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| Targets |
The target of the active isomer (1S,3R)-RSL3 is glutathione peroxidase 4 (GPX4). GPX4 is a key enzyme that reduces lipid hydroperoxides, protecting cells from ferroptosis (a form of iron‑dependent, non‑apoptotic cell death). The active isomer inhibits GPX4 by covalently binding to its active site selenocysteine residue, leading to the accumulation of lipid peroxides and ferroptotic cell death. In contrast, (1R,3R)-RSL3, being the inactive stereoisomer, does not bind effectively to GPX4 and does not induce ferroptosis.
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| ln Vitro |
(1R,3R)-RSL3 is the inactive isomer of the ferroptosis activator RSL3. It serves as a negative control to confirm the specificity of GPX4 inhibition and ferroptosis induction. In vitro, treatment with (1R,3R)-RSL3 should not cause a reduction in GPX4 protein expression or lead to the accumulation of lipid peroxides. It is used in head and neck cancer cells and other cell lines to differentiate on‑target effects from off‑target effects.
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| ln Vivo |
(1R,3R)-RSL3 is not used as an active therapeutic agent in vivo. Its primary use is as a negative control in animal studies to validate the anti‑tumor effects observed with the active (1S,3R)-RSL3 isomer. In a mouse xenograft model, the inactive isomer should not cause tumor growth inhibition or lipid peroxidation.
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| Enzyme Assay |
Non-cell-based (cell-free) experiments for (1R,3R)-RSL3 are used to measure its binding affinity and inhibitory activity against GPX4. A standard assay uses recombinant GPX4 protein. The enzyme is incubated with a reducing substrate (e.g., glutathione, GSH) and a lipid hydroperoxide substrate (e.g., phosphatidylcholine hydroperoxide, H2O2) in the presence of increasing concentrations of the compound (1-1000 nM). The activity of GPX4 is measured by monitoring the consumption of NADPH (in a coupled assay) or by directly measuring the reduction in lipid hydroperoxides. The active (1S,3R)-RSL3 isomer has an IC₅0 in the low nanomolar range, while the (1R,3R)-RSL3 isomer has an IC₅0 that is orders of magnitude higher, confirming its inactivity.
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| Cell Assay |
Cell-based assays for (1R,3R)-RSL3 are conducted to demonstrate its lack of effect on ferroptosis. Cells (e.g., head and neck cancer cells, fibrosarcoma HT‑1080 cells) are seeded in 96‑well plates and treated with varying concentrations of (1R,3R)-RSL3 (1 nM to 10 uM) for 24-48 hours. Cell viability is measured by MTT or CellTiter‑Glo assay. The EC₅0 for cell death is determined. In contrast to the active isomer, which typically has an EC₅0 in the low nanomolar range, the (1R,3R)-RSL3 isomer should have an EC₅0 >10 uM or be completely non‑toxic. Lipid peroxidation is measured using a fluorescent probe like C11‑BODIPY⁵⁸¹/⁵⁹¹. Cells are treated with the compound for 6-8 hours, stained with the probe, and analyzed by flow cytometry. The active isomer causes an increase in fluorescence (lipid peroxidation), while the (1R,3R)-RSL3 isomer should not.
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| Animal Protocol |
In vivo animal experiments for (1R,3R)-RSL3 are conducted as a negative control. In a mouse xenograft model (e.g., with HT‑1080 fibrosarcoma cells), mice are treated with the compound intraperitoneally at doses of 5-15 mg/kg daily for 10-20 days. Tumor volume is measured by calipers. The active (1S,3R)-RSL3 isomer causes significant tumor growth inhibition (TGI), while the (1R,3R)-RSL3 isomer should have no effect on tumor growth compared to a vehicle control. This validates that the tumor regression is specifically due to GPX4 inhibition and ferroptosis induction.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for (1R,3R)-RSL3 are not available. The compound has a molecular formula of C23H21ClN2O₅ and a molecular weight of 440.88 g/mol. It appears as a solid at room temperature (melting point not reported). The compound is stored at -20degC (stable for up to 3 years) or in a solvent at -80degC. Purity is ≥98% (HPLC).
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| Toxicity/Toxicokinetics |
The toxicity of (1R,3R)-RSL3 is expected to be significantly lower than that of the active isomer. As an inactive stereoisomer, it should not induce GPX4‑mediated ferroptosis and therefore should not cause the acute toxicity associated with ferroptosis (e.g., liver and kidney damage). As a research chemical, standard safety precautions apply.
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| References | |
| Additional Infomation |
Additional information: The compound has a CAS number of 1219810-15-7. It is also known as 1R,3R-RSL3 and RSL3 isomer (inactive). The compound is for research use only and is not for human or veterinary use. It is used as a negative control in ferroptosis research.
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| Molecular Formula |
C23H21CLN2O5
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| Molecular Weight |
440.88
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| CAS # |
1219810-15-7
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| Appearance |
Typically exists as solids at room temperature
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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 | 2.2682 mL | 11.3410 mL | 22.6819 mL | |
| 5 mM | 0.4536 mL | 2.2682 mL | 4.5364 mL | |
| 10 mM | 0.2268 mL | 1.1341 mL | 2.2682 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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