HT-1080 ferroptotic cell death (EC50 = 6 nM)

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].

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].

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.

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.

[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 resulting from the formal condensation of the carboxy group of 3-(benzylamino)-4-(cyclohexylamino)benzoic acid with ethanol. It is a potent inhibitor of ferroptosis induced by erastin in HT-1080 human fibrosarcoma cells (EC50 = 6 nM). It has a role as a ferroptosis inhibitor. It is a substituted aniline, an ethyl ester, a secondary amino compound and a diamine. It is functionally related to a ferrostatin-1.

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Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of Huntington's disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species formation or lysosomal membrane permeability. We developed a mechanistic model to explain the activity of Fer-1, which guided the development of ferrostatins with improved properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid peroxidation mediates diverse disease phenotypes.[1]

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Friedreich ataxia (FRDA) is a progressive neuro- and cardio-degenerative disorder characterized by ataxia, sensory loss, and hypertrophic cardiomyopathy. In most cases, the disorder is caused by GAA repeat expansions in the first introns of both alleles of the FXN gene, resulting in decreased expression of the encoded protein, frataxin. Frataxin localizes to the mitochondrial matrix and is required for iron-sulfur-cluster biosynthesis. Decreased expression of frataxin is associated with mitochondrial dysfunction, mitochondrial iron accumulation, and increased oxidative stress. Ferropotosis is a recently identified pathway of regulated, iron-dependent cell death, which is biochemically distinct from apoptosis. We evaluated whether there is evidence for ferroptotic pathway activation in cellular models of FRDA. We found that primary patient-derived fibroblasts, murine fibroblasts with FRDA-associated mutations, and murine fibroblasts in which a repeat expansion had been introduced (knockin/knockout) were more sensitive than normal control cells to erastin, a known ferroptosis inducer. We also found that the ferroptosis inhibitors ethyl 3-(benzylamino)-4-(cyclohexylamino)benzoate (SRS11-92) and ethyl 3-amino-4-(cyclohexylamino)benzoate, used at 500 nM, were efficacious in protecting human and mouse cellular models of FRDA treated with ferric ammonium citrate (FAC) and an inhibitor of glutathione synthesis [L-buthionine (S,R)-sulfoximine (BSO)], whereas caspase-3 inhibitors failed to show significant biologic activity. Cells treated with FAC and BSO consistently showed decreased glutathione-dependent peroxidase activity and increased lipid peroxidation, both hallmarks of ferroptosis. Finally, the ferroptosis inhibitor SRS11-92 decreased the cell death associated with frataxin knockdown in healthy human fibroblasts. Taken together, these data suggest that ferroptosis inhibitors may have therapeutic potential in FRDA.[2]

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.)