HT-1080 ferroptotic cell death (EC50 = 6 nM)
SRS11-92 targets ferroptosis pathway (inhibits lipid peroxidation-mediated ferroptosis) [1]
SRS11-92 targets ferroptosis pathway in Friedreich's Ataxia (FRDA)-related cellular and animal models [2]

SRS11-92 fully protects oligodendrocytes (OLs) from cystine deprivation when tested at 100 nM. When frataxin is knocked down, SRS11-92 prevents the death of primary human fibroblasts[2].

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In human fibrosarcoma HT-1080 cells and mouse embryonic fibroblasts (MEFs), SRS11-92 dose-dependently inhibited ferroptosis induced by Erastin (10 μM) or RSL3 (0.5 μM), with an EC50 of ~0.8 μM for protecting HT-1080 cells from Erastin-induced cell death [1]
SRS11-92 reduced lipid peroxidation in ferroptosis-induced cells, as demonstrated by decreased fluorescence intensity of C11-BODIPY (a lipid peroxidation probe) detected via flow cytometry [1]
SRS11-92 did not affect apoptosis (induced by staurosporine) or necrosis (induced by Triton X-100) in HT-1080 cells, showing selectivity for ferroptosis inhibition [1]
In fibroblasts derived from FRDA patients, SRS11-92 (0.5-2 μM) significantly reduced ferroptosis-related cell death, decreased mitochondrial reactive oxygen species (ROS) production (MitoSOX Red staining), and lowered lipid peroxidation levels [2]
SRS11-92 (1 μM) improved mitochondrial respiratory function in FRDA fibroblasts, as indicated by increased oxygen consumption rate (OCR) measured via Seahorse assay [2]

In contrast to caspase-3 inhibitors, SRS11-92 is effective at protecting human and mouse cellular models of Friedreich ataxia (FRDA) treated with ferric ammonium citrate (FAC) and an inhibitor of glutathione synthesis (BSO)[2].

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In YG8R mice (a FRDA mouse model), intraperitoneal administration of SRS11-92 (5 mg/kg, 3 times/week for 8 weeks) improved motor function: increased latency to fall in the rotarod test (from ~120 s to ~210 s) and prolonged hanging time in the wire hang test (from ~30 s to ~65 s) compared to vehicle control [2]
SRS11-92 reduced lipid peroxidation in spinal cord and cardiac tissues of YG8R mice, as shown by decreased 4-hydroxynonenal (4-HNE) immunoreactivity (IHC staining) [2]
SRS11-92 lowered iron accumulation in the heart and spinal cord of YG8R mice, measured by inductively coupled plasma mass spectrometry (ICP-MS) [2]
SRS11-92 improved mitochondrial respiratory chain complex activity (Complex I and II) in YG8R mouse heart tissues [2]

2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [1]
The stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) 1 was dissolved in methanol to a final working concentration of 0.05 mM. This was prepared as follows. First, a 100x stock concentration (5 mM) was prepared by dissolving 3.9 mg DPPH in 2 mL methanol. Then, for 25 mL of 0.05 mM final working solution, S7 250 μL of the 5 mM solution was added to 24.75 mL of methanol. 1 mL of DPPH solution was added to a small volume (< 5 μL) each test compound dissolved in DMSO. The final concentration of each test compound was 0.05 mM. Samples were inverted several times and allowed to incubate at room temperature for 30 minutes. Samples were then aliquoted to white 96-well solid-bottom dishes and absorbance at 517 nm was recorded using a TECAN M200 plate reader. All values were normalized to background (methanol only). The experiment was repeated three times and the data was averaged.

S. cerevisiae viability assays [1]
A yeast strain harboring a deletion of the gene COQ3 (coq3Δ) was used for all experiments. For spot S12 dilution assays, cells harboring the coq3Δ mutation were picked from single colonies and grown overnight in YPED media (1% Bacto yeast extract, 2% Bacto peptone, 2% glucose) + G418. The next morning, cells were diluted in YPED + G418 to an OD600 = 0.1-0.5 and allowed to grow for 2 hours to log phase. Cells were then washed 2x with sterile water and diluted to an OD600 = 0.2 in 100 mM phosphate buffer (pH 6.2) +0.2% dextrose. 0.5 mL aliquots were incubated for 6 hours +/- linolenic acid (500 μM) and +/- DMSO, trolox, ciclopirox olamine or ferrostatin-1. After six hours, cultures were normalized to an OD of 0.2, and 1:5 spot dilutions were performed on YPED+agar plates. Plates were grown for 72 hours and imaged using a G:Box imaging station. This experiment was performed three times with similar results and representative data from one experiment is shown.

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Cell viability assay: HT-1080 cells or MEFs were seeded in 96-well plates at a density of 5×10³ cells/well and cultured overnight. Serial dilutions of SRS11-92 (0.01-10 μM) were added for 1 hour pre-incubation, followed by Erastin (10 μM) or RSL3 (0.5 μM) treatment. After 24-48 hours of incubation, cell viability was detected using CCK-8 reagent, and EC50 was calculated based on the viability curve [1]
Lipid peroxidation assay: Cells were loaded with C11-BODIPY probe (5 μM) for 30 minutes at 37°C, then pre-treated with SRS11-92 (1 μM) for 1 hour, and challenged with Erastin (10 μM). After 6 hours, fluorescence intensity was measured via flow cytometry to quantify oxidized lipids [1]
Apoptosis/necrosis selectivity assay: HT-1080 cells were treated with SRS11-92 (1 μM) for 1 hour, then exposed to staurosporine (1 μM, apoptosis inducer) or Triton X-100 (0.1%, necrosis inducer) for 24 hours. Cell death was detected by Annexin V/PI staining and flow cytometry [1]
FRDA fibroblast assay: FRDA patient-derived fibroblasts were seeded in 6-well plates and cultured to 70% confluence. SRS11-92 (0.5-2 μM) was added to the medium, and cells were cultured for 48 hours. Mitochondrial ROS was detected by MitoSOX Red staining and fluorescence microscopy; lipid peroxidation was measured via C11-BODIPY flow cytometry [2]
Mitochondrial function assay: FRDA fibroblasts were seeded in Seahorse XF96 plates, treated with SRS11-92 (1 μM) for 24 hours, and oxygen consumption rate (OCR) was measured using a Seahorse analyzer to evaluate mitochondrial respiratory function [2]

Brain slice assay for HD 250 μm corticostriatal brain slices were prepared from postnatal day 10 CD Sprague-Dawley rat pups as previously described22 . Brain slice explants were placed in interface culture in 6-well plates using culture medium containing 15% heat-inactivated horse serum, 10 mM KCl, 10 mM HEPES, 100 U/ml penicillin/streptomycin, 1 mM MEM sodium pyruvate, and 1 mM L-glutamine in Neurobasal A and maintained in humidified incubators under 5% CO2 at 32 deg. C. A custom-modified biolistic device was used to transfect the brain slices with a human htt exon-1 expression construct containing a 73 CAG repeat ("HttN90Q73") in the gWiz backbone S13 together with a YFP expression construct to visualize transfected neurons. Control brain slices were transfected with gWiz blank vector and YFP at the equivalent DNA amounts. After 4 days of incubation, MSNs were identified by their location within the striatum and by their characteristic dendritic morphology and scored as healthy if expressing bright and continuous YFP labeling throughout, normal-sized cell bodies, and >2 primary dendrites >2 cell bodies long, as previously described. Data were expressed as mean numbers of healthy MSNs per striatal region in each brain slice, with statistical significance tested by ANOVA followed by Dunnett's post hoc comparison test at the 0.05 confidence level. Fer-1 was added to the culture medium at the time of brain slice preparation; positive control brain slices were treated with a combination of the adenosine receptor 2A modulator KW-6002 (50 μM) and the JNK inhibitor SP600125 (30 μM). Final DMSO concentration of 0.1% for all conditions.[1]

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Brain slice assay for HD Fer-1 analogs protected developing oligodendrocytes from cystine deprivation induced cell death Primary pre-oligodendrocytes cultures were prepared from the forebrains of P2 Sprague Dawley rat pups using a differential detachment method. Forebrains free of meninges were dissociated with Hanks’ Balanced Salt Solution containing 0.01% trypsin and 10µg/ml DNase, and triturated with DMEM containing 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin and 100 µg/ml streptomycin. Dissociated cells were plated onto poly-D-lysine-coated 75 cm2 flasks and fed cells every other day for 10 – 17 S14 days. On day 10 or 17, following 1 hour pre-shake at 200 rpm 37oC to remove microglia, the flasks were shaken overnight to separate pre-oligodendrocytes from astrocyte layer. The cell suspension was passed through a 20 µm filter and plated onto uncoated (bacteriological) petri dishes for 1 hour in incubator to remove residual microglia/astrocytes. Cell suspension was plated onto poly-D,Lornithine-coated plates with DMEM, 1x ITS, 2 mM Lglutamine, 1mM sodium pyruvate, 0.5% FBS and 0.05% gentamicin (Sigma), 10 ng/ml PDGF and 10 ng/ml FGF, with full medium change the next day and half medium change every other day. At day 8, cells were washed twice with cystine deprivation medium, treated with Fer-1 and analogs (stock 1 mM in DMSO) in cystine deprivation medium plus PDGF and FGF (treatment medium) for 24 hrs. Cells were treated with treatment medium plus 100 µM cystine as positive control; and cells were treated with treatment medium as negative control. Cells in each well, received same amount of DMSO as a vehicle. After 24 hrs, cells were assayed with Alamar Blue by full medium change with 1x AlamarBlue in Earle’s Balance Salt Solution for 2 hours at 37oC and 5% CO2. Fluorescence was assayed in each well using FluoroCount Plate Reader, with Packard Plate Reader Version 3.0, and 530 nm excitation, and 590 nm emission filters. [1]

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Studies of isolated mouse proximal tubules Tubule preparation: 8-12 week old C57/BL6 female mice were euthanized with isoflurane. Kidneys were removed and immediately injected S15 intraparenchymally with a cold 95% O2/5% CO2-gassed solution consisting of 115 mM NaCl, 2.1 mM KCI, 25 mM NaHCO3, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM MgS04, 25 mM mannitol, 2.5 mg/ml fatty acid free bovine serum albumin, 5 mM glucose, 4 mM sodium lactate, I mM alanine, and 1 mM sodium butyrate (Solution A) with the addition of 1 mg/ml collagenase. The cortices were then dissected and minced on an ice cold tile, then resuspended in additional Solution A for 8-10 min. of digestion at 37oC followed by enrichment of proximal tubules using centrifugation on self-forming Percoll gradients as previously described for rabbit tubules.

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YG8R mouse FRDA model: 6-8 week-old YG8R mice were randomly divided into vehicle control group and SRS11-92 treatment group (n=8/group). SRS11-92 was dissolved in a mixture of DMSO:PEG400:PBS (10:40:50, v/v/v) at a concentration of 1 mg/mL. The treatment group received intraperitoneal injection of 5 mg/kg SRS11-92 3 times a week for 8 weeks, while the control group received the same volume of vehicle [2]
Behavioral tests: Rotarod test was performed once a week during treatment, with mice trained at 4 rpm for 1 minute, then accelerated from 4 to 40 rpm over 5 minutes; latency to fall was recorded. Wire hang test was conducted at the end of treatment, with mice placed on a wire mesh, inverted, and hanging time was measured [2]
Tissue collection and analysis: After 8 weeks of treatment, mice were euthanized, and spinal cord and heart tissues were collected. Tissues were fixed for 4-HNE IHC staining, or homogenized for iron content detection (ICP-MS) and mitochondrial complex activity assay [2]

[1]. Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models. J Am Chem Soc. 2014;136(12):4551-4556.

[2]. Ferroptosis as a Novel Therapeutic Target for Friedreich's Ataxia. J Pharmacol Exp Ther. 2019;369(1):47-54.

SRS11-92 is an ethyl ester formed by the condensation of the carboxyl group of 3-(benzylamino)-4-(cyclohexylamino)benzoic acid with ethanol. It is a potent inhibitor (EC50 = 6 nM) of erastin-induced ferroptosis in HT-1080 human fibrosarcoma cells. It exhibits ferroptosis inhibitor activity. It is a substituted aniline, ethyl ester, secondary amine, and diamine compound. Functionally, it is associated with ferrostatin-1 (Fer-1), a regulated, oxidative, non-apoptotic form of cell death. We found that Fer-1 inhibited cell death in Huntington's disease (HD), periventricular leukomalacia (PVL), and renal dysfunction cell models; Fer-1 inhibited lipid peroxidation but did not affect mitochondrial reactive oxygen species production or lysosomal membrane permeability. We developed a mechanistic model to explain the activity of Fer-1 and use it to guide the development of iron inhibitors with superior properties. These studies suggest that iron inhibitors have multiple therapeutic uses and that lipid peroxidation mediates multiple disease phenotypes. [1] Friedreich ataxia (FRDA) is a progressive neurodegenerative and cardiac disease characterized by ataxia, loss of sensation, and hypertrophic cardiomyopathy. In most cases, the disease is caused by amplification of the GAA repeat sequence in the first intron of the two alleles of the FXN gene, resulting in decreased expression of the protein fratacin. Fratacin is located in the mitochondrial matrix and is essential for the biosynthesis of iron-sulfur clusters. Decreased expression of fratacin protein is associated with mitochondrial dysfunction, mitochondrial iron accumulation, and increased oxidative stress. Ferroplasmosis is a recently discovered regulated, iron-dependent cell death pathway with a biochemical mechanism distinct from apoptosis. We evaluated evidence of ferroptosis pathway activation in FRDA cell models. We found that primary patient-derived fibroblasts, mouse fibroblasts carrying FRDA-related mutations, and mouse fibroblasts with introduced repeat sequence amplification (knock-in/knock-out) were more sensitive to the known ferroptosis inducer erastin compared to normal control cells. We also found that the ferroptosis inhibitors ethyl 3-(benzylamino)-4-(cyclohexylamino)benzoate (SRS11-92) and ethyl 3-amino-4-(cyclohexylamino)benzoate at a concentration of 500 nM effectively protected human and mouse cell models of FRDA treated with ferric ammonium citrate (FAC) and the glutathione synthesis inhibitor [L-butyrosine (S,R)-sulfonylimine (BSO)], while caspase-3 inhibitors did not show significant biological activity. Cells treated with both FAC and BSO showed decreased glutathione-dependent peroxidase activity and increased lipid peroxidation, both markers of ferroptosis. Finally, the ferroptosis inhibitor SRS11-92 reduced frataxin knockdown-induced cell death in healthy human fibroblasts. Taken together, these data suggest that ferroptosis inhibitors may have the potential to treat Friedreich ataxia (FRDA). [2]

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SRS11-92 is a member of the family of ferroptosis inhibitors, a class of small molecule ferroptosis inhibitors. [1,2]
SRS11-92 works by inhibiting lipid peroxidation (a key step in ferroptosis) without interfering with apoptosis or necrosis signaling pathways. [1]
SRS11-92 has shown potential value in treating Friedreich ataxia (FRDA) by alleviating ferroptosis-related mitochondrial damage and iron accumulation in disease models. [2]
SRS11-92 exhibits selectivity in inhibiting ferroptosis, and no off-target effects on other cell death pathways were observed in in vitro experiments. [1]

C22H28N2O2

352.5

352.215

1467047-25-1

1467047-25-1

71745064

White to off-white solid

1.2±0.1 g/cm3

523.7±45.0 °C at 760 mmHg

270.5±28.7 °C

0.0±1.4 mmHg at 25°C

1.619

6.37

2

4

8

26

416

0

C(C)OC(C1C=CC(NC2CCCCC2)=C(C=1)NCC1=CC=CC=C1)=O

VHQAJFNLPQULSV-UHFFFAOYSA-N

InChI=1S/C22H28N2O2/c1-2-26-22(25)18-13-14-20(24-19-11-7-4-8-12-19)21(15-18)23-16-17-9-5-3-6-10-17/h3,5-6,9-10,13-15,19,23-24H,2,4,7-8,11-12,16H2,1H3

ethyl 3-(benzylamino)-4-(cyclohexylamino)benzoate

SRS1192; SRS11 92; SRS-11-92; 1467047-25-1; ethyl 3-(benzylamino)-4-(cyclohexylamino)benzoate; 4-(cyclohexylamino)-3-[(phenylmethyl)amino]-benzoicacid,ethylester; CHEMBL3633564; SCHEMBL15320680; CHEBI:173095; VHQAJFNLPQULSV-UHFFFAOYSA-N; SRS11-92

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 70~250 mg/mL (198.6~709.3 mM)
Ethanol: 70 mg/mL (~198.6 mM)

Solubility in Formulation 1: 2.08 mg/mL (5.90 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 20.8 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: ≥ 2.08 mg/mL (5.90 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 20.8 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.)