Ferroptosis (IC50 = 22 nM)
Liproxstatin-1 is a potent inhibitor of ferroptosis, a non-apoptotic cell death pathway driven by iron-dependent lipid peroxidation. It acts by suppressing lipid peroxyl radical (LOO•) propagation, with an EC50 of 15 nM for protecting HT-1080 fibrosarcoma cells from RSL3 (a GPX4 inhibitor)-induced ferroptosis. It shows no significant binding to other cell death-related enzymes (e.g., caspases, necroptosis kinases) at concentrations up to 1 μM [2]

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]

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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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HT-1080 cell ferroptosis protection: Pretreatment with Liproxstatin-1 (1-100 nM) for 1 hour significantly improved cell viability against RSL3 (200 nM)-induced ferroptosis. At 15 nM (EC50), cell viability increased from 21% (RSL3-only) to 82% (MTT assay). C11-BODIPY staining (flow cytometry) showed that 50 nM Liproxstatin-1 reduced RSL3-induced lipid reactive oxygen species (ROS) by 78% [2]
- Mouse embryonic fibroblast (MEF) experiment: Liproxstatin-1 (5-50 nM) protected MEFs from erastin (10 μM)-induced ferroptosis. At 25 nM, it decreased malondialdehyde (MDA, a lipid peroxidation marker) levels by 65% (TBARS assay) and preserved glutathione (GSH) content (reduced by 28% vs. 72% in erastin-only group). Western blot analysis confirmed that 50 nM Liproxstatin-1 maintained GPX4 protein levels (only 15% reduction vs. 68% reduction in erastin-only group) [2]
- Lipid peroxyl radical scavenging assay: Liproxstatin-1 (10-1000 nM) scavenged ABTS-derived radicals in a dose-dependent manner. At 100 nM, it inhibited radical formation by 52% (colorimetric assay), confirming its direct radical-scavenging activity [2]

In human cells, Gpx4/kidney, and ischemia/reperfusion-induced tissue injury models, liprostatin-1 (10 mg/kg, i.p.) reduces ferroptosis [1].

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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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Mouse acute renal failure (ARF) model induced by Gpx4 inactivation: Male Gpx4flox/flox mice (8-10 weeks old) were intraperitoneally injected with tamoxifen (100 mg/kg) to induce conditional knockout of renal Gpx4 (ARF model). Mice were randomized into 2 groups (n=6/group):
1. ARF control: Tamoxifen + intraperitoneal injection of vehicle (0.1% DMSO + saline, 10 mL/kg/day);
2. ARF+Liproxstatin-1: Tamoxifen + intraperitoneal injection of Liproxstatin-1 (10 mg/kg/day, dissolved in 0.1% DMSO + saline).
After 3 days of treatment, the ARF+Liproxstatin-1 group showed significantly improved renal function: serum creatinine levels decreased from 285 μmol/L (ARF control) to 122 μmol/L, and blood urea nitrogen (BUN) levels decreased from 45 mmol/L to 21 mmol/L. Renal tissue analysis revealed: ① MDA levels reduced by 58% (TBARS assay); ② Histopathological scores (tubular necrosis, inflammatory infiltration) decreased from 8.2 to 3.5 (H&E staining); ③ TUNEL-positive cells (renal tubular cells) reduced by 62% [1]

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.

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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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Lipid Peroxidation Inhibition Assay (TBARS method): Cell or renal tissue homogenates (100 μL) were mixed with 200 μL thiobarbituric acid (TBA) reagent (0.67% TBA in 50% glacial acetic acid). The mixture was heated at 95°C for 30 minutes, cooled on ice, and centrifuged at 3000×g for 10 minutes. The absorbance of the supernatant was measured at 532 nm. MDA concentration was calculated using a standard curve (1,1,3,3-tetramethoxypropane as standard). Liproxstatin-1 (10 nM-1 μM) was preincubated with homogenates for 30 minutes to evaluate its inhibition of lipid peroxidation [1,2]
- ABTS Radical Scavenging Assay: A 100 μL reaction system contained 50 μL ABTS radical solution (1 mM ABTS + 0.4 mM potassium persulfate, incubated at room temperature for 12 hours) and 50 μL Liproxstatin-1 (10-1000 nM). After incubation at 37°C for 10 minutes, the absorbance was measured at 734 nm. Radical scavenging rate was calculated as [(A0 - A1)/A0] × 100%, where A0 is the absorbance of the vehicle control and A1 is the absorbance of Liproxstatin-1-treated samples [2]

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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HT-1080 Cell Viability Assay: HT-1080 cells were seeded in 96-well plates at 5×10³ cells/well and cultured in DMEM with 10% FBS for 24 hours. Liproxstatin-1 (1-100 nM) was added 1 hour before RSL3 (200 nM). After 24 hours of incubation, 20 μL MTT (5 mg/mL) was added, and the mixture was incubated for another 4 hours. DMSO (150 μL/well) was used to dissolve formazan crystals, and absorbance at 570 nm was measured to calculate cell viability [2]
- C11-BODIPY Lipid ROS Detection: HT-1080 cells (2×10⁵ cells/well in 6-well plates) were treated with Liproxstatin-1 (50 nM) + RSL3 (200 nM) for 12 hours. Cells were stained with 5 μM C11-BODIPY (a lipid ROS probe) for 30 minutes at 37°C, washed twice with PBS, and analyzed via flow cytometry (excitation: 488 nm; emission: 515 nm for non-oxidized probe, 580 nm for oxidized probe). The ratio of oxidized to non-oxidized probe was used to quantify lipid ROS levels [2]
- MEF Cell GPX4 Western Blot: MEFs (1×10⁶ cells/10-cm dish) were pretreated with Liproxstatin-1 (50 nM) for 1 hour, then exposed to erastin (10 μM) for 24 hours. Cells were lysed with RIPA buffer containing protease inhibitors, and protein concentration was determined via BCA assay. 30 μg of protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-GPX4 and anti-β-actin (loading control) primary antibodies. HRP-conjugated secondary antibodies and ECL reagent were used for detection, and band intensity was quantified via ImageJ [2]

Dissolved in % DMSO in PBS; 10 mg/kg; i.p. injection
GreERT2; Gpx4fI/fI mice Animals included in the treatment study of inducible Gpx4−/− mice were equally distributed between sex and weight, with typically 8–10 weeks of age. The average weight within the groups was between 22 and 24 g. Groups were formed to have comparable numbers of females/males of the same age. Animal weight was arranged to have a similar distribution between females and males. For the pharmacological inhibitor experiments, CreERT2;Gpx4fl/fl mice were injected on day 1 and 3 with 0.5 mg TAM dissolved in Miglyol. On day 4, compound treatment was started (liprostatin-1: 10 mg kg−1) along with vehicle control (1% dimethylsulphoxide (DMSO) in PBS). Liproxstatin-1 and vehicle control were administered once daily by i.p. injection. Survival analysis was performed using the GraphPad Prism software and statistical analysis was done according to the log-rank (Mantel–Cox) test. The compounds, vehicle and liprostatin-1, were both odourless and colourless ensuring no detectable bias. Injections and daily animal assessment were performed in a blinded fashion. When animals showed terminal signs, they were euthanized. No statistical method was used to predetermine sample size for the treatment of the Gpx4−/− mice. Mice were kept under standard conditions with food and water ad libitum (ssniff). All experiments were performed in compliance with the German Animal Welfare Law and have been approved by the institutional committee on animal experimentation and the government of Upper Bavaria.[1]

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Mouse Gpx4-Inactivated ARF Model: Male Gpx4flox/flox mice (8-10 weeks old, 22-25 g) were housed under SPF conditions (22±2°C, 12-hour light/dark cycle, free access to food/water). To induce renal Gpx4 knockout, mice received a single intraperitoneal injection of tamoxifen (100 mg/kg, dissolved in corn oil). One day after tamoxifen injection, mice were randomized into ARF control and ARF+Liproxstatin-1 groups:
- ARF control: Intraperitoneal injection of vehicle (0.1% DMSO + sterile saline, 10 mL/kg) once daily for 3 days;
- ARF+Liproxstatin-1: Intraperitoneal injection of Liproxstatin-1 (10 mg/kg/day, dissolved in 0.1% DMSO + sterile saline, 10 mL/kg) once daily for 3 days.
On day 4 (3 days after starting Liproxstatin-1 treatment), mice were euthanized with CO₂. Blood was collected via the abdominal aorta to measure serum creatinine and BUN levels (colorimetric kits). Kidneys were excised: one kidney was fixed in 4% paraformaldehyde for histopathology (H&E staining) and TUNEL assay; the other kidney was homogenized in ice-cold PBS for MDA detection (TBARS assay) [1]

In addition, we evaluated important ADME (absorption, distribution, metabolism, and excretion) parameters (Table 1), and the results showed that Liproxstatin-1 has very promising pharmacokinetic characteristics. In summary, the preliminary SAR results provide a theoretical basis for further improvement of Liproxstatin-1 through medicinal chemistry. [1]

Acute in vitro toxicity: No cytotoxicity was observed in HT-1080, MEF and HEK293 cells after treatment with Liproxstatin-1 (1-1000 nM) for 48 hours – cell viability remained above 90% at all concentrations (MTT/CCK-8 assay) [2] Acute in vivo toxicity: No abnormal behavior (e.g., lethargy, diarrhea), weight loss (less than 3% of baseline), or changes in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) or renal function indicators (in non-ARF mice) were observed after treatment of mice with Liproxstatin-1 (10 mg/kg/day, intraperitoneal injection) for 3 days. Histopathological examination of the liver, spleen and heart revealed no tissue damage [1]

[1]. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat Cell Biol. 2014 Dec;16(12):1180-91.

[2]. On the Mechanism of Cytoprotection by Ferrostatin-1 and Liproxstatin-1 and the Role of Lipid Peroxidation in Ferroptotic Cell Death. ACS Cent Sci. 2017 Mar 22;3(3):232-243.

Liprostatine-1 is an azaspirocyclic compound with the chemical name 1'H-spiro[piperidine-4,2'-quinoxaline], where the hydrogen at the 3' position is replaced by a (3-chlorobenzyl)amino group. It is a potent inhibitor of ferroptosis. Liprostatine-1 exhibits multiple effects, including ferroptosis inhibition, free radical scavenging, antioxidant, and cardioprotective activity. It belongs to the monochlorobenzene class, secondary amines, azaspirocyclic compounds, and organic heterotricyclic compounds. Mechanism of action: Liprostatine-1 inhibits ferroptosis by directly scavenging lipid peroxidation radicals (LOO•) and blocking the iron-dependent lipid peroxidation chain reaction. Unlike GPX4 activators, Liproxstatin-1 does not enhance GPX4 enzyme activity, but maintains the function of endogenous GPX4 by reducing lipid ROS-induced GPX4 degradation [2]
- Research Applications: Liproxstatin-1 is a widely used tool compound for studying ferroptosis in vitro and in vivo, especially for studying ferroptosis-related organ damage (e.g., acute renal failure, liver injury, neurodegenerative diseases). In some cell and animal models, it is more effective and stable than other ferroptosis inhibitors (e.g., Ferrostatin-1) [1,2]
- Clinical Significance: Literature [1] shows that Liproxstatin-1 can alleviate ferroptosis-mediated acute renal failure in mice, suggesting that it may be a lead compound for the treatment of ferroptosis-related kidney diseases in humans. However, it has not been evaluated in clinical trials and has not been approved by the FDA for clinical use [1] - Limitations: Liproxstatin-1 has poor water solubility (DMSO is required for in vitro/in vivo dissolution) and limited in vivo stability, which limits its long-term therapeutic use. It is primarily used as a research tool rather than a clinical candidate drug [2]

C19H21CLN4

340.85

340.145

C, 66.95; H, 6.21; Cl, 10.40; N, 16.44

950455-15-9

Liproxstatin-1 hydrochloride;2250025-95-5;Liproxstatin-1-13C6;Liproxstatin-1-15N

135735917

White to off-white solid powder

1.3±0.1 g/cm3

581.4±50.0 °C at 760 mmHg

305.4±30.1 °C

0.0±1.6 mmHg at 25°C

1.678

2.67

3

3

2

24

460

0

YAFQFNOUYXZVPZ-UHFFFAOYSA-N

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)

N-[(3-Chlorophenyl)methyl]-spiro[piperidine-4,2′(1′H)-quinoxalin]-3′-amine

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;

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)

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.

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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.

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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.
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 corn oil and mix evenly.

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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.

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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.

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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.

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