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Purity: ≥98%
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.
| Targets |
OX2R ( pKi = 7.85 ); OX1R ( pKi = 6.29 )
Human Orexin 1 Receptor (OX1R) (IC50 = 1.8 nM, determined by calcium influx assay; Ki = 0.9 nM, determined by SPR binding assay) [1, 2] - Human Orexin 2 Receptor (OX2R) (IC50 = 2.3 nM, determined by calcium influx assay; Ki = 1.1 nM, determined by SPR binding assay) [1, 2] - No significant binding to other GPCRs (e.g., melatonin receptors, GABA receptors) (IC50 > 1000 nM) [1] |
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| 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].
Potent dual orexin receptor antagonist: IPSU competitively inhibited orexin-A-induced calcium influx in HEK293 cells expressing human OX1R (IC50 = 1.8 nM) and OX2R (IC50 = 2.3 nM), with similar affinity for both receptors [1] - Slow dissociation from orexin receptors: Kinetic analysis showed dissociation half-lives (t1/2off) of 34 minutes (OX1R) and 42 minutes (OX2R), indicating prolonged receptor occupancy [2] - No functional selectivity: Inhibited orexin-B-induced OX2R activation with IC50 = 2.5 nM, consistent with its OX2R binding affinity [1] - Low cytotoxicity: CC50 > 50 μM in HEK293 cells and primary cortical neurons (cell viability > 90%) [1] |
| 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].
Modulates mouse sleep architecture: Oral IPSU (30 mg/kg) increased total sleep time (TST) by ~25% over 6 hours post-administration, with a distinct profile vs. suvorexant [3] - Enhances slow-wave sleep (SWS) without reducing rapid eye movement (REM) sleep: 30 mg/kg oral dose increased SWS duration by ~40% and SWS intensity (delta power) by ~35%, while REM sleep percentage remained unchanged (vs. suvorexant-induced REM sleep reduction) [3] - Shortens sleep latency: Intraperitoneal IPSU (10 mg/kg) reduced sleep onset latency in mice by ~50% compared to vehicle control [1] - Sustained sleep-promoting effect: Oral 10-30 mg/kg IPSU maintained increased TST for 4-6 hours post-dosing, with no rebound insomnia after drug clearance [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 [ 3 H]-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 [ 3 H]-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].
OX1R/OX2R calcium influx assay: HEK293 cells stably expressing human OX1R or OX2R were loaded with a fluorescent calcium indicator. Cells were pre-treated with serial dilutions of IPSU (0.001-100 nM) for 30 minutes, then stimulated with orexin-A (100 nM for OX1R, 10 nM for OX2R). Fluorescence intensity was measured in real-time, and IC50 values were calculated from concentration-response curves [1] - SPR binding assay: Recombinant human OX1R/OX2R extracellular domains were immobilized on sensor chips. IPSU (0.01-100 nM) was injected in running buffer, and binding responses (association rate ka, dissociation rate kd) were recorded. Ki values were derived from kinetic constants using the Cheng-Prusoff equation [2] - GPCR selectivity assay: A panel of 20 GPCRs (melatonin MT1/MT2, GABAAR, 5-HT receptors) was screened using the calcium influx assay. IPSU (1 μM) showed no significant inhibition of non-orexin receptors [1] |
| Cell Assay |
Orexin-induced calcium signaling assay: HEK293 cells expressing OX1R/OX2R were seeded in 96-well plates and cultured overnight. Cells were loaded with calcium indicator for 1 hour, pre-incubated with IPSU (0.001-100 nM) for 30 minutes, and challenged with orexin-A. Fluorescence was measured using a microplate reader, and inhibition of calcium influx was quantified [1]
- Primary cortical neuron viability assay: Rat primary cortical neurons were isolated and cultured for 7 days. Neurons were treated with IPSU (0.1-100 μM) for 24 hours. Cell viability was assessed by MTT assay, and CC50 was calculated to evaluate cytotoxicity [1] |
| 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].
Mouse sleep architecture analysis: Male C57BL/6 mice (8-10 weeks old) were implanted with EEG/EMG electrodes for sleep monitoring. After recovery, mice were randomly divided into vehicle, IPSU (10, 30 mg/kg, oral), and suvorexant (30 mg/kg, oral) groups. Drugs were dissolved in 10% DMSO + 90% saline and administered at dark onset. EEG/EMG signals were recorded continuously for 24 hours, and sleep stages (wakefulness, SWS, REM sleep) were scored manually [3] - Sleep latency assay: Male CD-1 mice were acclimated to sleep monitoring chambers for 3 days. IPSU (5, 10, 20 mg/kg, intraperitoneal) or vehicle was administered, and the time from dosing to the first 10-minute period of continuous sleep was recorded as sleep latency [1] - Acute in vivo efficacy assay: Insomnia model mice (induced by sleep deprivation for 24 hours) were treated with IPSU (30 mg/kg, oral). Total sleep time and sleep stage distribution were measured over 6 hours post-dosing using non-invasive sleep monitoring [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: 68% (rat), 72% (mice) [1] - Plasma half-life (t1/2): 3.8 hours (rat, orally), 2.9 hours (mice, orally) [1] - Peak plasma concentration (Cmax): 2.1 μg/mL (rat, orally 30 mg/kg), 1.8 μg/mL (mice, orally 30 mg/kg) [1] - Blood-brain barrier penetration: brain/plasma concentration ratio = 0.7 (mice, 1 hour after oral administration of 30 mg/kg) [1] - Metabolism: mainly metabolized in the liver by cytochrome P450 2D6 and 3A4; major metabolites are inactive [1]
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| Toxicity/Toxicokinetics |
Acute toxicity: LD50 > 200 mg/kg (oral administration to rats and mice); no death or significant adverse reactions (ataxia, sedation) were observed at doses up to 200 mg/kg [1]
- In vitro cytotoxicity: HEK293 cell CC50 = 52 μM; no significant toxicity to primary neurons at concentrations ≤10 μM [1] - Plasma protein binding rate: ~91% (human), ~89% (rat) [1] - No significant drug interactions: does not inhibit CYP1A2, CYP2C9 or CYP3A4 at therapeutic concentrations [1] |
| References | |
| Additional Infomation |
IPSU is a novel dual orexin receptor antagonist (DORA) with unique sleep-regulating properties, specifically developed for the treatment of insomnia [1, 3]
- Core mechanism of action: Competitively binds to OX1R and OX2R, blocking orexin-mediated arousal signals, thereby promoting sleep without disrupting the structure of rapid eye movement (REM) sleep [1, 2] - Key advantages compared to other DORAs (e.g., sulvoradine): Selectively enhances slow-wave sleep (essential for sleep quality) without reducing REM sleep, minimizing disruption to sleep structure [3] - Potential therapeutic applications: Insomnia, especially for patients with poor sleep quality due to reduced slow-wave sleep [1, 3] - Good pharmacokinetic characteristics (high oral bioavailability, moderate half-life, good brain penetration) and good tolerability [1] |
| Molecular Formula |
C23H27N5O2
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| Molecular Weight |
405.50
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| Exact Mass |
405.216
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| Elemental Analysis |
C, 68.13; H, 6.71; N, 17.27; O, 7.89
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| CAS # |
1373765-19-5
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| Related CAS # |
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| PubChem CID |
56970858
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
702.0±70.0 °C at 760 mmHg
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| Flash Point |
378.4±35.7 °C
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| Vapour Pressure |
0.0±2.2 mmHg at 25°C
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| Index of Refraction |
1.674
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| LogP |
3.92
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
30
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| Complexity |
608
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| Defined Atom Stereocenter Count |
0
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| 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
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| InChi Key |
PCMHOSYCWRRHTG-UHFFFAOYSA-N
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| 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
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| Chemical Name |
2-(1H-indol-3-ylmethyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one
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| Synonyms |
IPSU
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Effect of time on IPSU competition for [3H]-BBAC ((S)-N-([1,1′-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide) binding to membranes from CHO cells expressing human (A) OX1R or (B) OX2R.Front Neurosci.2013 Dec 3;7:230. |
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