SRS11-92

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]

在 100 nM 浓度下进行测试时，SRS11-92 可充分保护少突胶质细胞 (OL) 免受胱氨酸剥夺的影响。当 frataxin 被击倒时，SRS11-92 可防止原代人成纤维细胞死亡[2]。

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在人纤维肉瘤HT-1080细胞和小鼠胚胎成纤维细胞（MEFs）中，SRS11-92 剂量依赖性抑制Erastin（10 μM）或RSL3（0.5 μM）诱导的铁死亡，保护HT-1080细胞免受Erastin诱导死亡的EC50约为0.8 μM [1]
SRS11-92 可减少铁死亡诱导细胞中的脂质过氧化水平，通过流式细胞术检测脂质过氧化探针C11-BODIPY的荧光强度降低证实 [1]
SRS11-92 不影响星形孢菌素诱导的HT-1080细胞凋亡或曲拉通X-100诱导的坏死，显示出对铁死亡抑制的选择性 [1]
在弗里德赖希共济失调（FRDA）患者来源的成纤维细胞中，SRS11-92（0.5-2 μM）显著减少铁死亡相关细胞死亡，降低线粒体活性氧（ROS）产生（MitoSOX Red染色），并下调脂质过氧化水平 [2]
SRS11-92（1 μM）可改善FRDA成纤维细胞的线粒体呼吸功能，通过Seahorse实验检测到氧消耗率（OCR）升高证实 [2]

与 caspase-3 抑制剂相比，SRS11-92 可有效保护经柠檬酸铁铵 (FAC) 和谷胱甘肽合成抑制剂 (BSO) 处理的人类和小鼠弗里德赖希共济失调 (FRDA) 细胞模型[2]。

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在YG8R小鼠（FRDA疾病模型）中，腹腔注射 SRS11-92（5 mg/kg，每周3次，持续8周）可改善运动功能：与溶媒对照组相比，转棒实验中跌落潜伏期从约120秒延长至210秒，悬线实验中悬挂时间从约30秒延长至65秒 [2]
SRS11-92 减少YG8R小鼠脊髓和心脏组织中的脂质过氧化，通过4-羟基壬烯醛（4-HNE）免疫组织化学（IHC）染色显示阳性信号降低证实 [2]
SRS11-92 降低YG8R小鼠心脏和脊髓中的铁积累，通过电感耦合等离子体质谱法（ICP-MS）检测证实 [2]
SRS11-92 改善YG8R小鼠心脏组织中的线粒体呼吸链复合物（复合物I和II）活性 [2]

酿酒酵母活力测定[1]
所有实验均使用携带COQ3基因缺失（COQ3Δ）的酵母菌株。对于点S12稀释试验，从单个菌落中挑选携带coq3Δ突变的细胞，并在YPED培养基（1%Bacto酵母提取物、2%Bacto蛋白胨、2%葡萄糖）+G418中生长过夜。第二天早上，将细胞在YPED+G418中稀释至OD600=0.1-0.5，并使其生长2小时至对数期。然后用无菌水洗涤细胞2次，并在100mM磷酸盐缓冲液（pH 6.2）+0.2%葡萄糖中稀释至OD600=0.2。将0.5 mL等分试样孵育6小时+/-亚麻酸（500μM）和+/-DMSO、trolox、环吡酮胺或ferrostatin-1。6小时后，将培养物标准化至OD为0.2，并在YPED+琼脂平板上进行1:5的点稀释。将板生长72小时，并使用G:Box成像站进行成像。该实验进行了三次，结果相似，并显示了一次实验的代表性数据。

2,2-二苯基-1-苦肼基（DPPH）测定[1]
将稳定的自由基2,2-二苯基-1-苦肼基（DPPH）1溶解在甲醇中，使其最终工作浓度为0.05 mM。首先，通过将3.9mg DPPH溶解在2mL甲醇中制备100倍储备浓度（5mM）。然后，对于25 mL 0.05 mM的最终工作溶液，将S7 250μL的5 mM溶液加入24.75 mL甲醇中。将1 mL DPPH溶液加入到溶解在DMSO中的小体积（<5μL）的每种试验化合物中。每种测试化合物的最终浓度为0.05 mM。将样品倒置几次，并在室温下孵育30分钟。然后将样品等分到白色96孔固体底皿中，并使用TECAN M200平板读数器记录517nm处的吸光度。所有值均归一化为背景值（仅甲醇）。实验重复三次，取平均值。

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细胞活力实验：HT-1080细胞或MEFs以5×10³个/孔的密度接种于96孔板，过夜培养。加入系列浓度 SRS11-92（0.01-10 μM）预处理1小时，随后加入Erastin（10 μM）或RSL3（0.5 μM）处理。孵育24-48小时后，使用CCK-8试剂检测细胞活力，根据活力曲线计算EC50值 [1]
脂质过氧化实验：细胞用5 μM C11-BODIPY探针负载30分钟（37°C），经1 μM SRS11-92 预处理1小时后，用Erastin（10 μM）刺激。6小时后，通过流式细胞术检测荧光强度以量化氧化脂质水平 [1]
凋亡/坏死选择性实验：HT-1080细胞经1 μM SRS11-92 预处理1小时，随后暴露于星形孢菌素（1 μM，凋亡诱导剂）或曲拉通X-100（0.1%，坏死诱导剂）24小时。通过Annexin V/PI染色和流式细胞术检测细胞死亡情况 [1]
FRDA成纤维细胞实验：FRDA患者来源成纤维细胞接种于6孔板，培养至70%汇合度。加入 SRS11-92（0.5-2 μM）至培养基中，培养48小时。通过MitoSOX Red染色和荧光显微镜检测线粒体ROS；通过C11-BODIPY流式细胞术检测脂质过氧化 [2]
线粒体功能实验：FRDA成纤维细胞接种于Seahorse XF96板，经1 μM SRS11-92 处理24小时后，使用Seahorse分析仪检测氧消耗率（OCR），评估线粒体呼吸功能 [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 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]

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SRS11-92 is a member of the Ferrostatins family, a class of small-molecule ferroptosis inhibitors [1,2]
SRS11-92 exerts its effects by inhibiting lipid peroxidation, a key step in ferroptosis, without interfering with apoptotic or necrotic signaling pathways [1]
SRS11-92 shows potential therapeutic value for Friedreich's Ataxia (FRDA) by alleviating ferroptosis-related mitochondrial damage and iron accumulation in disease models [2]
SRS11-92 demonstrates selectivity for ferroptosis inhibition, with no observable off-target effects on other cell death pathways in vitro [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

50.4

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

注意： 本产品在运输和储存过程中需避光。

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)

配方 1 中的溶解度: 2.08 mg/mL (5.90 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (5.90 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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