and the evaluation of the obtained 29 suggested that the sulfonamide moiety is one of the most important structural features for the OX2R agonistic activity. In addition, we also developed the dimethylamino derivative 30 and its dihydrochloride salt, which could be dissolved in saline at 1.3 M. The in vivo assay of the dihydrochloride salt showed a definite wake-promoting effect. These results would be helpful for further design of OXR agonists.[1]

C32H34N4O5S

586.70

586.224

C, 65.51; H, 5.84; N, 9.55; O, 13.63; S, 5.46

1796565-52-0

91810287

White to off-white solid powder

1.3±0.1 g/cm3

1.629

3.4

3

7

11

42

986

0

S(C1C(=CC=C(C2C=CC=C(C(N(C)C)=O)C=2)C=1)OC)(NC1C=CC=C(C=1)NCCNC(C1C=CC=C(C)C=1)=O)(=O)=O

RHLMXWCISNJNDH-UHFFFAOYSA-N

InChI=1S/C32H34N4O5S/c1-22-8-5-10-25(18-22)31(37)34-17-16-33-27-12-7-13-28(21-27)35-42(39,40)30-20-24(14-15-29(30)41-4)23-9-6-11-26(19-23)32(38)36(2)3/h5-15,18-21,33,35H,16-17H2,1-4H3,(H,34,37)

N-[2-[3-[[5-[3-(dimethylcarbamoyl)phenyl]-2-methoxyphenyl]sulfonylamino]anilino]ethyl]-3-methylbenzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)