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Purity: ≥98%
CYM-5442 is anovel, potent and highly-selective S1P1 (Spingosine 1-Phosphate Receptor 1) agonist which reduces the severity of acute GVHD by inhibiting macrophage recruitment. CYM-5442 prevented aGVHD but only markedly inhibited it. Tissue macrophages originate from monocytes, which migrate less when treated with CYM-5442 because it downregulates the expression of CCL2 and CCL7 in endothelial cells. CYM-5442 could be a possible course of treatment for aGVHD.
| Targets |
Sphingosine 1-phosphate (S1P1) receptor ( EC50 = 1.35 nM )
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]. |
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| ln Vitro |
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]. Treatment with CYM5442 (0.5 μM; 0-60 min; HEK293 cells) stimulates S1P1 phosphorylation in a time-dependent manner in P32 orthophosphate-labeled cells [1]. Medium/p44-MAPK phosphorylation, EC50 is 46 nM. The alanine R120 (R120A) mutant retained p42/p44-MAPK activity (EC50 of 67 nM) when stained with CYM5442. CYM5442 activates p42/p44-MAPK in E121A S1P1 cells in a concentration-dependent manner, with an average EC50 value of 134 nM [1]. Western Blot Analysis[1] Cell line: HEK293 cells stably expressing S1P1 fused to GFP at the carboxyl terminus Concentration: 0.5 µM Incubation time: 0 min, 2 min, 5 min, 10 min, 30 min, 60 min Results: in time Stimulates S1P1 phosphorylation in a dependent manner. |
| ln Vivo |
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]. CYM5442 (1 mg/kg; intraperitoneal injection; daily; for 5 days; male albinism Wistar status) treatment showed preservation of courtyard function at the courtyard evoked potential (VEP). CYM-treated animals compared to vehicle The fibrous layer (RNFL) is significantly thicker [2]. 1 mg/kg Administration: intraperitoneal injection; daily; continued for 5 days Results: Visual evoked potential (VEP) showed preservation of visual function. The number of retinal ganglion cells (RGC) increased significantly. |
| Cell Assay |
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]. Cell Line: HEK293 cells stably expressing S1P1 fused to GFP on the carboxy-terminus Concentration: 0.5 µM Incubation Time: 0 minutes, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 60 minutes Result: Stimulated S1P1 phosphorylation in a time-dependent manner. |
| Animal Protocol |
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]. Adult male albino Wistar rats (8-10 weeks old; 180-230 g) infected ocular endothelin-1 (ET-1) 1 mg/kg Intraperitoneal injection; daily; for 5 days |
| ADME/Pharmacokinetics |
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]. |
| Toxicity/Toxicokinetics |
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].
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| References |
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| Additional Infomation |
CYM5442 is an oxadiazole and cyclic compound.
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| Molecular Formula |
C23H27N3O4
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| Molecular Weight |
409.486
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| Exact Mass |
409.2
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| Elemental Analysis |
C, 67.46; H, 6.65; N, 10.26; O, 15.63
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| CAS # |
1094042-01-9
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| Related CAS # |
CYM5442 hydrochloride; 1783987-80-3
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| PubChem CID |
25110406
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| Appearance |
Light yellow to light brown solid powder
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| LogP |
4.161
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
30
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| Complexity |
525
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O1C(C2C([H])=C([H])C(=C(C=2[H])OC([H])([H])C([H])([H])[H])OC([H])([H])C([H])([H])[H])=NC(C2C([H])=C([H])C([H])=C3C=2C([H])([H])C([H])([H])C3([H])N([H])C([H])([H])C([H])([H])O[H])=N1
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| InChi Key |
NUIKTBLZSPQGCP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H27N3O4/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
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| Chemical Name |
2-[[4-[5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl]-2,3-dihydro-1H-inden-1-yl]amino]ethanol
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| Synonyms |
CYM 5442; CYM-5442; 1094042-01-9; CYM5,442; CYM-5,442; RefChem:130105; CYM 5,442; CYM5442
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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) |
DMSO: ~2.6 mg/mL (~6.4 mM）
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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 | 2.4421 mL | 12.2103 mL | 24.4206 mL | |
| 5 mM | 0.4884 mL | 2.4421 mL | 4.8841 mL | |
| 10 mM | 0.2442 mL | 1.2210 mL | 2.4421 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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