PF-5274857 HCl is a specific, orally bioactive and BBB (blood-brain barrier) permeable (penetrable) Smo antagonist (inhibitor) with IC50 of 5.8 nM and a Ki of 4.6 nM. PF-5274857 HCl may be used to be utilized in the research of a variety of tumors such as brain tumors and brain metastases driven by activated Hh pathways.

Physicochemical Properties

Molecular Formula	C20H26CL2N4O3S
Molecular Weight	473.42
Exact Mass	472.11
CAS #	1613439-62-5
Related CAS #	PF-5274857;1373615-35-0
PubChem CID	90489027
Appearance	Typically exists as solid at room temperature
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	5
Heavy Atom Count	30
Complexity	663
Defined Atom Stereocenter Count	0
SMILES	ClC1=CN=C(C=C1C1C(C)=CC(C)=CN=1)N1CCN(C(CCS(C)(=O)=O)=O)CC1.Cl
InChi Key	GULNFUQAVPTTQV-UHFFFAOYSA-N
InChi Code	InChI=1S/C20H25ClN4O3S.ClH/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;1H
Chemical Name	1-[4-[5-chloro-4-(3,5-dimethylpyridin-2-yl)pyridin-2-yl]piperazin-1-yl]-3-methylsulfonylpropan-1-one;hydrochloride
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	IC50: 5.8 nM (Smo); Ki: 4.6 nM (Smo)[1]
ln Vitro	The transcriptional activity of Smo downstream gene Gli1 in MEF cells indicates that PF-5274857 totally suppresses Shh-induced Hh pathway activity, with an IC50 of 2.7±1.4 nM[1]. When used at 1 μM, PF-5274857 exhibits less than 20% inhibition against a wide range of protein kinases[1].
ln Vivo	In medulloblastoma allograft mouse models, PF-5274857 (1-30 mg/kg; po once daily for 6 days) demonstrates strong antitumor efficaciousness and association between PK and PD[1]. In rats, PF-5274857 (10 mg/kg; ih) in the plasma can pass through the blood-brain barrier four hours after administration[1]. Targeting Smo in the brain, PF-5274857 (10–100 mg/kg; po once daily for 4 days) can cause the brain tumor's Hh pathway activity to be downregulated[1]. Mice with primary Ptch+/− p53−/− medulloblastoma have higher survival rates when treated with PF-5274857 (30 mg/kg; po once daily for 34 days)[1]. (5-30 mg/kg; po) has 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].
Animal Protocol	Animal/Disease Models: Severe combined immunodeficient (SCID )-beige mice (6-8 weeks old) are genetically engineered[1] Doses: 0, 1, 5, 10, 30 mg/kg Route of Administration: Po one time/day for 6 days Experimental Results: demonstrated robust antitumor activity with an in vivo IC50 of 8.9±2.6 nM. Animal/Disease Models: Severe combined immunodeficient (SCID)-beige mice (6-8 weeks old)[1] Doses: 0, 5, 10, 30 mg/kg (pharmacokinetic/PK Analysis) Route of Administration: A single po Experimental Results: The apparent volume of distribution of 5.6±0.5 L/kg; the half-life (T1/2) of 1.7±0.1 hrs (hours).
References	[1]. Rohner A, et, al. 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 Jan;11(1):57-65.

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	2.1123 mL	10.5614 mL	21.1229 mL
	5 mM	0.4225 mL	2.1123 mL	4.2246 mL
	10 mM	0.2112 mL	1.0561 mL	2.1123 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.