Necrostatin 2 (also known as Necrostatin-2, Necrostatin-2 racemate, (±)-Necrostatin-2, 7-Cl-O-Nec-1 and Nec-1s) is a powerful necroptosis inhibitor with an EC50 of 50 nM. It is a racemic mixture that prevents the autophosphorylation of the receptor-interacting protein kinase 1 (RIPK1). A controlled caspase-independent cell death mechanism called necroptosis produces morphological characteristics that resemble necrosis. It can be brought on by TNF-alpha-treated human Jurkat T cells with a FADD-deficient variant. Additionally effective in an animal model of ischemic stroke, necrostatin 2 was. After being administered intravenously, necrostatin 2 showed moderate pharmacokinetic properties and quickly entered the central nervous system.

Physicochemical Properties

Molecular Formula	C13H12CLN3O2
Molecular Weight	277.71
Exact Mass	277.062
Elemental Analysis	C, 56.23; H, 4.36; Cl, 12.77; N, 15.13; O, 11.52
CAS #	852391-19-6
Related CAS #	Necrostatin 2 racemate;852391-15-2;Necrostatin 2 S enantiomer;852391-20-9
PubChem CID	11288934
Appearance	White to light yellow solid powder
LogP	1.491
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	2
Heavy Atom Count	19
Complexity	403
Defined Atom Stereocenter Count	1
SMILES	C([C@H]1NC(=O)N(C)C1=O)C1=CNC2C(=CC=CC1=2)Cl
InChi Key	WIKGAEMMNQTUGL-SNVBAGLBSA-N
InChi Code	InChI=1S/C13H12ClN3O2/c1-17-12(18)10(16-13(17)19)5-7-6-15-11-8(7)3-2-4-9(11)14/h2-4,6,10,15H,5H2,1H3,(H,16,19)/t10-/m1/s1
Chemical Name	(5R)-5-[(7-chloro-1H-indol-3-yl)methyl]-3-methylimidazolidine-2,4-dione
Synonyms	Necrostatin-2 R-isomer; Necrostatin 2; (±)-Necrostatin-2; Necrostatin-2 racemate; 7-Cl-O-Nec-1; Nec-1S
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

Targets	Necroptosis; RIPK1
ln Vitro	Nec-1s is a potent inhibitor of RIPK1 and cellular necroptosis while lacking IDO inhibitory activity[1].
ln Vivo	Necrostatin 2 R-isomer(Nec-1s) is effective at minimizing brain injuries. It is a superior inhibitor that can be used in vivo and does not have a paradoxical sensitizing effect in TNF-induced lethality[1]. Necrostatin 2 R-isomer(Nec-1s) differs from Nec-1 in that it has a number of favorable pharmacokinetic and pharmacodynamic properties. Because of their lower in vivo and in vitro toxicity, Nec-1s have a safer safety profile than Nec-1[2].
Enzyme Assay	Necrostatin 2 (also known as Necrostatin-2, Necrostatin-2 racemate, (±)-Necrostatin-2, 7-Cl-O-Nec-1 and Nec-1s, is a potent necroptosis inhibitor with EC50 of 50 nM.
Cell Assay	Cells were treated with indicated concentrations of drug.
Animal Protocol
References	[1]. Cell Death Dis . 2012 Nov 29;3(11):e437. [2]. Molecules . 2018 Aug 3;23(8):1946. [3]. Cell Rep . 2022 Oct 4;41(1):111434.

Solubility Data

Solubility (In Vitro)

DMSO: ~10 mM Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (9.00 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 25.0 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (9.00 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.6009 mL	18.0044 mL	36.0088 mL
	5 mM	0.7202 mL	3.6009 mL	7.2018 mL
	10 mM	0.3601 mL	1.8004 mL	3.6009 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.