| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg | |||
| 500mg | |||
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| Other Sizes |
| Targets |
W146 is a selective antagonist of the Sphingosine 1-Phosphate Receptor Subtype 1 (S1P1). It is the (R)-enantiomer of 3-amino-4-(3-hexylphenylamino)-4-oxobutyl phosphonic acid, while its inactive enantiomer is W140 [2,3].
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| ln Vitro |
In rat bone marrow-derived endothelial progenitor cells (EPCs), pretreatment with W146 (10 μM) for 30 minutes significantly inhibited S1P-induced cell proliferation, as measured by MTS assay. It also blocked S1P-induced Akt phosphorylation (detected by Western blot) and S1P-induced VEGF secretion and expression. In addition, W146 (10 μM) pretreatment significantly reversed the protective effect of S1P against H₂O₂-induced apoptosis, as evidenced by increased TUNEL labeling index and increased cleaved caspase-3 levels [3].
W146 is an antagonist of S1PR1 with a Ki of roughly 70–80 nM[1]. The W146 pretreatment markedly raised the amounts of activated cleaved caspase-3. After receiving W146 therapy, the S1P-induced decrease in EPC apoptosis totally vanished [2]. |
| ln Vivo |
In C57BL/6 mice, pretreatment with W146 (5 mg/kg body weight, intraperitoneal injection) 1 hour prior to AMD3100 administration (5 mg/kg, subcutaneous injection) significantly augmented AMD3100-stimulated mobilization of Kit+/Sca-1+/Lin- (KSL) hematopoietic stem progenitor cells (HSPCs) into peripheral blood. The W146-mediated augmentation was approximately 8-fold as measured by CFU-G/M colony forming assays. Treatment with W146 alone had no effect on KSL-HSPC mobilization [2].
W146-mediated enhancement of KSL-HSPC mobilization was specific, as pretreatment of mice with W140 failed to have any effect on AMD3100-stimulated KSL-HSPC mobilization. Pretreatment with W146 (5 mg/kg, i.p.) prior to AMD3100 administration demonstrated an approximately 8-fold increase in KSL-HSPC mobilization as measured by CFU-G/M colony formation assay compared to mice treated with AMD3100 alone [3]. Mice's basal WBC counts remain unchanged after injections of W146, W140, JTE013 or Cay10444 [3]. |
| Cell Assay |
Endothelial Progenitor Cell (EPC) Proliferation Assay (MTS): Rat bone marrow-derived EPCs were seeded in 96-well plates at 1×10⁴ cells/well and cultured in serum-starved medium for 12 hours. Cells were pretreated with W146 (10 μM) for 30 minutes, followed by stimulation with S1P (800 nM). After 8 hours, MTS reagent (20 μL/well) was added and incubated for another 8 hours. Optical density at 490 nm was measured using a plate reader [3].
EPC Apoptosis Assay (TUNEL): EPCs were pretreated with W146 (10 μM) for 30 minutes, followed by S1P (800 nM) for 30 minutes, then exposed to H₂O₂ (600 μM) for 6 hours. Cells were fixed and stained using the TUNEL assay kit according to manufacturer instructions. Nuclear staining was performed with DAPI. TUNEL-positive cells were counted under a confocal microscope, and the labeling index was calculated as the ratio of positive cells to total cells [3]. Western Blot Analysis: EPCs were cultured in 6-well plates to 80% confluence, then pretreated with W146 (10 μM) for 30 minutes, followed by stimulation with S1P (800 nM) for 30 minutes (for Akt phosphorylation) or 24 hours (for VEGF expression). Cell lysates were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against phospho-Akt, total Akt, cleaved caspase-3, VEGF, and GAPDH. Protein bands were visualized by enhanced chemiluminescence [3]. Apoptosis analysis [2] Cell Types: endothelial progenitor cells (EPC). Tested Concentrations: 10μM. Incubation Duration: 30 minutes before adding S1P. Experimental Results: Increased levels of activated cleaved caspase-3. |
| Animal Protocol |
HSPC Mobilization Study (Mice): C57BL/6 mice (6-8 weeks old, 5 mice per group) were injected intraperitoneally (i.p.) with W146 (5 mg/kg body weight) or vehicle. One hour later, animals received AMD3100 (5 mg/kg, subcutaneous injection). Two hours after AMD3100 administration, approximately 500 μL of peripheral blood was collected from the vena cava into EDTA tubes. Complete blood count was performed, and after red blood cell lysis, KSL-HSPCs were stained with lineage markers, Sca-1, and c-Kit antibodies, then analyzed by flow cytometry. CFU-G/M colony forming assays were performed using methylcellulose culture [2].
EPC Study (Rats): In vitro experiments were conducted using rat bone marrow-derived endothelial progenitor cells (EPCs) isolated from Sprague-Dawley rats. Cells were treated with W146 at a concentration of 10 μM for 30 minutes prior to S1P stimulation [3]. Animal/Disease Models: Mice (4-6 weeks old) [3]. Doses: 5 mg/kg. Management: IP, AMD3100 (ADM) 1 hour before management. Experimental Results: KSL-HSPC mobilization was Dramatically increased compared to mice pretreated with dextran and subsequently administered AMD3100. |
| Toxicity/Toxicokinetics |
In the mouse HSPC mobilization study, W146 was administered at 5 mg/kg via intraperitoneal injection. No overt toxicity was reported. The inactive enantiomer W140 was used as a control and showed no effect [2]. In the rat EPC study, W146 was used in vitro at 10 μM concentration without reported cytotoxicity [3].
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| References |
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| Additional Infomation |
[(3R)-3-amino-4-[(3-hexylphenyl)amino]-4-oxobutyl]-phosphonic acid is an amino acid amide.
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| Molecular Formula |
C16H27N2O4P
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|---|---|
| Molecular Weight |
342.37038
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| Exact Mass |
342.171
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| Elemental Analysis |
C, 56.13; H, 7.95; N, 8.18; O, 18.69; P, 9.05
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| CAS # |
909725-61-7
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| Related CAS # |
W146 TFA;909725-62-8
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| PubChem CID |
6857802
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| Appearance |
White to off-white solid powder
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| LogP |
3.992
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
23
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| Complexity |
400
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CCCCCCC1=CC(=CC=C1)NC(=O)[C@@H](CCP(=O)(O)O)N
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| InChi Key |
FWJRVGZWNDOOFH-OAHLLOKOSA-N
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| InChi Code |
InChI=1S/C16H27N2O4P/c1-2-3-4-5-7-13-8-6-9-14(12-13)18-16(19)15(17)10-11-23(20,21)22/h6,8-9,12,15H,2-5,7,10-11,17H2,1H3,(H,18,19)(H2,20,21,22)/t15-/m1/s1
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| Chemical Name |
[(3R)-3-amino-4-(3-hexylanilino)-4-oxobutyl]phosphonic acid
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| Synonyms |
909725-61-7; W-146; W 146; P-((3R)-3-Amino-4-((3-hexylphenyl)amino)-4-oxobutyl)phosphonic acid; P-[(3R)-3-Amino-4-[(3-hexylphenyl)amino]-4-oxobutyl]phosphonic acid; RefChem:367534; W146;
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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 |
| 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) |
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
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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 | 2.9208 mL | 14.6041 mL | 29.2082 mL | |
| 5 mM | 0.5842 mL | 2.9208 mL | 5.8416 mL | |
| 10 mM | 0.2921 mL | 1.4604 mL | 2.9208 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.
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