S1PR1 ( EC50 = 13.8 nM )

SEW2871 shows enhanced migration effects by upregulating smooth muscle α-actin, procollagen αI and αIII, and total collagen content in LX-2 cells, a human hepatic stellate cell line [2].

SEW2871 (20 mg/kg, gavage, once daily for 2 weeks) improves established experimental ductitis in IL-10–/– mice [3]. SEW2871 (0.5 mg/kg IP once daily) SEW2871 (0-0.3 mg/kg IV for 2 weeks) inhibits amyloid beta (Aβ1-42) in a trajectory model of Alzheimer's disease Induced spatial impairment memory and hippocampal neuron loss [2]. ) attenuates LPS-induced acute stress-induced lung injury in C57Bl/6 electrodes by producing the required alveolar and vascular septal protection [2]. SEW2871 reduces CD4+ T cell respiration inside the electrode and effectively protects cardiovascular short circuit animal models: IL-10–/– (interleukin (IL)-10 gene deficiency) mice, mouse model of Crohn’s disease (CD) [3] Dosage: 20 mg/kg Administration method: gavage. , once daily for 2 weeks Results: Colitis ameliorated in IL-10–/– mice, associated with reduced serum amyloid A concentration, reduced colonic MPO concentration, peripheral CD4+CD45+ T cell depletion, and T cell homing to colon LP. Inhibits typical cytokine and p-STAT-3 expression of type 1 helper T (Th1) and Th17 cells, and significantly reduces TNF-α, IFN-γ, IL-1β, and IL-17A mRNA levels.

Receptor Binding Assay[2]
[33P]-S1P (final concentration of 83 pM) was added to serial dilutions of S1P or AFD(R) in a 96-well plate. Assays were initiated with the addition of 25 μg membrane protein of S1P1-CHO cell membranes in a final volume of 200 μl assay buffer (50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM CaCl2, 15 mM sodium fluoride, and 1% fatty-acid-free bovine serum albumin). Binding mixtures were incubated for 60 min at room temperature and terminated by filtration over Packard GF/B filter plates as described.

---

Rac-Activation Assay[2]
Cells were serum starved for 16 hr and stimulated with S1P (50 nM, 500 nM, or vehicle control) or SEW2871 (500 nM, 5 μM, or vehicle control) in the serum-free medium with 0.1% fatty-acid-free BSA. At 2 min and 5 min, cells were lysed in 500 μl ice-cold lysis buffer (50 mM Tris [pH 8.0], 500 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM MgCl2, and protease inhibitor cocktail) containing 20 μg GST-tagged p21 binding domain of p21-activated protein kinase 1 (PAK1) [47]. The cell lysates were centrifuged at 16,000 × g at 4°C for 3 min, and 40 μl prewashed 50% GSH-Sepharose beads were added to the supernatant. The mixture was incubated at 4°C for 30 min, and the beads were washed twice in lysis buffer. The proteins bound to the beads were eluted with SDS sample buffer. Activated Rac GTPases were detected by Western blotting with a mouse monoclonal anti-Rac antibody.

---

Membrane Preparation and Receptor Activation Assay[2]
Membranes were prepared from CHO cells expressing human S1P1 for use in ligand and 35S-GTPγS binding studies as described. For 35S-GTPγS binding assay, serial dilutions of S1P, AFD(R), SEW2871, SEW2905, or SEW2898 were added to membranes (1 to 10 μg protein/well) and assayed as described.

Western Blotting[2]
Control CHO cells and the CHO cells stably transfected with human S1P1 or S1P3 were cultured to 50% confluence on a 6-well plate in complete RPMI1640 supplemented with 10% FBS. Cells were serum starved for 16 hr and stimulated with S1P, AFD(R), or SEW2871 diluted to various concentrations in the serum-free medium with 0.1% fatty-acid-free BSA. At 5 min, 1 hr, or 4.5 hr, cells were lysed in 50 mM Tris (pH 8.0), 125 mM NaCl, 20 mM CHAPS, 2 mM dithiothreitol, 1 mM EDTA, 2 mM Na3VO4, 10 mM NaF, 1 mM PMSF, and protease inhibitor cocktail. For the study of pertussis toxin (PTx) inhibition, cells were incubated with PTx at the final concentration of 100 ng/ml for 3 hr, prior to agonist stimulation. Cell lysates were analyzed by Western blotting after separation on 10% SDS-PAGE with mouse monoclonal anti-phospho-ERK1/2 antibody and rabbit polyclonal anti-phospho-Akt antibody (BD Biosciences). Total ERK1 and ERK2 were detected with a rabbit-affinity-purified polyclonal anti-ERK antibody, and total Akt was detected with a rabbit-affinity-purified polyclonal anti-Akt1 antibody. Band intensities corresponding to pERK1, pERK2, and pAkt were quantitated by image analysis. Amounts of pERK1/2 and pAkt were normalized for the total amounts of ERK1/2 and Akt.

---

Receptor Internalization and Recycling[2]
HEK293 cells that were stably transfected with GFP-tagged S1P1 were grown in DMEM medium with 2% charcoal-stripped serum for 2 days [28]. Cells were pretreated with cycloheximide (15 μg/ml) for 30 min to block the synthesis of new S1P1 and stimulated with 100 nM S1P, FTY720-phosphate, 500 nM SEW2871, or SEW2898 for 30 min. Cells were washed and replenished with plain DMEM and cycloheximide and incubated for indicated time points. The fluorescence of the recycled receptor at each time point was quantified and expressed as percent control.

IL-10–/– (interleukin (IL)-10 gene-deficient) mice, a murine model of Crohn's disease (CD)
20 mg/kg
Gavage, once daily for 2 weeks

---

SEW2871 was dissolved in 100% dimethyl sulphoxide and diluted with 50% Tween 20 for use. We chose a dose of SEW2871 (20 mg/kg/day for 2 weeks) by gavage for the treatment, which was well tolerated, as reported previously by Lien et al. 26. In brief, the mice were divided into three groups: (i) treatment group: IL-10–/– mice received SEW2871 by gavage (20 mg/kg/day); (ii) control group: IL-10–/– mice that received equal volumes of distilled water alone; and (iii) WT group: WT mice also received equal volumes of distilled water. After administration, the mice were killed by excessive anaesthesia with pentobarbital sodium and their blood and colons were collected for experiments[3].

[1]. Sphingosine 1-Phosphate Receptor Modulators and Drug Discovery. Biomol Ther (Seoul). 2017 Jan 1;25(1):80-90.

[2]. S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate. Chem Biol. 2005 Jun;12(6):703-15.

[3]. Oral treatment with SEW2871, a sphingosine-1-phosphate type 1 receptor agonist, ameliorates experimental colitis in interleukin-10 gene deficient mice. Clin Exp Immunol. 2014 Jul;177(1):94-101.

5-[4-phenyl-5-(trifluoromethyl)-2-thiophenyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole is an oxadiazole and a ring assembly.

---

SEW2871, a selective sphingosine-1-phosphate type 1 receptor (S1P1) agonist, has been shown to be effective in protecting kidneys against ischaemia-reperfusion injury by reducing CD4(+) T cell infiltration in mice. However, the effects of SEW2871 on colitis remain unclear. The aim of this study was to investigate the effects of SEW2871 on established colitis in interleukin (IL)-10 gene-deficient (IL-10(-/-)) mice, a murine model of Crohn's disease (CD). SEW2871 was administered by gavage at a dose of 20 mg/kg/day for 2 weeks to IL-10(-/-) mice. Severity of colitis, serum amyloid A, tissue myeloperoxidase (MPO), T cells in blood and colon lamina propria (LP) and proinflammatory cytokine productions were evaluated. Furthermore, the phospho-signal transducer and activator of transcription (STAT)-3 (p-STAT-3) expression in lymphocytes isolated from colon LP was also assessed. The 2-week administration of SEW2871 ameliorated established colitis in IL-10(-/-) mice, associated with a reduction of serum amyloid A concentration, a decreased colon MPO concentration, a depletion of the peripheral CD4(+) CD45(+) T cells and a reduction of the homing of T cells into colon LP. Moreover, typical cytokines of T helper type 1 (Th1) and Th17 cells and p-STAT-3 expression were also suppressed by SEW2871 treatment. SEW2871 treatment ameliorates established experimental colitis in IL-10(-/-) mice, which may provide a new therapeutic approach for human CD therapy.[1]

---

The essential role of the sphingosine 1-phosphate (S1P) receptor S1P(1) in regulating lymphocyte trafficking was demonstrated with the S1P(1)-selective nanomolar agonist, SEW2871. Despite its lack of charged headgroup, the tetraaromatic compound SEW2871 binds and activates S1P(1) through a combination of hydrophobic and ion-dipole interactions. Both S1P and SEW2871 activated ERK, Akt, and Rac signaling pathways and induced S1P(1) internalization and recycling, unlike FTY720-phosphate, which induces receptor degradation. Agonism with receptor recycling is sufficient for alteration of lymphocyte trafficking by S1P and SEW2871. S1P(1) modeling and mutagenesis studies revealed that residues binding the S1P headgroup are required for kinase activation by both S1P and SEW2871. Therefore, SEW2871 recapitulates the action of S1P in all the signaling pathways examined and overlaps in interactions with key headgroup binding receptor residues, presumably replacing salt-bridge interactions with ion-dipole interactions.[2]

---

Initial discovery on sphingosine 1-phosphate (S1P) as an intracellular second messenger was faced unexpectedly with roles of S1P as a first messenger, which subsequently resulted in cloning of its G protein-coupled receptors, S1P₁₋₅. The molecular identification of S1P receptors opened up a new avenue for pathophysiological research on this lipid mediator. Cellular and molecular in vitro studies and in vivo studies on gene deficient mice have elucidated cellular signaling pathways and the pathophysiological meanings of S1P receptors. Another unexpected finding that fingolimod (FTY720) modulates S1P receptors accelerated drug discovery in this field. Fingolimod was approved as a first-in-class, orally active drug for relapsing multiple sclerosis in 2010, and its applications in other disease conditions are currently under clinical trials. In addition, more selective S1P receptor modulators with better pharmacokinetic profiles and fewer side effects are under development. Some of them are being clinically tested in the contexts of multiple sclerosis and other autoimmune and inflammatory disorders, such as, psoriasis, Crohn's disease, ulcerative colitis, polymyositis, dermatomyositis, liver failure, renal failure, acute stroke, and transplant rejection. In this review, the authors discuss the state of the art regarding the status of drug discovery efforts targeting S1P receptors and place emphasis on potential clinical applications.[3]

C20H10F6N2OS

440.3616

440.041

C, 54.55; H, 2.29; F, 25.89; N, 6.36; O, 3.63; S, 7.28

256414-75-2

256414-75-2

4077460

White to light yellow solid powder

1.4±0.1 g/cm3

490.3±55.0 °C at 760 mmHg

94.5-95.3ºC

250.3±31.5 °C

0.0±1.2 mmHg at 25°C

1.535

8.42

0

10

3

30

583

0

S1C(C2=NC(C3C([H])=C([H])C([H])=C(C(F)(F)F)C=3[H])=NO2)=C([H])C(C2C([H])=C([H])C([H])=C([H])C=2[H])=C1C(F)(F)F

OYMNPJXKQVTQTR-UHFFFAOYSA-N

InChI=1S/C20H10F6N2OS/c21-19(22,23)13-8-4-7-12(9-13)17-27-18(29-28-17)15-10-14(11-5-2-1-3-6-11)16(30-15)20(24,25)26/h1-10H

5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole

SEW2871; SEW2871; 256414-75-2; SEW2871; SEW 2871; 5-[4-Phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole; SEW-2871; 5-(4-Phenyl-5-(trifluoromethyl)thiophen-2-yl)-3-(3-(trifluoromethyl)phenyl)-1,2,4-oxadiazole; 5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole; MFCD00096600; SEW2871

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: ~25 mg/mL (~56.8 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.72 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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 (4.72 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.)