AZD4547

 

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Biological Activity

Description	AZD4547 is a novel selective FGFR inhibitor targeting FGFR1/2/3 with IC50 of 0.2 nM/2.5 nM/1.8 nM, weaker activity against FGFR4, VEGFR2(KDR), and little activity observed against IGFR, CDK2, and p38. Phase 1/2.
Targets	FGFR1	FGFR2	FGFR3
IC50	0.2 nM	2.5 nM	1.8 nM [1]
In vitro	Compared to FGFR1-3, AZD4547 displays weaker activity against FGFR4 with IC50 of 165 nM. AZD4547 only inhibits recombinant VEGFR2 (KDR) kinase activity with IC50 of 24 nM, in the in vitro selectivity test against a diverse panel of representative human kinases. AZD4547 at 0.1 μM exhibits no activity against a range of recombinant kinases including ALK, CHK1, EGFR, MAPK1, MEK1, p70S6K, PDGFR, PKB, Src, Tie2, and PI3-kinase. Consistently, the potent selectivity of AZD4547 for FGFR1-3 over FGFR4, IGFR, and KDR is also observed in cellular phosphorylation assays. AZD4547 has potent in vitro antiproliferative activity only against tumor cell lines expressing deregulated FGFRs such as KG1a, Sum52-PE, and KMS11 with IC50 of 18-281 nM, and is inactive against MCF7 as well as more than 100 additional tumor cell lines. AZD4547 treatment potently inhibits FGFR and MAPK phosphorylation in human tumor cell lines in a dose-dependent manner. AZD4547 also potently inhibits the phosphorylation of FRS2 and PLCγ, downstream markers of FGFR signaling. Notably, AZD4547 affects the AKT phosphorylation in the breast cell lines, MCF7 and Sum52-PE but not in KG1a and KMS11 lines. AZD4547 treatment significantly induces apoptosis in Sum52-PE and KMS11 cells, dramatically increases G1 arrest but not apoptosis in KG1a cells, and has no effect on cell cycle distribution or apoptosis in MCF7 cells. [1]
In vivo	Oral administration of AZD4547 at 3 mg/kg twice daily in mice bearing KMS11 tumors results in significant tumor growth inhibition of 53% when compared with vehicle-treated controls, and AZD4547 at 12.5 mg/kg once daily or 6.25 mg/kg twice daily leads to complete tumor stasis, which is associated with dose proportional pharmacodynamic modulation of phospho-FGFR3 and reduced KMS11 tumor cell proliferation. Moreover, oral administration of AZD4547 at 12.5 mg/kg once daily results in 65% tumor growth inhibition in the FGFR1-fusion KG1a xenograft model. At efficacious dose levels, AZD4547 does not exhibit antiangiogenic effects. AZD4547 has no significant effect on blood pressure and therefore lacks in vivo anti-KDR activity. Consistently, dosing of 6.25 mg/kg orally twice daily AZD4547 is inactive in the cediranib-sensitive xenograft models including Calu-6, HCT-15 and LoVo. [1]
Features	Greater selectivity for FGFR1-3 over FGFR4. AZD4547 is active against the tyrosine kinase activity of both the wild-type and mutant forms of FGFR.

Protocol(Only for Reference)

Kinase Assay: ^[1]

AZD4547 kinase activity	The ability of AZD4547 to inhibit the human recombinant kinase activities of FGFR1-3 is tested using ATP concentrations at, or just below, the respective Km.

Cell Assay: ^[1]

Cell lines	KG1a, Sum52-PE, KMS11, and MCF7
Concentrations	Dissolved in DMSO, final concentrations ~1 μM
Incubation Time	72 hours
Method	Cells are exposed to various concentrations of AZD4547 for 72 hours. The antiproliferative IC50 values are obtained by MTS proliferation assay. For fluorescence-activated cell sorting (FACS), cells are fixed with 70% ethanol and then incubated with propidium iodide/RNase A labeling solution. Cell cycle profiles are assessed with a FACSCalibur instrument and CellQuest analysis software. For apoptotic analysis, cells and media are gently harvested and centrifuged, followed by washing of cell pellets. Cells are then processed for Annexin V-fluorescein isothiocyanate (FITC) staining and propidium iodide uptake. The proportion of cells staining positive for Annexin V are then assessed with a FACSCalibur instrument and quadrant sorting is done by CellQuest analysis software.

Animal Study: ^[1]

Animal Models	Female swiss derived nude (nu/nu) and severe combined immunodeficient mice (SCID) injected s.c. with LoVo, HCT-15, Calu-6, KMS11 or KG1a
Dosages	1.5-50 mg/kg
Administration	Oral gavage once daily or twice daily
Solubility	30% PEG400/0.5% Tween80/5% propylene glycol, 30 mg/mL

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References

Clinical Trial Information( data from http://clinicaltrials.gov)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT00979134	Recruiting	Cancer|Advanced Solid Malignancies	AstraZeneca	2009-10	Phase 1
NCT01202591	Recruiting	FGFR Inhibition, Pharmacokinetics, Biomarkers|ER+ Breast Cancer	AstraZeneca	2010-12	Phase 1|Phase 2
NCT01213160	Active, not recruiting	Cancer|Advanced Solid Malignancies	AstraZeneca	2011-11	Phase 1
NCT01457846	Recruiting	Gastro-oesophageal Junction Cancer|Gastric Cancer	AstraZeneca	2011-11	Phase 2
NCT01824901	Not yet recruiting	Recurrent Non-small Cell Lung Cancer|Squamous Cell Lung Cancer	Eastern Cooperative Oncology Group|National Cancer Institute (NCI)	2012-11	Phase 1|Phase 2

Chemical Information

Download AZD4547 SDF

Chemical Name	N-(5-(3,5-dimethoxyphenethyl)-1H-pyrazol-3-yl)-4-((3S,5R)-3,5-dimethylpiperazin-1-yl)benzamide

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.46357	4.6357	9.2714	13.9071