IPSU is a novel, potent, selective, orally bioavailable and brain penetrant orexin receptor (OX2R) antagonist with potential to be used for insomnia. It inhibits OX2R with a pKi of 7.85. IPSU binds quickly and reaches equilibrium in assays for binding and/or functionality very quickly. In general, IPSU has a slow rate of equilibrium convergence and a tendency to be relatively unselective in non-equilibrium situations. Every ligand exhibits a selectivity profile when equilibrium is reached, which differs from the non-equilibrium state. The 'dual' antagonists tested have slow kinetics, which implies that the duration of in vitro receptor occupancy might be longer than anticipated. This raises concerns about the accuracy of pharmacokinetic studies measuring these antagonists' brain or plasma levels as indicators of receptor occupancy in vivo.

Physicochemical Properties

Molecular Formula	C23H27N5O2
Molecular Weight	405.50
Exact Mass	405.216
Elemental Analysis	C, 68.13; H, 6.71; N, 17.27; O, 7.89
CAS #	1373765-19-5
Related CAS #	1373765-19-5
PubChem CID	56970858
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Boiling Point	702.0±70.0 °C at 760 mmHg
Flash Point	378.4±35.7 °C
Vapour Pressure	0.0±2.2 mmHg at 25°C
Index of Refraction	1.674
LogP	3.92
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	4
Heavy Atom Count	30
Complexity	608
Defined Atom Stereocenter Count	0
SMILES	O=C1C2(C([H])([H])C([H])([H])N(C3=NC([H])=C([H])C(=N3)OC([H])([H])[H])C([H])([H])C2([H])[H])C([H])([H])C([H])([H])C([H])([H])N1C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12
InChi Key	PCMHOSYCWRRHTG-UHFFFAOYSA-N
InChi Code	InChI=1S/C23H27N5O2/c1-30-20-7-11-24-22(26-20)27-13-9-23(10-14-27)8-4-12-28(21(23)29)16-17-15-25-19-6-3-2-5-18(17)19/h2-3,5-7,11,15,25H,4,8-10,12-14,16H2,1H3
Chemical Name	2-(1H-indol-3-ylmethyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one
Synonyms	IPSU
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	OX2R ( pKi = 7.85 ); OX1R ( pKi = 6.29 )
ln Vitro	The development of medications to treat insomnia is attracted to orexin receptor antagonists as potential targets. IPSU exhibits rapid binding and rapid equilibrium in binding and/or functional assays [2].
ln Vivo	IPSU exhibits high maximal blood exposure, low blood clearance, and high AUC following oral dosage. It displays favorable brain penetration and an acceptable absolute oral bioavailability as well as a brain/blood concentration ratio. When administered to mice during their active phase (with their lights off), IPSU results in increased sleep.This is mainly because it increases NREM sleep. IPSU exhibits a rapid onset of action, with a discernible rise in the overall amount of time spent sleeping in the hour following dosage. After the effect wears off, which is about 4-5 hours, each hour of sleep is the same as it is on a vehicle day [1].
Enzyme Assay	One concentration of radioligand and six concentrations of competitors (unlabeled ligands such as BBAC, almorexant, SB-649868, suvorexant, filorexant, or IPSU) are used in competition experiments. Membranes (150 μL/well) are filled to capacity with 4.6 nM [3H]-BBAC and different concentrations of unlabeled ligand (0.1 nM–10 μM) in 50 μL/well of assay buffer, for a total volume of 250 μL/well. At room temperature, the amount of [3H]-BBAC bound to receptors is measured at various time intervals (from 15 min to 4 h), and the process is concluded by liquid scintillation counting and fast vacuum filtration[2].
Animal Protocol	Mice: C57Bl/6 mice that are allowed to roam freely and have permanent electrode implantations are accustomed to the experiment boxes and have unlimited access to food and drink. Just before lights out and recording begins, the test compounds (IPSU) or vehicle are given one at a time as a suspension in 0.5% methylcellulose. Infrared sensors installed in the box's roof record movement. To categorize 10 s epochs into wake, NREM sleep, and REM sleep, EEG/EMG signals and motility data are utilized. By applying and recording the vehicle the day before compound (IPSU) dosing, each animal acted as its own control[1].
References	[1]. Identification of a novel series of orexin receptor antagonists with a distinct effect on sleeparchitecture for the treatment of insomnia. J Med Chem. 2013 Oct 10;56(19):7590-607. [2]. Kinetic properties of "dual" orexin receptor antagonists at OX1R and OX2R orexin receptors. Front Neurosci. 2013 Dec 3;7:230. [3]. Distinct effects of IPSU and suvorexant on mouse sleep architecture.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 30mg/mL (~74.0 mM) Water: N/A Ethanol: N/A

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.4661 mL	12.3305 mL	24.6609 mL
	5 mM	0.4932 mL	2.4661 mL	4.9322 mL
	10 mM	0.2466 mL	1.2330 mL	2.4661 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.