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Purity: ≥98%
Liproxstatin-1 (Lip-1) is a novel and potent inhibitor of ferroptosis with important biological activity. It inhibits ferroptosis with an IC50 value of 22 nM in cell free assays. Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells that overexpress oncogenic RAS. Liproxstatin-1 showed inhibition against ferroptosis-inducing agent (FIN) -triggered cell death. In Gpx4-/- cells, liproxstatin-1 inhibited RSL3-induced BODIPY 581/591 C11 oxidation.
| Targets |
Ferroptosis (IC50 = 22 nM)
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| ln Vitro |
In mouse embryonic fibroblasts, lipostatin-1 has anti-ferroptosis action with an IC50 of about 38 nM[2].Fer-1 and Lip-1 Are Inherently Good, but Not Great, Radical-Trapping Antioxidants; Fer-1 and Lip-1 Are Excellent Radical-Trapping Antioxidants in Phospholipid Bilayers; Fer-1 and Lip-1 Are Poor Inhibitors of 15-LOX-1 at Best, As Is α-TOH. [2]
Supporting the involvement of ferroptosis, treatment with Liproxstatin-1 was able to protect HRPTEpiCs from RSL3-induced cell death (Fig. 7a). Similar findings were obtained in the immortalized human renal proximal tubule epithelial cell line, HK-2 (Supplementary Fig. 7b). Next, we knocked down Gpx4 in HK-2 cells using a pool of siRNAs, revealing a small yet significant decrease in cell viability sensitive to αToc treatment (Supplementary Fig. 7c). Inducing cell death through Gpx4 knockdown, however, turned out to be challenging for the high expression levels of Gpx4 in kidney tubular epithelial cells (Supplementary Fig. 7d). Nonetheless, the Gpx4 knockdown rendered cells more sensitive to ferroptosis-inducing agents (Supplementary Fig. 7e), indicating a Gpx4-regulated ferroptotic machinery in human proximal tubular epithelial cells. Moreover, RSL3-induced BODIPY 581/591 C11 oxidation could be blocked by Liproxstatin-1 (Fig. 7b), demonstrating that Liproxstatin-1 prevents ferroptotic cell death also in humans.[1] |
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| ln Vivo |
In human cells, Gpx4/kidney, and ischemia/reperfusion-induced tissue injury models, liprostatin-1 (10 mg/kg, i.p.) reduces ferroptosis [1].
Next, researchers assessed the in vivo potential of liprostatin-1 to prevent the consequences of inducible Gpx4 disruption in animals. On TAM treatment of CreERT2;Gpx4fl/fl mice, mice were injected daily with liprostatin-1 intraperitoneally (i.p.) until the mice showed signs of acute renal failure (ARF), at which point they were euthanized (Fig. 7c). Notably, Liproxstatin-1 remarkably extended survival compared with the vehicle-treated group. TUNEL staining at day 9 after TAM treatment showed a strongly reduced number of TUNEL+ cells in Liproxstatin-1 compared with the vehicle-treated group (Fig. 7d), suggesting that Liproxstatin-1 delays ferroptosis in tubular cells. The discrepancy between death of mice due to ARF in Fig. 1c and vehicle-treated animals in Fig. 7c is explained by the mode of TAM administration, feeding versus i.p. injection. As an independent proof-of-concept, we analysed the in vivo efficacy of Liproxstatin-1 in a bona fide model of hepatic ischaemia/reperfusion injury, providing evidence that liprostatin-1 mitigated tissue injury in ischaemia/reperfusion-induced liver injury (Fig. 7e). Hence, these data implicate ferroptosis as a contributor in ischaemia/reperfusion-induced tissue injury and hold great promise for the development of therapeutics to treat related pathologies.[1] |
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| Enzyme Assay |
Inhibited Autoxidation of Styrene[2]
These experiments were carried out in a manner similar to that described in our previous work.36 In brief, styrene was washed thrice with 1 M aqueous NaOH, dried over MgSO4, filtered, distilled under vacuum, and purified by percolating through silica, then basic alumina. To a cuvette of 1.25 mL of styrene was added 1.18 mL of chlorobenzene, and the solution equilibrated for 5 min at 37 °C. The cuvette was blanked, 12.5 μL of 2 mM PBD-BODIPY in 1,2,4-trichlorobenzene was added followed by 50 μL of 0.3 M AIBN in chlorobenzene, and the solution was thoroughly mixed. After 20 min, an aliquot of liprostatin-1, Fer-1, C15-THN, PMHC, or α-TOH stock solution (1 mM) in chlorobenzene was added and the loss of absorbance at 591 nm followed. The inhibition rate constant (kinh) and stoichiometry (n) were determined for each experiment according to Figure 1B (see the Supporting Information for complete details). Autoxidations were carried out with three technical replicates at each concentration, and kinetics are reported as the mean ± standard deviation. Inhibited Autoxidation of PC Liposomes[2] To a cuvette of 2.34 mL of 10 mM PBS at pH 7.4 were added liposomes (125 μL of 20 mM stock in PBS at pH 7.4),32 and the solution was equilibrated for 5 min at 37 °C. The cuvette was blanked, 10 μL of 2 mM STY-BODIPY in DMSO was added followed by 10 μL of 0.05 M MeOAMVN in acetonitrile, and the solution was thoroughly mixed. After 5 min, an aliquot of liprostatin-1, Fer-1, C15-THN, PMHC, or α-TOH stock solution (1 mM) in DMSO was added and the loss of absorbance at 565 nm followed. The inhibition rate constant (kinh) and stoichiometry (n) were determined for each experiment according to Figure 3B (see the Supporting (see the Supporting Information for complete details). Autoxidations were carried out with three technical replicates at each concentration, and kinetics are reported as the mean ± standard deviation. Indistinguishable results were obtained in select control experiments where the antioxidant was added prior to liposome extrusion. |
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| Cell Assay |
Phenotypic screening for ferroptosis inhibitors[1]
In brief, compound seeding onto 96-well plates (1,000 cells per well) was carried out simultaneously with 1 μM TAM administration (leading to Gpx4 inactivation) obviating multiple medium changes, followed by incubation for 72 h. Cell viability was assessed subsequently using the live/dead assay dye AquaBluer. In the primary screening round, all compounds were tested at a single concentration of 10 μM and positive hits were selected from wells with >80% cell viability. To confirm primary hits, compounds were re-screened in the same assay and dose-dependent survival as well as toxicity curves were obtained using concentrations of 0–100 μM. IC50 and TC50 values were calculated using the GraphPad Prism software. Validated hits were then evaluated based on efficacy, selectivity for ferroptosis, therapeutic range and physicochemical properties. In addition, an in silico ADME-Tox screening was implemented to exclude compounds with potential in vivo side effects. To further validate liprostatin-1, SAR studies were performed using commercially available derivatives. |
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| Animal Protocol |
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| ADME/Pharmacokinetics |
In addition, we assessed important ADME (absorption, distribution, metabolism and excretion) parameters (Table 1), indicating a very promising pharmacokinetic profile for Liproxstatin-1. In summary, the initial SAR results provide the rationale for further improvement of Liproxstatin-1 by medicinal chemistry.[1]
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| References |
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| Additional Infomation |
Liproxstatin-1 is an azaspiro compound that is 1'H-spiro[piperidine-4,2'-quinoxaline] in which the hydrogen at position 3' is replaced by a (3-chlorobenzyl)amino group. It is a potent inhibitor of ferroptosis. It has a role as a ferroptosis inhibitor, a radical scavenger, an antioxidant and a cardioprotective agent. It is a member of monochlorobenzenes, a secondary amino compound, an azaspiro compound and an organic heterotricyclic compound.
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| Molecular Formula |
C19H21CLN4
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|---|---|
| Molecular Weight |
340.85
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| Exact Mass |
340.145
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| Elemental Analysis |
C, 66.95; H, 6.21; Cl, 10.40; N, 16.44
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| CAS # |
950455-15-9
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| Related CAS # |
Liproxstatin-1 hydrochloride;2250025-95-5;Liproxstatin-1-13C6;Liproxstatin-1-15N
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| PubChem CID |
135735917
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
581.4±50.0 °C at 760 mmHg
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| Flash Point |
305.4±30.1 °C
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| Vapour Pressure |
0.0±1.6 mmHg at 25°C
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| Index of Refraction |
1.678
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| LogP |
2.67
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
24
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| Complexity |
460
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
YAFQFNOUYXZVPZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H21ClN4/c20-15-5-3-4-14(12-15)13-22-18-19(8-10-21-11-9-19)24-17-7-2-1-6-16(17)23-18/h1-7,12,21,24H,8-11,13H2,(H,22,23)
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| Chemical Name |
N-[(3-Chlorophenyl)methyl]-spiro[piperidine-4,2′(1′H)-quinoxalin]-3′-amine
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| Synonyms |
Liproxstatin-1; 950455-15-9; Liproxstatin 1; Lip-1; liproxstatin1; N-(3-Chlorobenzyl)-1'H-spiro[piperidine-4,2'-quinoxalin]-3'-amine; CHEBI:173097; N-[(3-CHLOROPHENYL)METHYL]-1'H-SPIRO[PIPERIDINE-4,2'-QUINOXALIN]-3'-AMINE;
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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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.33 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 25.0 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: ≥ 2.5 mg/mL (7.33 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 25.0 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: ≥ 2.5 mg/mL (7.33 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 2.5 mg/mL (7.33 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: ≥ 2.5 mg/mL (7.33 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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. Solubility in Formulation 6: ≥ 0.5 mg/mL (1.47 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9338 mL | 14.6692 mL | 29.3384 mL | |
| 5 mM | 0.5868 mL | 2.9338 mL | 5.8677 mL | |
| 10 mM | 0.2934 mL | 1.4669 mL | 2.9338 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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