Pifithrin-HBr (PFT hydrobromide; PFT-) is a brand-new and powerful p53 inhibitor that works by preventing the transcription of the p53 protein and the p53-dependent transactivation of p53-responsive genes. It also has aryl hydrocarbon receptor (AhR) agonist properties. First, it was discovered that pifithrin- inhibited p53-responsive lacZ activation in ConA cells and decreased the activation of endogenous cellular p53-responsive genes. Pifithrin- can also cause embryonic stem cells to experience cell cycle arrest and growth arrest. The protein lever of Nanog (a pluripotency marker) is significantly downregulated after treatment of Pifithrin-. It has been demonstrated that Pifithrin-induced p53 activity inhibition has no impact on the viability of ES cells.

Physicochemical Properties

Molecular Formula	C16H18N2OS.HBR
Molecular Weight	367.3
Exact Mass	366.04
Elemental Analysis	C, 52.32; H, 5.21; Br, 21.75; N, 7.63; O, 4.36; S, 8.73
CAS #	63208-82-2
Related CAS #	63208-82-2
PubChem CID	9929138
Appearance	White to light yellow solid powder
Density	1.28g/cm3
Boiling Point	456.8ºC at 760 mmHg
Melting Point	192.1-192.5ºC(lit.)
Flash Point	230.1ºC
Index of Refraction	1.666
LogP	4.157
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	3
Heavy Atom Count	21
Complexity	449
Defined Atom Stereocenter Count	0
SMILES	Br[H].S1/C(=N\[H])/N(C([H])([H])C(C2C([H])=C([H])C(C([H])([H])[H])=C([H])C=2[H])=O)C2=C1C([H])([H])C([H])([H])C([H])([H])C2([H])[H]
InChi Key	HAGVCKULCLQGRF-UHFFFAOYSA-N
InChi Code	InChI=1S/C16H18N2OS.BrH/c1-11-6-8-12(9-7-11)14(19)10-18-13-4-2-3-5-15(13)20-16(18)17;/h6-9,17H,2-5,10H2,1H3;1H
Chemical Name	2-(2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3-yl)-1-(4-methylphenyl)ethanone;hydrobromide
Synonyms	PFT-alpha; Pifithrin-alpha; PFTalpha; Pifithrin alpha; Pifithrin-alpha hydrobromide; Pifithrin-; A hydrobromide; Pifithrin-; PFT alpha
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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
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	p53; AhR
ln Vitro	Pifithrin (PFT) hydrobromide is a water-soluble substance that has the potential to inhibit the transcription of the p53 protein. Pifithrin-α can prevent the p53 protein from increasing in whole cell lysates when glucose oxidase (GOX) is activated, but cyclosporine A (CsA) cannot. Notably, Pifithrin-α can prevent the reduction of Bcl-2 protein caused by GOX. In a similar vein, Pifithrin-α rather than CsA, is able to stop the Bax protein from growing in whole cell lysates[1]. Through an unknown mechanism, Pifithrin-α prevents p53-dependent apoptosis. Aryl hydrocarbon receptor (AhR) agonist activity is another function of Pifithrin-α. According to its capacity to bind the AhR, trigger the formation of its DNA binding complex, stimulate reporter activity, and up-regulate the traditional AhR target gene CYP1A1, Pifithrin-α is a potent AhR agonist.
ln Vivo	The experiment's percentage of annexin V-positive Foxe3-/- SMCs drops to WT levels when Pifthirin-α(PFT-α) hydrobromide, a pharmacological p53 inhibitor, is used. In Foxe3-/- mice, transverse aortic constriction (TAC) significantly lowers the incidence of aortic rupture and intramural hematomas (50% to 17%, P<0.05). The average diameter of the ascending aorta and the proportion of TUNEL-positive cells in the aortic media are also normalized to WT levels in surviving Foxe3-/- animals after Pifthirin-α treatment (P<0.05).[3].
Enzyme Assay	Assays for ligand binding competition are carried out. HEDG buffer [25 mM Hepes, 1 mM EDTA, 1 mM dithiothreitol, and 10% (v/v) glycerol, pH 7.5] containing 0.4 mM leupeptin, 4 mg/mL aprotinin, and 0.3 mM phenylmethylsulfonyl fluoride is used to create cytosolic cell extracts from Hepa-1 cells. Aliquots of the supernatant (120 g) are incubated with the indicated concentrations of Pifithrin-α while being exposed to 3 nM [3H]TCDD in HEDG buffer for 2 hours at room temperature. HEDG buffer containing 0.5% Tween 80 is added after 30 minutes of hydroxyapatite incubation on ice. Scintillation counting is performed on the samples after they have been centrifuged, cleaned twice, and resuspended in 0.2 mL of scintillation fluid. A 150-fold molar excess of TCDF is used to calculate nonspecific binding, which is then subtracted from the total binding to get the specific binding. According to [3H]TCDD alone, the precise binding is reported[2].
Cell Assay	The human hepatoma cell lines HepG2 (p53++) are cultured in RMPI 1640 medium with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 37°C in a 5% CO2 environment. Cells are exposed to GOX (0–5 0U) for 0–8 hours either with or without Pifithrin-α (20 μM/L), Pifithrin-μ (5 μM/L), CsA (10μM/L), Sanglifehrin A (20μM/L), and NAC (5 mM/L), for an hour each. Cells are gathered and prepared for further experiments after treatment[1].
Animal Protocol	Mice: The Foxe3-null (Foxe3-/-) mice are employed. Pifithrin-α is dissolved in PBS one hour prior to TAC and then given every 48 hours in order to study the role of p53 in Foxe3-related apoptosis. Two weeks after the operation, the animals are put to sleep, and the ascending aortic tissues are collected for RNA, total protein, histomorphometric analysis, or TUNEL assay.
References	[1]. Int J Biol Sci. 2016 Jan 1;12(2):198-209. [2]. J Pharmacol Exp Ther. 2005 Aug;314(2):603-10. [3]. J Clin Invest. 2016 Mar 1;126(3):948-61.
Additional Infomation	Pifithrin-? is an aromatic ketone.

Solubility Data

Solubility (In Vitro)

DMSO: ~67 mg/mL (~182.4 mM) Water: <1 mg/mL (slightly soluble or insoluble) Ethanol: ~2 mg/mL (~6.1 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.81 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 (6.81 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 (6.81 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. Solubility in Formulation 4: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.7226 mL	13.6129 mL	27.2257 mL
	5 mM	0.5445 mL	2.7226 mL	5.4451 mL
	10 mM	0.2723 mL	1.3613 mL	2.7226 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.