LSN2424100 (LSN-2424100) is a novel and potent orexin-2 receptor antagonist with antidepressant-like activity.
Physicochemical Properties
| Exact Mass | 407.11037 |
| Elemental Analysis | C, 64.85; H, 4.45; F, 4.66; N, 10.31; O, 7.85; S, 7.87 |
| PubChem CID | 121231398 |
| Appearance | Typically exists as solid at room temperature |
| LogP | 4 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 6 |
| Heavy Atom Count | 29 |
| Complexity | 612 |
| Defined Atom Stereocenter Count | 0 |
| InChi Key | PCDMCECXKAELMI-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C22H18FN3O2S/c23-18-10-12-19(13-11-18)29(27,28)26(16-22-24-14-15-25-22)21-9-5-4-8-20(21)17-6-2-1-3-7-17/h1-15H,16H2,(H,24,25) |
| Chemical Name | 4-fluoro-N-(1H-imidazol-2-ylmethyl)-N-(2-phenylphenyl)benzenesulfonamide |
| Synonyms | LSN2424100; CHEMBL3597951; 4-fluoro-N-(1H-imidazol-2-ylmethyl)-N-(2-phenylphenyl)benzene-1-sulfonamide; GTPL9121; BDBM50108627; Q27081879; 4-fluoro-N-(1H-imidazol-2-ylmethyl)-N-(2-phenylphenyl)benzenesulfonamide |
| 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 | Orexin-2 (OX2)/hypocretin-2 receptor |
| ln Vitro | In this work, researchers describe a novel, potent and selective orexin-2 (OX2)/hypocretin-2 receptor antagonist with in vivo activity in an animal model predictive of antidepressant-like efficacy. N-biphenyl-2-yl-4-fluoro-N-(1H-imidazol-2-ylmethyl) benzenesulfonamide HCl (LSN2424100) binds with high affinity to recombinant human OX2 receptors (Ki = 4.5 nM), and selectivity over OX1 receptors (Ki = 393 nM). LSN2424100 inhibited OXA-stimulated intracellular calcium release in HEK293 cells expressing human and rat OX2 receptors (Kb = 0.44 and 0.83 nM, respectively) preferentially over cells expressing human and rat OX1 (Kb = 90 and 175 nM, respectively)[1]. |
| ln Vivo | LSN2424100 exhibits good exposure in Sprague-Dawley rats after IP, but not PO, administration of a 30 mg/kg dose (AUC0-6 h = 1300 and 269 ng(*)h/mL, respectively). After IP administration in rats and mice, LSN2424100 produces dose-dependent antidepressant-like activity in the delayed-reinforcement of low-rate (DRL) assay, a model predictive of antidepressant-like efficacy. Efficacy in the DRL model was lost in mice lacking OX2, but not OX1 receptors, confirming OX2-specific activity. Importantly, antidepressant-like efficacy of the tricyclic antidepressant, imipramine, was maintained in both OX1 and OX2 receptor knock-out mice. In conclusion, the novel OX2 receptor antagonist, LSN2424100, is a valuable tool compound that can be used to explore the role of OX2 receptor-mediated signaling in mood disorders[1]. |
| Enzyme Assay |
Radioligand binding[1] Recombinant human OX1 or OX2 receptors were stably expressed in HEK293 cells and grown in DMEM/F-12 (3:1) supplemented with 5% FBS, 20 mM HEPES, 100 ug/ml Penn/Strep, and 500 ug/ml geneticin. Briefly, the membranes were isolated by homogenizing cell pellet in 30 ml (w/v) 50 mM Tris buffer (pH 7.4) containing Roche Complete EDTA free protease tablets. Membranes were incubated with ~0.25 nM [125I]-Orexin A for 90 min at 22°C in polystyrene 96-deep well plates. All binding studies were conducted at a final volume of 200 μl. The assay buffer contained 25 mM HEPES, 2.5 mM CaCl2, 1.0 mM MgCl2, 0.5% BSA, and 0.125% BSA (pH 7.4). To generate binding affinity (Ki), 11 different compound concentrations were incubated with ~15 μg of OX1 membrane or ~50 μg of OX2 membrane in the presence of [125I]-Orexin A. Compounds were solubilized to make a 10 mM stock in DMSO then diluted to 40 μM by placing 4 μl into 996 μl binding assay buffer. Then 300 μl of this solution was placed in column B on a Nunc polypropylene 96-well plate. Serial dilutions were then made using a Biomek 2000 from a starting concentration of 40 μM. Non-specific binding was determined in these experiments using 10 μM SB-334867 and LSN2158312 for OX1 and OX2, respectively. All binding was terminated by rapid filtration using a TOMTEC 96-well cell harvester through GF/A filters that had been presoaked with 0.3% polyethyleneimine. The filters were washed with 5.0 ml ice-cold 50 mM Tris buffer (pH 7.4) and air-dried overnight. The dried filters were treated with MeltiLex A melt-on scintillator sheets, and the radioactivity retained on the filters counted using a Wallac 1205 Betaplate scintillation counter. Protein concentrations were measured using Coomassie Protein Plus Assay Reagent and serum albumin standards. Ki-values from displacement of [125I]-orexin-A binding were calculated based on 11-point dilution curves using ActivityBase templates. Reported values are shown as a mean ± the standard error of the mean (s.e.m.). |
| Cell Assay |
Intracellular calcium mobilization[1] Recombinant human OX1 and OX2 receptors were stably expressed in HEK293 cells, or rat recombinant OX1 and OX2 receptors stably expressed in AV12 cells, and assessed for intracellular calcium mobilization using Fluo-3 dye. Fluo-3 dye was made at 2.2 mM in equal parts of Pluronic f-127 and 100% DMSO. It was further diluted to 8 mM in 2.5 mM probenecid loading buffer which was made as described below. The human cell line was grown in DMEM/F-12 (3:1) supplemented with 10% FBS (heat-inactivated), 20 mM HEPES, 1% Penn/Strep, and 4 μg/ml Blasticidin (35,000 cells/well). The rat cell line was grown in DMEM supplemented with 10% FBS (heat-inactivated), 20 mM HEPES, 1% Penn/Strep, 4 μg/ml Blasticidin, and 1 mM sodium pyruvate (60,000 cells/well). Cells were plated in Biocoat black poly-d-lysine coated clear bottom 96-well plates, allowed to attach for 30 min at room temperature, and then grown overnight at 37°C and 5% CO2 in a humidified incubator. Probenecid loading buffer was made at 250 mM in equal parts of 1 N NaOH and HBSS. It was further diluted to 2.5 mM in HBSS containing calcium and magnesium +20 mM HEPES. The agonist used in this study was Orexin-A dissolved in de-ionized water at a stock concentration of 80 μM. An EC70 concentration of orexin A in 0.1% BSA in HBSS with calcium and magnesium, was used to challenge the antagonists. Compounds were serial diluted 1:3 from a 10 mM stock in DMSO on a Biomek and further diluted in HBSS containing calcium and magnesium +0.04% Bacitracin. The assay was performed with the following steps: (1) growth medium was removed and cells were washed with 30 μl HBSS (with calcium and magnesium) and then removed. Next, 30 μl of dye was added to the wells, and cell plates were incubated in the dark at room temperature for 60 min; dye was removed, cells were washed with 30 μl probenecid loading buffer and removed, and then 50 μl of probenecid loading buffer was added to the wells. Finally, plates were read on a fluorescence imaging plate reader instrument with a 50 μl addition of antagonist; cells were placed in the dark at room temperature for an additional 15 min; then read on FLIPR instrument with a 100 μl addition of an EC70 concentration of orexin A (for a total volume of 200 μl). Final concentrations of the test compounds were 20 μM in 1.25% DMSO and 10 μM in 0.625% DMSO for the two different FLIPR reads, respectively. Kb-values were calculated based on 10-point dilution curves using ActivityBase templates. Reported values are shown as a mean ± the standard error of the mean (s.e.m.). |
| Animal Protocol |
Drugs and formulations[1] LSN2424100 and SB334867 were suspended in a solution of 1% carboxymethylcellulose, 0.25% polysorbate 80, and 0.05% Dow antifoam and injected IP in a volume of 10 ml/kg body weight for mice and 1 ml/kg in rat (LSN2424100 was injected in a 2 ml/kg volume for c-fos studies). (S)-almorexant and (R)-almorexant were suspended in a solution of 1% carboxymethylcellulose, 0.25% polysorbate 80, and 0.05% Dow antifoam and dosed PO in a volume of 2 ml/kg in rat (dosed IP at 10 ml/kg for mice). Imipramine was dissolved in sterile water. Alprazolam was dissolved in 10% 2-hydroxypropyl-β-cyclodextrin. Drugs were mixed fresh on the day of the experiment and doses were calculated based on the free base weight. Rat exposure and unbound fraction[1] Two male cannulated rats were administered a single 30 mg/kg intraperitoneal (IP) or 30 mg/kg oral (PO) dose of LSN2424100 to determine the pharmacokinetic parameters. Plasma samples were collected at 0.5, 1, 1.5, 2, 4, and 6 h post-dose and analyzed by liquid chromatography coupled to tandem mass spectral detection (LC-MS/MS) to determine the concentrations of LSN2424100. Two cohorts of rats were used in each study, with samples taken from one cohort at 0.5, 1, and 2 h post-dose, and samples taken from the second cohort at 1.5, 4, and 6 h post-dose. The first cohort of animals was sacrificed at 2 h post-dose, and the second at 6 h post-dose to allow for collection of brain samples. The plasma and brain binding of LSN2424100 were determined by equilibrium dialysis at 1 μM which were used to calculate unbound concentrations in brain and plasma at 2 h post-dose (Zamek-Gliszczynski et al., 2011). Restraint-stress-induced c-Fos activation[1] Beginning at 0830 h on the day of the study, groups of rats (n = 8 per group) received IP injections of vehicle, 30 mg/kg LSN2424100, or 3 mg/kg alprazolam in a counter-balanced manner and were returned to the home cage. Thirty min later, rats were restrained in a rigid plastic flat-bottom restraint tube (Braintree Scientific, Inc.; Braintree, MA, USA) for 20 min. After the 20 min stress period, rats were moved to a new cage and housed individually. Rats were sacrificed 2 h after stress onset. Separate groups of control animals (n = 6 – 8 each) received either vehicle or 30 mg/kg LSN2424100 and were returned to their home cage until sacrifice. Immediately after sacrifice, brains were removed, the medial prefrontal cortex (mPFC), a brain area implicated in the pathophysiology of depression, was dissected from both hemispheres and frozen in 1.5 ml centrifuge tubes in dry ice, and stored at −80°C for later analysis. |
| References | [1]. LSN2424100: a novel, potent orexin-2 receptor antagonist with selectivity over orexin-1 receptors and activity in an animal model predictive of antidepressant-like efficacy. Front Neurosci. 2014 Jan 28;8:5. |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| 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.) |