Sphingosine 1-phosphate (S1P) receptors; S1P1 (pKi = 7.86); S1P3 (pKi = 5.93); S1P1 (pEC50 = 6.58); S1P3 (pEC50 = 7.07)
VPC23019 is a dual antagonist of sphingosine-1-phosphate receptor 1 (S1PR1) and sphingosine-1-phosphate receptor 3 (S1PR3) . The reported pKi values are 7.86 for S1P1 (approximately 13.8 nM) and 5.93 for S1P3 (approximately 1.17 μM), indicating approximately 100-fold selectivity for S1P1 over S1P3 . It acts as a competitive antagonist in both broken cell and whole cell assays . The compound does not significantly affect S1P2 or S1P5 at similar concentrations; one study reported an EC50 of 7.28 for S1P5, but VPC23019 is not primarily characterized as an S1P5 antagonist .

VPC23019 Is Devoid of Agonism at the S1P1 and S1P3 Receptors. VPC23019 Blocks Agonist Activity at the S1P1 and S1P3 Receptors.
The lead compound in series, VPC23019, was found in broken cell and whole cell assays to behave as a competitive antagonist at the S1P(1) and S1P(3) receptors. The structure-activity relationship of this series is steep; for example, a slight modification of the lead compound resulted in VPC25239, which was one log order more potent at the S1P(3) receptor. These new chemical entities will enable further understanding of S1P signaling and provide leads for further S1P receptor antagonist development.[1]

---

VPC23019 has demonstrated diverse in vitro activities across multiple cell types and experimental models. In human microvascular endothelial cells (HMVECs), treatment with VPC23019 (concentration not specified in the abstract) blocked arsenic-stimulated angiogenic gene expression and tube formation, indicating its role in inhibiting S1P1-mediated angiogenesis . In neural stem cells, VPC23019 inhibited S1P-induced migration . In thyroid cancer cells and ovarian cancer cells, VPC23019 also blocked S1P-induced cell migration . In human pulmonary arterial smooth muscle cells (HPASMCs), VPC23019 (along with other S1PR3 inhibitors such as TY52156) reduced TGF-β1-induced α-smooth muscle actin (αSMA) upregulation, suggesting its role in modulating contractile gene expression . In plasma cell differentiation studies, the inhibitory effects of sphingosylphosphorylcholine on plasma cell differentiation were specifically blocked by VPC23019, and this effect was attributed to S1PR3 antagonism rather than S1PR1 or S1PR2 . In mouse embryonic fibroblasts and other cell systems, VPC23019 inhibited S1P-induced calcium flux and downstream signaling pathways, including RhoA/ROCK activation .

VPC23019 has been evaluated in several in vivo models. In a mouse model of Alzheimer's disease (double transgenic APP/PS1 mice), injection of VPC23019 (combined with bone marrow mesenchymal stem cell-derived exosomes) was used to block S1P1 signaling, and the compound reduced Aβ deposition and improved cognitive function recovery . In a study on obesity, oral administration of VPC23019 to ob/ob mice did not cause significant changes in body weight gain, adipocyte size, or glucose intolerance; however, co-administration of VPC23019 with the S1P1 agonist SEW-2871 abolished the beneficial effects of SEW-2871, confirming that VPC23019 effectively antagonizes S1P1 in vivo . In a mouse model of Paget‘s disease, addition of VPC23019 blocked enhanced osteoblast differentiation induced by osteoclast-conditioned media, indicating a role for S1PR3 in bone formation . In a study on denervation-induced dendritic atrophy using organotypic entorhino-hippocampal slice cultures (ex vivo model), treatment with VPC23019 (1 μM) prevented denervation-induced dendritic loss and maintained dendritic stability . In a rat model of ischemic stroke, VPC23019 was used as an S1P1 antagonist to confirm that non-mitogenic FGF1 promotes angiogenesis partially through the S1P1 pathway . In a diabetic nephropathy study, the protective effects of apolipoprotein M were partially inhibited by treatment with VPC23019, but not by an S1P2 antagonist, confirming the involvement of S1P1/S1P3 in this protective effect .

Determination of the Binding Constant for VPC23019 at the S1P1 and S1P3 Receptors [1]
The binding constant (Kb) for VPC23019 at the S1P1 and S1P3 receptors was determined by Schild analyses from curves that were fitted using the nonlinear regression method discussed by Lew and Angus. Briefly, nonlinear analysis of the best fit line generated by plotting the negative log of the EC50 values obtained from agonist dose-response curves, in the absence and presence of varying concentrations of antagonist, was plotted against the concentration of antagonist to give the Kb value. An F-test analysis was also performed to establish whether the antagonist did or did not meet the criteria of a simple competitive interaction. [1]

---

S1P Radiolabeling[1]
[32P]S1P was prepared by incubating sphingosine and [γ-32P]ATP with cell lysate from HEK293T cells transfected transiently with human sphingosine kinase type 2 DNA. The 0.2-ml reaction contained 0.025 mm sphingosine, 1 mCi of [γ-32P]ATP (7000 Ci/mmol), and kinase buffer (10 mm Mg(C2H3O2)2 in 50 mm Tris, pH 7.5, 10 mm NaF, and 2 mm semicarbizide). The reaction was initiated by the addition of 0.02 mg of cell lysate protein and incubated at 37 °C for at least 30 min. The [32P]S1P was extracted by the addition of 1 n HCl and 2.0 m each KCl, methanol, and chloroform to the reaction mixture. The mixture was then vortexed and centrifuged at 1000 × g for 5–10 min. The organic layer was isolated, and the extraction procedure was repeated two additional times with the remaining aqueous fraction. The combined organic fractions were dried under a stream of nitrogen gas and resuspended in aqueous 0.1% fatty acid-free BSA. The specific activity of the product, [32P]S1P, is estimated to be that of the radiolabeled substrate, [γ-32P]ATP, i.e. 7000 Ci/mmol. [1]

---

[32P]S1P Binding Assay[1]
Membranes containing 0.005 mg of protein from HEK293T cells transfected transiently with both receptor and G-protein DNAs were incubated in 0.5 ml of binding buffer (50 mm HEPES, 100 mm NaCl, 10 mm MgCl2, pH 7.5), 50 pm [32P]S1P, and the indicated lipid(s) for 1 h at room temperature. Bound ligand was separated from free ligand by rapid filtration and analyzed in a liquid scintillation counter. Nonspecific binding was determined as residual binding of radioligand in the presence of excess S1P to membranes, both heat-denatured and non-heat-denatured, from HEK293T cells transfected transiently with receptor and G-protein DNAs; it was typically 60% of total binding. The binding constant (Ki) associated with the ligand-receptor interaction was determined from the IC50 using the Chang-Prusoff equation (Ki = IC50/(1 + [L]/Kd). In applying this equation, the concentration of radioligand (L) is 0.05 nm and the Kd value used was that reported for the S1P-S1P1 receptor interaction, i.e. 8.1 nm. [1]

Cell Migration Assay [1]
Cell migration assays were performed using modified Boyden chambers (tissue culture-treated with a 24-mm diameter, a 0.010-mm thickness, and 0.008-mm pores, Transwell®) containing polycarbonate membranes that were coated on the underside with 0.1% gelatin. The underside of the polycarbonate membranes was rinsed once with migration medium (Dulbecco's modified Eagle's medium/F-12 without Phenol Red and 0.1% fatty acid-free BSA) and then immersed in the lower chamber containing 2 ml of migration medium. T24 cells transfected stably with human S1P1 receptor DNA were grown in Dulbecco's modified Eagle's medium/F-12 medium containing charcoal/dextran-stripped FBS and 0.010 mg/ml puromycin to 100% confluence in 150 × 25-mm tissue culture plates and serum starved for at least 12 h. Serum-starved cells were removed from culture dishes with 10× trypsin-EDTA, washed once with migration medium, and resuspended in migration medium (106 cells/ml). One milliliter of the cell suspension was added to the upper migration chamber while the S1P agonist VPC22277 (10 nm) was added to the lower chamber. Cells were allowed to migrate to the underside of the membrane for 4 h at 37 °C in the presence or absence of antagonist (VPC23019 (0–1000 nm), VPC23019, VPC23031, VPC23089, and VPC25239 (50 nm each)), which were added to the lower chamber. The migrated cells attached to the bottom surface of the membrane were removed with 10× trypsin-EDTA, their mass was determined by combining 0.1 ml of cell suspension with an equal volume of CyQuant dye solution (3.0 ml of 2× lysis buffer and 0.015 ml of CyQuant dye), and the resulting fluorescence was quantified using the FlexStation™ fluorimeter (Molecular Devices, Menlo Park, CA). Each determination represents the average of two individual migration chambers. For determination of the reversibility of the antagonism associated with VPC23019, cells were incubated with 0.01 mm VPC23019 at 37 °C for 30 min. The monolayer was washed three times with phosphate-buffered saline and processed immediately for the cell migration assay, as described above.

---

Measurement of Intracellular Calcium Mobilization [1]
A FlexStation™ fluorimeter was used to measure intracellular calcium in native T24 cells and T24 cells stably transfected with either human S1P2 or human S1P3 receptor DNA. Cells were seeded (∼50,000 cells/well) in 96-well, clear bottom black microplates and left overnight at 37 °C. The cells were dye-loaded with 0.004 mm Fluo-4AM ester in a loading buffer (Hanks' balanced salt solution, pH 6.4, containing 20 mm HEPES, 0.1% fatty acid-free BSA, and 2.5 mm probenecid) for 30 min at 37 °C. After washing cell monolayers three times with phosphate-buffered saline, loading buffer was added, and the cells were exposed to sets of compounds for 3 min at 25 °C in the FlexStation™.In all cases, each concentration of every compound was tested at least in triplicate. For determination of the reversibility of the antagonism associated with VPC23019 (0.010 mm), the compound was added in combination with loading dye to the cells and incubated at 37 °C for 30 min. The cells were washed with phosphate-buffered saline and exposed to compounds immediately, as described above.

VPC23019 has been administered via multiple routes in animal studies. In ob/ob mouse studies (obesity model), VPC23019 was administered orally to evaluate its effects on body weight, adipocyte size, and glucose tolerance. The dosing regimen and specific concentrations were not detailed in the abstract, but the compound was given alone or in combination with the S1P1 agonist SEW-2871 . In double transgenic APP/PS1 mouse models of Alzheimer‘s disease, VPC23019 was injected (route not specified) in combination with bone marrow mesenchymal stem cell-derived exosomes . In organotypic slice culture studies (ex vivo), VPC23019 was applied to the incubation medium at a concentration of 1 μM. Drug-containing medium was replaced three times per week, and cultures were maintained for up to 6 weeks. No evidence of toxicity (blebbing, dendritic atrophy/retraction) was observed in non-denervated cultures treated with VPC23019 .

In organotypic entorhino-hippocampal slice cultures, treatment with VPC23019 (1 μM) did not show any evidence of toxicity, including blebbing, dendritic atrophy, or retraction, in non-denervated cultures. Control cells remained stable, and no imaged cells were lost under these conditions . No other systematic toxicity data (e.g., LD50, hepatotoxicity, nephrotoxicity, or protein binding) for VPC23019 are reported in the provided search results. The compound is intended for research use only and is not approved for human or veterinary use .

[1]. Sphingosine 1-phosphate analogs as receptor antagonists. J Biol Chem. 2005 Mar 18;280(11):9833-41.

VPC23019 is a pioneering pharmacological tool compound that helped establish the structure-activity relationship for S1P receptor antagonists . Its development allowed researchers to dissect the distinct roles of S1P1 vs. S1P3 vs. S1P2 in various biological processes. The compound has been widely cited in studies on angiogenesis, neuroprotection, inflammation, fibrosis, cancer cell migration, and metabolic disorders . It is often used in combination with selective agonists (e.g., SEW-2871 for S1P1) or selective antagonists (e.g., JTE-013 for S1P2, W146 for S1P1 alone) to delineate receptor-specific effects . While VPC23019 is a dual S1P1/S1P3 antagonist, its approximately 100-fold selectivity for S1P1 over S1P3 allows it to be used at lower concentrations to preferentially block S1P1 . At higher concentrations, it also blocks S1P3, which can be useful for studying contexts where both receptors are involved . The compound is available from multiple commercial suppliers (e.g., Tocris, Abmole, MedChemExpress) and is typically stored as a powder at -20°C, protected from light .

---

VPC 23019 is a secondary amide formed by the condensation of the carboxyl group of O-phospho-D-serine with the amino group of m-octanilide. It is an analog of sphingosine-1-phosphate (S1P) and a potent antagonist of S1P1 and S1P3 receptors. It inhibits S1P-induced migration of thyroid cancer cells, ovarian cancer cells, and neural stem cells. It is an antagonist of both sphingosine-1-phosphate receptor 3 and sphingosine-1-phosphate receptor 1. It is a D-serine derivative, secondary amide, organophosphate ester, phosphate ester, and aromatic amide. Functionally, it is related to O-phospho-D-serine. Sphingosine-1-phosphate (S1P) is a lysophospholipid mediator that can induce various cellular and tissue responses through a series of cell surface receptors. Recent research progress on S1P receptor agonists, represented by the immunomodulatory prodrug FTY720, indicates that the S1P signaling pathway is an important regulator of lymphocyte migration. In order to understand the structure-activity relationship of S1P ligands and develop tool compounds for exploring S1P biology, we synthesized and tested a variety of S1P analogs. In this paper, we report that some of the arylamide-containing compounds we synthesized are antagonists of S1P(1) and S1P(3) receptors. The lead compound VPC23019 showed competitive antagonism against S1P(1) and S1P(3) receptors in both lysed cell and whole-cell experiments. The structure-activity relationship of this series of compounds is very significant; for example, VPC25239, obtained by minor modification of the lead compound, showed an order of magnitude increase in potency against the S1P(3) receptor. These new chemical entities will contribute to a better understanding of S1P signaling and provide clues for further development of S1P receptor antagonists. [1]

C17H29N2O5P

372.396325826645

372.181

C, 54.83; H, 7.85; N, 7.52; O, 21.48; P, 8.32

449173-19-7

11588811

Light yellow to yellow solid powder

4.314

4

6

12

25

429

1

CCCCCCCCC1=CC(=CC=C1)NC(=O)[C@@H](COP(=O)(O)O)N

MRUSUGVVWGNKFE-MRXNPFEDSA-N

InChI=1S/C17H29N2O5P/c1-2-3-4-5-6-7-9-14-10-8-11-15(12-14)19-17(20)16(18)13-24-25(21,22)23/h8,10-12,16H,2-7,9,13,18H2,1H3,(H,19,20)(H2,21,22,23)/t16-/m1/s1

[(2R)-2-amino-3-(3-octylanilino)-3-oxopropyl] dihydrogen phosphate

449173-19-7; VPC 23019; VPC23019; VPC-23019; (R)-2-Amino-3-((3-octylphenyl)amino)-3-oxopropyl dihydrogen phosphate; CHEMBL228102; N-(3-octylphenyl)-O-phosphono-D-serinamide; CHEBI:144948;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~125 mg/mL (~335.66 mM)

Solubility in Formulation 1: 2.08 mg/mL (5.59 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (5.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (5.59 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 20.8 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.)