CYM-5442 is a highly selective agonist of the Sphingosine 1-Phosphate Receptor 1 (S1P1). It activates S1P1 in vitro with an EC50 of 1.35 ± 0.25 nM in a CRE reporter assay [1]. It exhibits no activity on S1P2, S1P3, and S1P4, and only 20% activity at 10 μM on S1P5 [1]. Unlike S1P, CYM-5442 does not require the headgroup interaction residues R120 and E121 on S1P1 for receptor activation [1].

In P32-orthophosphate-labeled cells, CYM5442 (0.5 µM; 0–60 min; HEK293 cells) treatment induces S1P1 phosphorylation in a time-dependent manner [1]. In S1P1-transfected CHO-K1 cells, CYM5442 with an EC50 of 46 nM stimulates S1P1-dependent p42/p44-MAPK phosphorylation. When CYM5442 was added to the R120 alanine (R120A) mutant, it was still possible to sustain p42/p44-MAPK activity (EC50 of 67 nM). In E121A S1P1 cells, CYM-5442 activates p42/p44-MAPK in a concentration-dependent manner, with an average EC50 value of 134 nM [1].

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CYM-5442 activated S1P1 in a concentration-dependent manner in a CRE reporter assay, with an EC50 of 1.35 ± 0.25 nM, showing higher potency than SEW2871 (EC50 = 29 nM) and CYM-5181 (EC50 = 3.4 nM) [1].
In HEK293 cells stably expressing S1P1-GFP, CYM-5442 (500 nM) induced time-dependent S1P1 phosphorylation (detected by P32-orthophosphate labeling) and S1P1 internalization from the plasma membrane to cytoplasmic vesicles within 45 minutes, as visualized by fluorescence microscopy. These effects were abolished by pre-incubation with the selective S1P1 antagonist W146 (10 μM) [1].
CYM-5442 also induced S1P1 polyubiquitination, with a similar magnitude to the full agonist AFD-R. This ubiquitination was blocked by W146 pre-incubation [1].
In CHO-K1 cells expressing wild-type S1P1, CYM-5442 activated p42/p44 MAPK phosphorylation with an EC50 of 46 nM (Emax = 70% of S1P). In contrast to S1P, CYM-5442 retained the ability to activate p42/p44 MAPK in S1P1 mutants (R120A and E121A), with EC50 values of 67 nM and 134 nM respectively, demonstrating that it does not require these headgroup interaction residues [1].

Adult male albino Wistar rats treated with CYM5442 (1 mg/kg; intraperitoneal; daily; for 5 days) demonstrated preservation of visual function at the visual evoked potential (VEP). When CYM was administered to mice instead of a vehicle, the retinal nerve fiber layer (RNFL) was noticeably thicker [2].

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Lymphopenia Induction: In C57BL/6 mice, intraperitoneal administration of CYM-5442 (10 mg/kg) induced acute lymphopenia within 5 hours, reducing white blood cell count by 64%, B220+ B-cells by 63%, CD4+ T-cells by 83%, and CD8+ T-cells by 84% compared to vehicle. The ED50 values were 0.5 mg/kg for CD4+, 1.0 mg/kg for CD8+, and 2.0 mg/kg for B220+ cells. Maximal lymphopenia was achieved at serum concentrations of 50-100 nM. The lymphopenic effect was reversible upon drug clearance and was blocked by the S1P1 antagonist W146 (20 mg/kg), confirming S1P1 dependence [1].
Neuroprotection in Retinal Ischemia: In adult male Wistar rats with endothelin-1 (ET-1)-induced retinal ganglion cell (RGC) loss, intraperitoneal administration of CYM-5442 (1 mg/kg/day for 5 consecutive days, starting 1 hour after ET-1 injection) significantly preserved visual function. Pattern visual evoked potential (VEP) amplitudes were 11.95 ± 0.86 μV in treated animals vs. 3.47 ± 1.20 μV in vehicle-treated animals. Retinal nerve fiber layer (RNFL) thickness was significantly greater in treated animals (93.62 ± 3.22 μm) compared to vehicle (77.72 ± 0.35 μm). Brn3a+ RGC counts were significantly higher in treated retinas (76,540 ± 303 cells/retina) than in vehicle-treated retinas (52,426 ± 1,932 cells/retina). CYM-5442 treatment was associated with significant reduction in retinal cleaved caspase-3-positive cells (1,073 ± 89 cells/mm²) compared to vehicle (13,299 ± 432 cells/mm²), indicating reduced apoptosis [2].

Western Blot Analysis[1]
Cell Types: HEK293 cells stably expressing S1P1 fused to GFP at the carboxyl terminus
Tested Concentrations: 0.5 µM
Incubation Duration: 0 min, 2 min, 5 min, 10 min, 30 min, 60 min
Experimental Results: Stimulated S1P1 is phosphorylated in a time-dependent manner.

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CRE Reporter Assay: CHO-K1 cells stably expressing human S1P1 and a CRE-β-lactamase reporter were used. Cells were incubated with forskolin (2 μM) and increasing concentrations of CYM-5442 for 2 hours. Agonist-mediated inhibition of forskolin-induced cAMP accumulation was measured by detecting β-lactamase expression via cleavage of the fluorogenic substrate CC4-AM, using SEW2871 as a positive control [1].
S1P1-GFP Internalization and Ubiquitination Assays: HEK293 cells stably expressing S1P1-GFP were used. For internalization, cells were treated with CYM-5442 (500 nM) for 45 minutes, fixed, and visualized by fluorescence microscopy. For ubiquitination, cells were treated with agonists for 1 hour, lysed, and S1P1-GFP was immunoprecipitated using GFP antibodies, followed by immunoblotting with ubiquitin antibodies [1].
p42/p44 MAPK Activation ELISA: CHO-K1 cells transiently transfected with wild-type or mutant (R120A, E121A) S1P1 cDNA were serum-starved for 4 hours. Cells were stimulated with increasing concentrations of CYM-5442 for 5 minutes (determined empirically as maximal). Cell lysates were analyzed using an ELISA kit specific for phosphorylated p42/p44 MAPK according to the manufacturer’s instructions [1].

Animal/Disease Models: Adult male albino Wistar rats (8-10 weeks old; 180-230 g) infected ocular endothelin-1 (ET-1) [2]
Doses: 1 mg/kg
Route of Administration: intraperitoneal (ip) injection; Routine; continued for 5 days
Experimental Results: Visual evoked potentials (VEP) demonstrated preservation of visual function. The number of retinal ganglion cells (RGC) increased Dramatically.

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Lymphopenia Studies (Mice): Male C57BL/6 mice (30 g) were injected intraperitoneally with CYM-5442 (0.3-10 mg/kg) or vehicle (sterile water) in a volume of 300 μL. For antagonist studies, mice received W146 (20 mg/kg) or vehicle (10% DMSO, 25% Tween-20 in sterile water) intraperitoneally 30 minutes prior to CYM-5442 (2 mg/kg) administration. Blood was collected at indicated time points (1, 3, 5, 8, 16 hours) in EDTA tubes. White blood cell counts were obtained using an automated veterinary hematoanalyzer. Lymphocyte subpopulations (B220+, CD4+, CD8+) were analyzed by flow cytometry [1].
Pharmacokinetic Studies (Rats): Sprague Dawley rats were dosed with CYM-5442 at 1 mg/kg intravenously (jugular) or 2 mg/kg by oral gavage. The compound was formulated at 1 mg/mL in 10:10:80 (DMSO:Tween-80:water, vol:vol:vol). Blood was collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h post-dose. Plasma was generated by centrifugation. Compound concentrations were determined by LC-MS/MS [1].
Brain Distribution Studies (Mice): C57BL/6 mice were dosed intraperitoneally with CYM-5442 (10 mg/kg). Blood and brains were collected 2 hours post-dose. Brain samples were weighed and extracted with acetonitrile. Compound concentrations in plasma and brain homogenates were determined by LC-MS/MS [1].
Retinal Neuroprotection Study (Rats): Adult male Wistar rats (8-10 weeks, 180-230 g) received a single intravitreal injection of endothelin-1 (ET-1) (2 μL of 10 pmol/μL) in the right eye. One hour later, CYM-5442 (1 mg/kg dissolved in PBS) or vehicle (PBS) was administered intraperitoneally once daily for five consecutive days. Animals were weighed daily for dose calculation. Electrophysiological recordings (VEP, PERG, ERG) and OCT imaging were performed after treatment completion (5-7 days post-ET-1). Retinas were collected for Brn3a immunohistochemistry and cleaved caspase-3 analysis [2].

In rats, following intravenous administration (1 mg/kg), CYM-5442 had a half-life (t1/2) of 3.05 ± 0.37 hours, clearance (Cl) of 72.90 ± 11.70 mL/min/kg, and volume of distribution (Vss) of 15.60 ± 3.50 L. Following oral administration (2 mg/kg), the half-life was 4.00 ± 1.45 hours, with a Cmax of 36 ± 7.2 nM, Tmax of 1.17 ± 0.76 hours, and oral bioavailability of 20.5 ± 4.67% [1].
In mice, following intraperitoneal administration (10 mg/kg for 2 hours), CYM-5442 showed significant central nervous system penetration, with brain concentrations of 13.7 ± 2.9 μM and plasma concentrations of 1.08 ± 0.3 μM, resulting in a brain-to-plasma ratio of approximately 13:1 [1].

The literature does not describe specific toxicity data (such as LD50, hepatotoxicity, nephrotoxicity) for CYM-5442. However, in the retinal neuroprotection study, CYM-5442 was administered daily for five days at 1 mg/kg intraperitoneally, and no adverse effects on overall health or behavior were reported [2]. In a mouse study, doses up to 10 mg/kg intraperitoneally were used without reported overt toxicity [1].

[1]. Full pharmacological efficacy of a novel S1P1 agonist that does not require S1P-like headgroup interactions. Mol Pharmacol. 2008 Nov;74(5):1308-18.

[2]. The S1P1 receptor-selective agonist CYM-5442 protects retinal ganglion cells in endothelin-1 induced retinal ganglion cell loss. Exp Eye Res. 2017 Nov;164:37-45.

Background: S1P1 is a G protein-coupled receptor that regulates lymphocyte recirculation and endothelial integrity. FTY720 (fingolimod), a non-selective S1P receptor modulator, is approved for multiple sclerosis but has side effects due to activity on other S1P receptors [1,2].
Mechanism of Action: CYM-5442 is a selective S1P1 agonist that binds to a hydrophobic pocket distinct from the orthosteric S1P binding site. It does not require the headgroup interaction residues R120 and E121 for receptor activation. It induces S1P1 internalization, phosphorylation, and ubiquitination, leading to sustained signaling in internalized vesicles [1].
Neuroprotective Mechanism: In the retinal ischemia model, the neuroprotective effect of CYM-5442 is associated with a reduction in cleaved caspase-3, indicating reduced apoptosis. It is hypothesized that S1P1 activation may counteract pro-apoptotic ceramide signaling [2].
CNS Penetration: CYM-5442 is highly central nervous system penetrant (brain:plasma ratio ~13:1 in mice), making it a useful tool for studying S1P1 functions in the brain and retina [1,2].

C23H28CLN3O4

445.939125061035

445.176

1783987-80-3

CYM5442;1094042-01-9

90488865

Typically exists as solid at room temperature

3

7

9

31

525

0

Cl.O1C(C2C=CC(=C(C=2)OCC)OCC)=NC(C2=CC=CC3=C2CCC3NCCO)=N1

KMZLVDAVGXYZDA-UHFFFAOYSA-N

InChI=1S/C23H27N3O4.ClH/c1-3-28-20-11-8-15(14-21(20)29-4-2)23-25-22(26-30-23)18-7-5-6-17-16(18)9-10-19(17)24-12-13-27;/h5-8,11,14,19,24,27H,3-4,9-10,12-13H2,1-2H3;1H

2-[[4-[5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl]-2,3-dihydro-1H-inden-1-yl]amino]ethanol;hydrochloride

CYM 5442 HCl; CYM-5442 HCl

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)

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

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)