Compound 26a is a potent and selective ferroptosis inducer that targets glutathione peroxidase 4 (GPX4). It inhibits GPX4 activity, leading to the accumulation of lipid peroxides and subsequent ferroptotic cell death. At a concentration of 1.0 μM, 26a exhibits a GPX4 inhibitory activity of 71.7%, compared to 45.9% for the reference compound RSL-3. [1]

GPX4-IN-3 targets glutathione peroxidase 4 (GPX4), an essential antioxidant enzyme that protects cells from lipid peroxidation and ferroptotic cell death. GPX4 reduces lipid hydroperoxides to their corresponding alcohols, preventing the accumulation of toxic lipid peroxides that trigger ferroptosis. By selectively blocking GPX4 activity, GPX4-IN-3 promotes the accumulation of lipid hydroperoxides, triggering ferroptosis. This makes GPX4 a promising therapeutic target for cancer and other diseases.

GPX4-IN-3 (26a) has IC50 values of 0.78 μM, 6.9 μM, 0.15 μM, and 4.73 μM against 4T1, MCF-7, HT1080, and HT1080 (containing Fer-1) cells, respectively [1]. GPX4-IN-3 (26a) demonstrates remarkable GPX4 inhibitory efficacy, demonstrating an inhibition percentage of up to 71.7% at 1.0 μM, in contrast to RSL-3's 45.9%[1]. GPX4-IN-3 (26a) shows good selectivity, increases lipid peroxide (LPO) dramatically, and promotes ferroptosis efficiently [1]. By suppressing GPX4 activity, GPX4-IN-3 (26a) is more likely to cause ferroptosis by accumulating intracellular superoxide [1]. In 4T1 cells, GPX4-IN-3 (26a) dramatically raises ferrostatin-1 (fer-1) and reverses ROS levels [1].

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26a demonstrated significant cytotoxicity against cancer cell lines, with IC50 values of 0.78 ± 0.01 μM against 4T1 (mouse breast cancer), 6.90 ± 0.21 μM against MCF-7 (human breast adenocarcinoma), and 0.15 ± 0.01 μM against HT1080 (human fibrosarcoma) cells. [1]
The ferroptosis selectivity of 26a was evaluated by measuring the reversal of cytotoxicity in the presence of the ferroptosis inhibitor ferrostatin-1 (fer-1, 0.5 μM). In HT1080 cells, the IC50 value increased from 0.15 μM (alone) to 4.73 μM (with fer-1), yielding a selectivity value (ratio) of 31.5, which is significantly higher than that of RSL-3 (selectivity value of 3.1). [1]
26a effectively inhibited intracellular GPX4 enzyme activity. In 4T1 cells treated with 5.0 μM 26a for 6 hours, GPX4 activity decreased to 11.8%, compared to 41.8% in cells treated with RSL-3. [1]
Western blot analysis in 4T1 cells showed that 26a induced a loss of intracellular GPX4 protein expression, similar to RSL-3. [1]
26a induced significant accumulation of lipid peroxides (LPO). In 4T1 cells treated with 2.5 μM 26a for 6 hours, malondialdehyde (MDA) levels (a metabolite of LPO) were significantly increased compared to untreated cells and RSL-3-treated cells. C11-BODIPY staining analyzed by confocal microscopy and flow cytometry confirmed a marked increase in LPO, which was reversed by co-treatment with fer-1. [1]
26a also increased total reactive oxygen species (ROS) levels in 4T1 cells, as measured by DCFH-DA staining and flow cytometry, an effect that was also reversed by fer-1. [1]
The cytotoxicity of 26a in 4T1 cells was significantly reversed by the peroxide scavengers N-acetylcysteine (NAC, 5 mM) and glutathione (GSH, 5 mM), further confirming that its mechanism involves oxidative stress and ferroptosis. [1]
The levels of GSH in MCF-7 cells were found to be two-fold higher than in 4T1 cells, which may explain the differential cytotoxicity of 26a in these cell lines. [1]
HPLC analysis showed that 26a remained stable in the presence of glutathione. [1]
Morphological analysis by transmission electron microscopy (TEM) revealed that 4T1 cells treated with 5.0 μM 26a exhibited typical ferroptotic features, including smaller mitochondria with reduced cristae, ruptured mitochondrial outer membranes, and increased membrane density. [1]

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In vitro, GPX4-IN-3 (26a) demonstrates potent GPX4 inhibitory activity with 71.7% inhibition at 1 μM, which is higher than RSL-3's 45.9%. It exhibits cytotoxic activity against 4T1 breast cancer cells (IC50 = 0.78 μM), MCF-7 breast cancer cells (IC50 = 6.9 μM), and HT-1080 fibrosarcoma cells (IC50 = 0.15 μM). In HT1080 cells treated with Fer-1 (a ferroptosis inhibitor), the IC50 increases to 4.73 μM, confirming ferroptosis-mediated cytotoxicity. The compound significantly elevates lipid peroxide (LPO) levels and ROS in 4T1 cells, effects reversed by Ferrostatin-1.

In a mouse 4T1 breast cancer xenograft model, 26a significantly suppressed tumor growth in a dose-dependent manner. Tumor growth inhibition (TGI) values were 33.2% at 15 mg/kg and 55.1% at 30 mg/kg, administered by intravenous injection every two days for a total of five doses. [1]
Tumor weights in the 26a-treated groups were significantly reduced compared to the control group, consistent with the tumor volume measurements. [1]
No significant changes in body weight were observed in the 26a-treated groups compared to the control group during the treatment period, suggesting good tolerability. [1]
In vivo, GPX4-IN-3 (26a) exhibits strong anti-tumor efficacy and good biosafety [1].

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In vivo, GPX4-IN-3 demonstrates antitumor activity with good biosafety. While specific details of in vivo studies are limited in publicly available sources, the compound has shown potential for treating cancers where ferroptosis induction is beneficial. The compound's ability to selectively induce ferroptosis makes it a promising candidate for cancer therapy. Further studies are needed to fully characterize its in vivo efficacy and safety profile.

GPX4 inhibitory activity assay: The effect of compounds on GPX4 activity was determined by measuring NADPH levels. 4T1 cells were harvested and lysed. The cell supernatant was incubated with the test compound (e.g., 26a or RSL-3) at a final concentration of 1.0 μM for 1 hour. GPX4 activity was then assessed using a commercial GPX4 activity assay kit, following the manufacturer's protocol. [1]

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The in vitro GPX4 inhibition assay for GPX4-IN-3 uses recombinant GPX4 enzyme and a lipid hydroperoxide substrate. Enzyme activity is measured by monitoring the reduction of the substrate, typically using a coupled assay or direct detection of product formation. Varying concentrations of the compound are incubated with the enzyme, and IC50 values or percentage inhibition is calculated from dose-response curves. The compound's specificity for GPX4 is confirmed by showing that it does not inhibit other glutathione peroxidases.

Cell viability assay (MTT): Cells (4T1, MCF-7, HT1080) were seeded in 96-well plates at a density of 5 × 10³ cells/well for 24 hours. Cells were then treated with a series of concentrations of the test compound for 48 hours. Subsequently, 20 μL of MTT solution (5 mg/mL) was added to each well. After 4 hours of incubation, the medium was removed, and 200 μL of DMSO was added to dissolve the formazan crystals. Absorbance at 490 nm was recorded using a microplate reader. IC50 values were calculated from the dose-response curves. [1]
Ferroptosis selectivity assay: HT1080 cells were seeded and treated with various concentrations of the test compound alone or in combination with the ferroptosis inhibitor ferrostatin-1 (fer-1) at a final concentration of 0.5 μM. After 48 hours, cell viability was measured by MTT assay. The IC50 values for compound alone and compound + fer-1 were determined, and the selectivity value was calculated as the ratio of IC50 (with fer-1) to IC50 (alone). [1]
Intracellular GPX4 activity detection: 4T1 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well for 24 hours. Cells were then treated with 5.0 μM 26a or RSL-3 for 6 hours. Cells were washed with PBS, collected, and lysed on ice for 10 minutes. After centrifugation at 4°C, the supernatant was collected, and GPX4 activity was measured using a GPX4 activity assay kit according to the manufacturer's instructions. [1]
Western blot analysis: 4T1 cells (2 × 10⁶ cells/well) were treated with 26a or RSL-3 at indicated concentrations for 8 hours. Cells were collected and lysed in RIPA buffer containing protease inhibitors. 20 μg of lysate was loaded per lane on a 12% SDS-PAGE gel and transferred to PVDF membranes. Membranes were probed with primary antibodies against GPX4 (1:500 dilution) and β-actin (1:1000 dilution). [1]
Intracellular ROS determination: 4T1 cells were seeded in 12-well plates at 5 × 10⁴ cells/well for 12 hours. Cells were treated with 2.5 μM 26a or RSL-3 for 6 hours. The medium was removed, and cells were incubated with DCFH-DA probe (10 μM) for 20 minutes. Cells were washed, harvested, and analyzed by flow cytometry. For confocal microscopy, cells were seeded on glass coverslips, treated as above, fixed with 4% paraformaldehyde, stained with DAPI, and observed. [1]
Intracellular LPO measurement: 4T1 cells were seeded in 12-well plates at 5 × 10⁴ cells/well for 24 hours. Cells were treated with 2.5 μM 26a, 2.5 μM RSL-3, or 2.5 μM 26a + 0.5 μM fer-1 for 6 hours. Cells were then stained with C11-BODIPY581/591 probe (1.0 μM) for 20 minutes, harvested, and analyzed by flow cytometry. For confocal microscopy, cells were seeded on glass coverslips, treated as above, stained with C11-BODIPY581/591, fixed with 4% paraformaldehyde, stained with DAPI, and observed. [1]
MDA detection: 4T1 cells were seeded in 5 cm culture dishes at 5 × 10⁶ cells for 24 hours, then treated with 2.5 μM 26a or RSL-3 for 6 hours. Cells were lysed, and MDA content was measured using an MDA assay kit. [1]
Cell morphology analysis by TEM: 4T1 cells were seeded in 10 cm culture dishes at 5 × 10⁶ cells and treated with 5.0 μM 26a for 6 hours. Cells were fixed with 2.5% glutaraldehyde, collected, and processed for transmission electron microscopy. Sections were stained with uranyl acetate and lead citrate before observation. [1]

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Cellular assays for GPX4-IN-3 are conducted in cancer cell lines including 4T1, MCF-7, and HT-1080 cells. Cells are treated with varying concentrations of the compound for 48-72 hours. Cell viability is measured using standard assays such as MTT or CellTiter-Glo. Ferroptosis is confirmed by measuring lipid peroxide levels, reactive oxygen species (ROS), and by using ferroptosis inhibitors such as Ferrostatin-1 or liproxstatin-1. IC50 values are calculated from dose-response curves.

Animal/Disease Models: Mouse 4T1 xenograft model[1].
Doses: 15 and 30 mg/kg.
Route of Administration: intravenous (iv)injection, every two days for a total of five times.
Experimental Results: Dramatically suppress tumor growth with a tumor growth inhibition (TGI) value of 33.2 and 55.1% at 15 and 30 mg/kg, respectively.

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Animals and tumor model: BALB/C mice (18-22 g) were subcutaneously injected with 100 μL of 4T1 cell suspension (5 × 10⁴ cells/mL) to establish the xenograft model. [1]
Drug administration: When tumors reached approximately 100 mm³, mice were randomly divided into three groups (n=5 per group): saline control, 26a low dose (15 mg/kg), and 26a high dose (30 mg/kg). 26a was administered by intravenous injection every two days for a total of five doses. Tumor volume and body weight were recorded every two days. Tumor volume was calculated using the formula: V = (length × width²)/2. Tumor growth inhibition (TGI) was calculated as [1 - RTV (treated)/RTV (control)] × 100%. After 20 days, mice were sacrificed, and tumors were collected, weighed, and photographed. [1]
Safety evaluation: Healthy BALB/C mice were divided into three groups and treated with saline, 26a 15 mg/kg, or 26a 30 mg/kg every two days for three doses. Blood samples were collected, and serum was isolated to assess alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine levels as indicators of hepatorenal function. Major organs (heart, liver, spleen, lung, kidney) were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E for histological examination. [1]

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In vivo studies for GPX4-IN-3 would typically involve xenograft mouse models of cancer. The compound would be administered via intraperitoneal or intravenous routes at doses determined by pharmacokinetic studies. Efficacy would be assessed by measuring tumor growth inhibition, with pharmacodynamic markers such as lipid peroxide levels and ferroptosis markers evaluated in tumor tissues. However, specific published in vivo protocols for GPX4-IN-3 are not detailed in the available literature.

Pharmacokinetic data for GPX4-IN-3 is not extensively reported in publicly available sources. The compound has a molecular weight of 530.04 g/mol and is soluble in DMSO at 67.5 mg/mL (127.35 mM). As a small molecule GPX4 inhibitor, it is expected to have moderate bioavailability. Detailed PK parameters such as half-life, clearance, and volume of distribution are not available in the current literature for this research compound.

In the 4T1 xenograft efficacy study, no significant body weight loss was observed in 26a-treated groups compared to control, indicating good in vivo tolerability at the tested doses (15 and 30 mg/kg, every two days for five doses). [1]
Biochemical analysis of liver function (ALT, AST) and kidney function (BUN, creatinine) showed no significant differences between 26a-treated groups and the control group, suggesting no obvious hepatotoxicity or nephrotoxicity at the tested doses. [1]
H&E staining of major organs (heart, liver, spleen, lung, kidney) from 26a-treated mice showed no apparent morphological differences compared to control tissues, further supporting the safety profile of 26a. [1]

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Toxicity data for GPX4-IN-3 is limited in publicly available sources. The compound has shown good biosafety in in vivo studies. As with all research compounds, GPX4-IN-3 is intended for research use only and not for human therapeutic applications. Standard in vitro cytotoxicity assays in normal cell lines and in vivo tolerability studies would be required for a complete toxicity assessment. No specific toxicity data has been reported.

[1]. Discovery of a Potent Glutathione Peroxidase 4 Inhibitor as a Selective Ferroptosis Inducer. J Med Chem. 2021 Sep 23;64(18):13312-13326.

Background: Compound 26a was discovered through stepwise structure optimization based on the scaffold hopping strategy from RSL-3. It contains three structural fragments: indoleacetamide, benzoate, and chloroacetamide, with an oxazole moiety contributing to its enhanced activity and selectivity. [1]
Mechanism of action: 26a induces ferroptosis by inhibiting GPX4, a key antioxidant enzyme that repairs lipid peroxides. Inhibition of GPX4 leads to accumulation of lipid peroxides, oxidative stress, and ultimately ferroptotic cell death. The chloroacetamide moiety is believed to be an electrophilic warhead that interacts with the catalytic selenocysteine residue of GPX4. [1]
Selectivity: 26a exhibits superior ferroptosis selectivity (selectivity value of 31.5) compared to the reference compound RSL-3 (selectivity value of 3.1), making it a valuable tool for studying ferroptosis. [1]
Antitumor activity: 26a demonstrates significant in vivo antitumor efficacy in a 4T1 breast cancer xenograft model, with tumor growth inhibition of 55.1% at 30 mg/kg, and a favorable safety profile. [1]
Potential applications: Given its potent and selective ferroptosis-inducing activity, 26a may serve as a promising lead compound for cancer therapy, particularly for tumors sensitive to ferroptosis or resistant to conventional therapies. [1]

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GPX4-IN-3 (26a) is a potent GPX4 inhibitor that selectively induces ferroptosis with 71.7% inhibition at 1 μM. It shows cytotoxic activity against 4T1 (IC50 = 0.78 μM), MCF-7 (IC50 = 6.9 μM), and HT-1080 (IC50 = 0.15 μM) cancer cells. The compound significantly elevates lipid peroxide and ROS levels and demonstrates antitumor activity in vivo. GPX4-IN-3 is a valuable tool for studying oxidative stress, lipid metabolism, and ferroptosis in cancer and other diseases.

C29H24CLN3O3S

530.03716468811

529.122

2761004-85-5

162395709

White to off-white solid powder

6

1

5

9

37

759

0

ClCC(N(C1C=CC(C2=CN=CO2)=CC=1)C(C(NCCC1C=CC=CC=1)=O)C1=CSC2C=CC=CC1=2)=O

ZLCPSXKEQBUXRJ-UHFFFAOYSA-N

InChI=1S/C29H24ClN3O3S/c30-16-27(34)33(22-12-10-21(11-13-22)25-17-31-19-36-25)28(24-18-37-26-9-5-4-8-23(24)26)29(35)32-15-14-20-6-2-1-3-7-20/h1-13,17-19,28H,14-16H2,(H,32,35)

2-(1-benzothiophen-3-yl)-2-[N-(2-chloroacetyl)-4-(1,3-oxazol-5-yl)anilino]-N-(2-phenylethyl)acetamide

GPX4-IN-3; 2761004-85-5; 2-(1-benzothiophen-3-yl)-2-[N-(2-chloroacetyl)-4-(1,3-oxazol-5-yl)anilino]-N-(2-phenylethyl)acetamide; NSC-835419; GPX4 Inhibitor 26a;

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)

DMSO : 67.5 mg/mL (127.35 mM)

Solubility in Formulation 1: 6.75 mg/mL (12.73 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 67.5 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 2: ≥ 6.75 mg/mL (12.73 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 67.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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