NSC319726 is a novel and potent p53(R175) mutant reactivator with with potential anticancer activity. It exhibits growth inhibition in cells expressing mutant p53 (IC50 for p53(R175) mutant is 8 nM), but it has no effect on p53 wild-type cells.

Physicochemical Properties

Molecular Formula	C11H14N4S
Molecular Weight	234.32
Exact Mass	234.093
Elemental Analysis	C, 56.38; H, 6.02; N, 23.91; S, 13.68
CAS #	71555-25-4
Related CAS #	71555-25-4
PubChem CID	5351307
Appearance	Off-white to light yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	367.7±34.0 °C at 760 mmHg
Flash Point	176.2±25.7 °C
Vapour Pressure	0.0±0.8 mmHg at 25°C
Index of Refraction	1.659
LogP	0.17
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	2
Heavy Atom Count	16
Complexity	286
Defined Atom Stereocenter Count	0
SMILES	S=C(N([H])/N=C(\C([H])([H])[H])/C1=C([H])C([H])=C([H])C([H])=N1)N1C([H])([H])C([H])([H])C1([H])[H]
InChi Key	XDHBUMNIQRLHGO-UKTHLTGXSA-N
InChi Code	InChI=1S/C11H14N4S/c1-9(10-5-2-3-6-12-10)13-14-11(16)15-7-4-8-15/h2-3,5-6H,4,7-8H2,1H3,(H,14,16)/b13-9+
Chemical Name	N-[(E)-1-pyridin-2-ylethylideneamino]azetidine-1-carbothioamide
Synonyms	NSC319726; NSC-319726; NSC 319726
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	R175 (p53 = 8 nM)
ln Vitro	NSC319726 is a p53(R175) mutant reactivator, exhibits growth inhibition in cells expressing mutant p53, with an IC50 for p53(R175) mutant of 8 nM, showing no inhibition for p53 wild-type cells, and exhibiting selectivity of 10- to 100-fold over other hotspot p53 mutants. p53(R175)-dependent apoptosis is induced by NSC319276. The p53(R175) mutant protein undergoes a conformational change similar to that of the WT after being treated with NSC319726, which restores sequence-specific p53 transcription. NSC319726's ability to chelate zinc ions and undergo redox changes are both necessary for the compound to be active. [1]
ln Vivo	NSC319726 (5mg/kg, 7days) exhibits greater toxicity for p53(R172H/R172H) mice, with only 30% survival, as opposed to 100% survival for p53+/+ and p53-/- mice. NSC319726 inhibits the growth of TOV112D-p53(R175H) xenografts, but not H460 (p53+/+) and MDAMB468-p53(R273W) xenografts.[1]
Enzyme Assay	After two days of serial compound dilution treatment, 5,000 TOV112D cells (5,000 cells/well, in a 100 ml culture) are cultured in a 96-well plate and reach a confluence of 50% to 60%. Following a 3-day incubation period, the growth is assessed using the MTS reagent and a Victor Plate reader instrument.
Cell Assay	Viability assays are done. The cells are cultured in a 12-well plate at a density of 5×104 cells per well in a 1 ml of culture, and are treated with serial dilutions of the compound on the second day to reach a confluence of 50% to 60%. Guava ViaCount reagent and Guava PCA instrument are used to measure the growth after three days of incubation.
Animal Protocol	5-10 mg/kg/day, up to 7 days DMSO p53+/+, p53-/- and p53R172H knockin mice
References	[1]. Cancer Cell . 2012 May 15;21(5):614-625.

Solubility Data

Solubility (In Vitro)

DMSO: ~19 mg/mL (~81.1 mM) Water: <1 mg/mL (slightly soluble or insoluble) Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.88 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 20.8 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: ≥ 2.08 mg/mL (8.88 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	4.2677 mL	21.3383 mL	42.6767 mL
	5 mM	0.8535 mL	4.2677 mL	8.5353 mL
	10 mM	0.4268 mL	2.1338 mL	4.2677 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.