PF-5274857 (PF5274857; PF 5274857) is a novel, potent, orally bioavailable and selective Smoothened (Smo) antagonist with potential antineoplastic activity. Its IC50s and Ki values for the Hedgehog (Hh) signaling pathway are 5.8 nM and 4.6 nM, respectively.

Physicochemical Properties

Molecular Formula	C20H25CLN4O3S
Molecular Weight	436.96
Exact Mass	436.133
Elemental Analysis	C, 54.98; H, 5.77; Cl, 8.11; N, 12.82; O, 10.98; S, 7.34
CAS #	1373615-35-0
Related CAS #	PF-5274857 hydrochloride; 1613439-62-5
PubChem CID	56956240
Appearance	Light yellow to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	686.0±55.0 °C at 760 mmHg
Flash Point	368.7±31.5 °C
Vapour Pressure	0.0±2.1 mmHg at 25°C
Index of Refraction	1.590
LogP	1.44
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	5
Heavy Atom Count	29
Complexity	663
Defined Atom Stereocenter Count	0
SMILES	ClC1=C([H])N=C(C([H])=C1C1=C(C([H])([H])[H])C([H])=C(C([H])([H])[H])C([H])=N1)N1C([H])([H])C([H])([H])N(C(C([H])([H])C([H])([H])S(C([H])([H])[H])(=O)=O)=O)C([H])([H])C1([H])[H]
InChi Key	BBVNTTZIOTWDSV-UHFFFAOYSA-N
InChi Code	InChI=1S/C20H25ClN4O3S/c1-14-10-15(2)20(23-12-14)16-11-18(22-13-17(16)21)24-5-7-25(8-6-24)19(26)4-9-29(3,27)28/h10-13H,4-9H2,1-3H3
Chemical Name	1-[4-[5-chloro-4-(3,5-dimethylpyridin-2-yl)pyridin-2-yl]piperazin-1-yl]-3-methylsulfonylpropan-1-one
Synonyms	PF-5274857; PF 5274857; PF5274857
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	Smo ( IC50 = 5.8 nM ); Smo ( Ki = 4.6 nM )
ln Vitro	In vitro activity: PF-5274857 completely suppresses Shh-induced Hh pathway activity withan IC50 of 2.7±1.4 nM based on the transcriptional activity of the Smo downstream gene Gli1 in MEF cells[1]. PF-5274857 exhibits less than 20% inhibition at 1 μM against a wide range of protein kinases[1].
ln Vivo	PF-5274857 (1-30 mg/kg; p.o. once daily for 6 days) demonstrates strong antitumor efficacy and correlation between PK and PD in models of medulloblastoma allograft mice[1]. PF-5274857 (10 mg/kg; i.h.) in the plasma can pass through the blood-brain barrier four hours after dosing in rats[1]. PF-5274857 (10-100 mg/kg; p.o. once daily for 4 days) is able to target Smo in the brain, which causes the brain tumor's Hh pathway activity to be downregulated[1]. PF-5274857 (30 mg/kg; p.o. once daily for 34 days) raises the primary Ptch+/− p53−/− medulloblastoma mice's survival rates[1]. PF-5274857 (5-30 mg/kg; p.o.) displays a half-life (T1/2) of 1.7±0.1 hours and an apparent volume of distribution of 5.6±0.5 L/kg[1].
Enzyme Assay	In Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% FBS, Pen–Strep, and 0.1 mg/mL hygromycin, HEK293 cells overexpressing human Smo (amino acids 181–787) are grown to 90% confluence. The cell pellet is resuspended in membrane preparation buffer (50 mM Tris-HCl, pH 7.5, 250 mM sucrose with Roche complete protease cocktail) and homogenized following washing with cold Dulbeccos PBS. After centrifuging the homogenate, the cell pellet is resuspended in assay buffer, which contains 0.1% protease-free bovine serum albumin, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 25 mM MgCl2, and 1 mM EDTA. The homogenization process is carried out in a glass tissue grinder. The Pierce BCA protein assay is used to determine the total protein in the membrane preparation that contains Smo. In order to perform the competitive binding assay, 96-well GF/B filter plates are pre-wetted with 100 μL of assay buffer for ten minutes, after which the filter is removed. After that, the following reagents are added: 50 μL of membrane preparation (40 μg total protein), 10 μL serial dilutions of compound, 20 μL of 3H-Smo antagonist (3 nM final concentration), and 20 μL of assay buffer. After two hours of room temperature incubation, the plates are cleaned and vacuum dried. Following an hour-long drying process in an oven set at 60°C, 45 μL of Microscint 20 is added to the plates, which are then incubated for 30 to 1 hour at room temperature before being counted using a TopCount scintillation counter. Software called GraphPad Prism is used to analyze the data.
Cell Assay	The knockout DMEM containing 10% heat-inactive FBS, 2 mM l-glutamine, and 0.55 mM β-mercaptoethanol is added to Gli-Luc/MEF cells during growth until 90% confluence. Day 1: Trypsinized cells are seeded at a concentration of 7,500 cells per well in white 384-well plates using 20 μL of OptiMEM media per well, supplemented with 1% heat-inactive FBS and 1 mM sodium pyruvate. Overnight, plates are incubated at 37 °C with 5% CO2. On day 2, recombinant mouse Sonic Hedgehog is added to the cells at a final concentration of 2 μg/mL, after PF-5274857 is added at a final concentration ranging from 3 μM to 50 pM at a 3-fold serial dilution. PF-5274857 and Shh are added to the cells and incubated for 48 hours at 37 °C with 5% CO2. Day 4 luciferase assays using the Bright-Glo Luciferase Assay System are carried out. To summarise, 25 μL of Bright-Glo luciferase reagent is introduced into every well of the 384-well plate holding media. After five minutes of room temperature storage, plates are read using a luminescence plate reader. PF-5274857's IC50 value is computed.
Animal Protocol	Dissolved in 0.5% methylcellulose; 30 mg/kg; Oral administration SCID-beige mice bearing primary Ptch+/ p53+/ or Ptch+/ p53 / medulloblastoma tumor
References	[1]. Effective targeting of Hedgehog signaling in a medulloblastoma model with PF-5274857, a potent and selective Smoothened antagonist that penetrates the blood-brain barrier. Mol Cancer Ther. 2012, 11(1), 57-65.

Solubility Data

Solubility (In Vitro)

DMSO: 93~125 mg/mL (212.8~286.1 mM) Water: <1 mg/mL Ethanol: ~93 mg/mL (~212.8 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.76 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 (4.76 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 20.8 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: ≥ 2.08 mg/mL (4.76 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. Solubility in Formulation 4: Saline: 30 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2885 mL	11.4427 mL	22.8854 mL
	5 mM	0.4577 mL	2.2885 mL	4.5771 mL
	10 mM	0.2289 mL	1.1443 mL	2.2885 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.