Choose Your Country or Region

PF-477736

Name: PF-477736
Brand: Selleck Chemicals
SKU: S2904
Rating: 5 (34 reviews)

Synonyms: PF-736, PF-00477736

Catalog No.S2904 Purity:99.80%

PF-477736 (PF-736, PF-00477736) is a selective, potent and ATP-competitive Chk1 inhibitor with K_i of 0.49 nM in a cell-free assay and also inhibits VEGFR2, Aurora-A, FGFR3, Flt3, Fms (CSF1R), Ret and Yes. It shows ~100-fold selectivity for Chk1 than Chk2.

PF-477736 Chemical Structure

CAS No. 952021-60-2

Purity & Quality Control

Batch: Purity: 99.80%

99.80

PF-477736 Related Products

Related Targets	Chk1 Chk2	Click to Expand
Related Products	AZD7762 Rabusertib (LY2603618) MK-8776 (SCH 900776) CHIR-124 Prexasertib HCl (LY2606368) BML-277 (Chk2 Inhibitor II) Prexasertib (LY2606368) CCT245737 (SRA737) GDC-0575	Click to Expand
Related Compound Libraries	Kinase Inhibitor Library PI3K/Akt Inhibitor Library MAPK Inhibitor Library DNA Damage/DNA Repair compound Library Cell Cycle compound library	Click to Expand

Signaling Pathway

Chk Signaling Pathway

Cell Data

Cell Lines	Assay Type	Activity Description	PMID
TC32	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for TC32 cells	29435139
U-2 OS	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for U-2 OS cells	29435139
A673	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for A673 cells	29435139
Saos-2	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for Saos-2 cells	29435139
RD	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for RD cells	29435139
SK-N-SH	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for SK-N-SH cells	29435139
OHS-50	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for OHS-50 cells	29435139
SJ-GBM2	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for SJ-GBM2 cells	29435139
SK-N-MC	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for SK-N-MC cells	29435139
NB-EBc1	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for NB-EBc1 cells	29435139
LAN-5	qHTS assay	qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for LAN-5 cells	29435139
Click to View More Cell Line Experimental Data

Biological Activity

Description

Targets

Chk1 ^[1] (Cell-free assay)	VEGFR2 ^[1] (Cell-free assay)	Fms ^[1] (Cell-free assay)	YES ^[1] (Cell-free assay)	Chk2 ^[1] (Cell-free assay)
0.49 nM(Ki)	8 nM(Ki)	10 nM(Ki)	14 nM(Ki)	47 nM(Ki)

In vitro
In vitro	PF-477736 (128 nM) abrogates the camptothecin-induced DNA damage checkpoint in a dose-dependent manner in CA46 and HeLa cells. PF-477736 effectively abrogates the -induced S-phase arrest with a corresponding increase in apoptotic cell populations in HT29 cells. PF-477736 (540 nM) enhances -induced cytotoxicity in a time- and dose-dependent manner in HT29 cells. PF-477736 potentiates the growth-inhibitory activity of a panel of chemotherapeutic agents across a broad spectrum of p53-deficient human cancer cell lines in the MTT assay. Addition of PF-477736 (360 nM) to -arrested cells induces a dramatic increase in the intensity of H2AX phosphorylation, reflecting a greater number of γ-H2AX molecules near sites of DNA damage. ^[1] PF-477736 (0.5 nM) selectively blocks p73 and P53 phosphorylation in presence of curcumin in HL-60 cells. ^[2] PF-477736 (360 nM) suppresses -induced phosphorylation of histone H3 (Ser10) and Cdc25C (Ser216) and potentiates apoptosis in COLO205 cells. ^[3] PF-477736 (250 nM) combined with MK-1775 has marked synergistic cytotoxic activity in OVCAR-5 cells. PF-477736 (250 nM) combined with MK-1775 causes accumulation of cells with a DNA content between 2N and 4N in OVCAR-5 cells. PF-477736 (250 nM) combined with MK-1775 causes premature mitosis before the end of DNA replication, with damaged DNA leading to apoptotic cell death in OVCAR-5 cells. ^[4]
Kinase Assay	Binding assay
Kinase Assay	The assay is performed in a 96-well plate for 20 minutes at 30℃ in 0.1 mL of assay buffer containing 50 mM TRIS pH 7.5, 0.4 M NaCl, 4 mM PEP, 0.15 mM NADH, 28 units of lactate dehydrogenase/mL, 16 units of pyruvate kinase/mL, 3 mM DTT, 0.125 mM Syntide-2, 0.15 mM ATP and 25 mM magnesium chloride. Assays are initiated with 1 nM of CHK1 kinase domain. The inhibition of CHK1 activity is determined by measuring initial velocities in the presence of varying concentrations of PF-477736. The data is analyzed using Enzyme Kinetic and Excel software and fit to a kinetic model for competitive inhibition to obtain a Ki value. The kinase selectivity of PF-477736 is evaluated by screening the compound at 1 μM or 10 μM against a panel 2 of about 100 protein kinases.
Cell Research	Cell lines	HT29, Colo205, PC-3, MDA-MB-231 and K562 cells
	Concentrations	~1 μM
	Incubation Time	96 hours
	Method	The IC50 assay measures the antiproliferative effects of PF-477736 on p53-defective human cancer cell lines. Cells in each line are seeded in complete medium at an exponentially growing density in 96-well assay plate and allowed to attach for 16 hours. Serial dilutions of PF-477736 are then done, and appropriate controls are added to each plate. Cells are incubated with drug for 96 hours. After incubation, MTT working stock diluted in complete medium is added to each well, and cells are incubated for 4 hours. After centrifugation and supernatant removal, DMSO is added to each well and plates are read on SpectraMax plate reader at 540 nm.
Experimental Result Images	Methods	Biomarkers	Images	PMID
	Western blot	p-53 / p-p53 / p21 / MDM2 p-ATM / γ-H2AX		31362335
	Growth inhibition assay	Cell viability		29167438
	Immunofluorescence	p-p53		31362335

In Vivo
In vivo	PF-477736 (4 mg/kg i.v.) results in terminal half-life (T1/2) of 2.9 hours, AUC of 5.72 μg×hr/mL and CLp of 11.8 mL/min/kg in rats. PF-477736 dose-dependently enhances the antitumor activity of a maximum tolerated dose in the Colo205 xenograft mouse model. PF-477736 (12 mg/kg) induces an increase in the phosphorylation of histone H3 (Ser10) and of phospho-histone H2AX in the Colo205 xenograft mouse model. ^[1] PF-477736 (15 mg/kg i.p.) enhances induced tumor growth inhibition and tumor growth delay in COLO205 and MDA-MB-231 xenograft models. ^[3] PF 477736 (10 mg/kg once daily i.p.) combined with MK-1775 (30 mg/kg twice a day oral) leads to greater tumor growth inhibition in mice bearing OVCAR-5 xenografts. ^[4]
Animal Research	Animal Models	Colo205 xenograft mouse model
	Dosages	40 mg/kg
	Administration	intravenous injection

NCT Number	Recruitment	Conditions	Sponsor/Collaborators	Start Date	Phases
Clinical Trial Information (data from https://clinicaltrials.gov, updated on 2024-05-22)
NCT00437203	Terminated	Neoplasms	Pfizer	December 2006	Phase 1

References

[4] Carrassa L, et al. Cell Cycle, 2012, 11(13), 2507-2517.

+ Expand

Chemical Information & Solubility

Molecular Weight	419.48	Formula	C₂₂H₂₅N₇O₂
CAS No.	952021-60-2	SDF	Download PF-477736 SDF
Smiles	CN1C=C(C=N1)C2=C3C=NNC(=O)C4=C3C(=CC(=C4)NC(=O)C(C5CCCCC5)N)N2
Storage (From the date of receipt)	3 years-20°Cpowder	Storage of Stock Solutions Aliquot the stock solutions to avoid repeated freeze-thaw cycles	1 year-80°Cin solvent
Storage (From the date of receipt)	3 years-20°Cpowder		1 month-20°Cin solvent

In vitro
Batch:

DMSO : 84 mg/mL ( (200.24 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Water : Insoluble

Ethanol : Insoluble

Molecular Weight Calculator

In vivo Batch: Add solvents to the product individually and in order.				In vivo Formulation Calculator
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	7.500mg/ml (17.88mM)	Taking the 1 mL working solution as an example, add 50 μL of 150 mg/ml clarified DMSO stock solution to 400 μL of PEG300, mix evenly to clarify it; add 50 μL of Tween80 to the above system, mix evenly to clarify; then continue to add 500 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.

Preparing Stock Solutions

Molarity Calculator

Mass	Concentration	Volume	Molecular Weight
=	×	×

Dilution Calculator Molecular Weight Calculator

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

Dosage: mg/kg Average weight of animals: g Dosing volume per animal:μL Number of animals:

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO + % + % Tween 80 + % ddH₂O

%DMSO+ %

Calculation results:

Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
If you have any other enquiries, please leave a message.

* Indicates a Required Field

Frequently Asked Questions

Question 1:
Can you advise the vehicle suggested for S2904: 2% DMSO/40% PEG 300 at 5mg/ml, is it a suspension or clear solution? I wanted to know how to dissolve this compound for an i.p. injection in mice

Answer:
S2904 can dissolve in 2% DMSO/40% PEG 300 at 5mg/ml as a clear solution. If you are going to use this kind of vehicle, please dissolve the compound in DMSO clearly first, then add PEG300, then water.

C1=C0/X	C1:	LOG(C1):
C2=C1/X	C2:	LOG(C2):
C3=C2/X	C3:	LOG(C3):
C4=C3/X	C4:	LOG(C4):
C5=C4/X	C5:	LOG(C5):
C6=C5/X	C6:	LOG(C6):
C7=C6/X	C7:	LOG(C7):
C8=C7/X	C8:	LOG(C8):