FA16 is a specific, metabolically stable ferroptosis inducer (IC50=1.26 μM; HT1080 cells) and is an analogue of 2-(trifluoromethyl) benzimidazole. Cystine/glutamate antiporter (system Xc-) mediates the exchange of intracellular glutamate and extracellular cystine, and FA16 functions by inhibiting system Xc-. FA16 also significantly inhibited tumor growth in the HepG2 xenograft tumor model.

Physicochemical Properties

Molecular Formula	C22H25F3N4O2S
Molecular Weight	466.52
CAS #	3037775-42-8
Appearance	Typically exists as solid at room temperature
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	In rat and human liver microsomes, FA16 (1 μM; 5 min) exhibits adequate metabolic stability [1]. In HT1080 cells, FA16 (5 μM; 10 h) causes lipid ROS buildup and inhibits glutamate release [1]. In accordance with morphological traits associated with iron failure, FA16 (5 μM; 24 h) caused mitochondrial atrophy and increased membrane density. While apoptosis or necrosis inhibitors are ineffective, ferroptosis triggered by FA16 (10 μM; 24 h) can be saved by the iron shedding inhibitors Fer-1, Trolox, or DFO [1]. Specification Stability of microsomal (T1/2 min) Excretion rate (μL/min/mg protein) 15.6 88.6 Rat 10.4 132.8 Human
ln Vivo	FA16 (i.p.; 15 or 30 mg/kg; given every other day for 21 days) safe (did not cause weight loss), generated tumors, and greatly decreased tumor growth in a 786-O xenograft mice model. Tissues go through ferroptosis [1].
Cell Assay	Cell Viability Assay[1] Cell Types: Human cancer Cell Types: Clear-cell renal cell carcinoma cells(786-O ), breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa), hepatocellular carcinoma cells (HepG2), melanoma cells (A375), and prostate cancer cells (DU145); Human normal Cell Types: cardiomyocytes (AC16), colon mucosal epithelial cells(NCM460), embryonic kidney cells(293T), and hepatic cells(LO2) Tested Concentrations: 0-10 μM Incubation Duration: 48 hrs (hours) Experimental Results: Inhibited cell growth with IC50s of 0.7 μM(786-O), 4.34 μM(MDA-MB-231), 1.91 μM(HeLa), 1.33 μM(HepG2), 2.31 μM(A375), and 1.64 μM(DU145), respectively. Immunofluorescence[1] Cell Types: HT1080 cells Tested Concentrations: 5 μM Incubation Duration: 10 hrs (hours) Experimental Results: Dramatically induced lipid ROS accumulation, as indicated by the great enhancement in green fluorescence intensity. RT-PCR[1] Cell Types: HT1080 cells Tested Concentrations: 0.5 μM, 1 μM, and 5 μM Incubation Duration: 6 hrs (hours) and 18 hrs (hours) Experimental Results: Increased the system Xc-component SLC7A11, Cha
Animal Protocol	Animal/Disease Models: BALB/c nude mice bearing HepG2 tumors (sc)[1] Doses: 15 or 30 mg/kg Route of Administration: intraperitoneal (ip)injection; every other for 21 days Experimental Results: Dramatically inhibited tumor growth with a tumor growth inhibition (TGI) value of 47.6% and 77.1% at 15 and 30 mg/kg, respectively.
References	[1]. Discovery and optimization of 2-(trifluoromethyl)benzimidazole derivatives as novel ferroptosis inducers in vitro and in vivo. Eur J Med Chem. 2023 Jan 5;245(Pt 1):114905.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.1435 mL	10.7177 mL	21.4353 mL
	5 mM	0.4287 mL	2.1435 mL	4.2871 mL
	10 mM	0.2144 mL	1.0718 mL	2.1435 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.