Home MAPK Raf inhibitor RAF265 (CHIR-265)

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RAF265 (CHIR-265) Raf inhibitor

Cat.No.S2161

RAF265 (CHIR-265) is a potent selective inhibitor of C-Raf/B-Raf/B-Raf V600E with IC50 of 3-60 nM, and exhibits potent inhibition on VEGFR2 phosphorylation with EC50 of 30 nM in cell-free assays. This compound induces cell cycle arrest and apoptosis. Phase 2.
RAF265 (CHIR-265) Raf inhibitor Chemical Structure

Chemical Structure

Molecular Weight: 518.41

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Quality Control

Batch: Purity: 99.98%
99.98

Cell Culture, Treatment & Working Concentration

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
SK-MEL-28 Growth inhibition assay Growth inhibition of SK-MEL-28 cells, IC50=0.14μM. 21576023
MALME-3M Growth inhibition assay Growth inhibition of MALME-3M cells, IC50=0.14μM. 21576023
A375M Growth inhibition assay Growth inhibition of A375M cells, IC50=0.14μM. 21576023
A375 Function assay Inhibition of B-RAF V600E mutant in human A375 cells assessed as phosphorylation of ERK, IC50=0.04μM. 26396681
Malme-3M Function assay Inhibition of B-RAF V600E mutant in human Malme-3M cells assessed as phosphorylation of ERK, IC50=0.04μM. 26396681
WM-1799 Function assay Inhibition of B-RAF V600E mutant in human WM-1799 cells assessed as phosphorylation of ERK, IC50=0.04μM. 26396681
MALME-3M Antiproliferative assay Antiproliferative activity against human MALME-3M cells harboring B-RAF V600E mutant, IC50=0.04μM. 26396681
A375 Antiproliferative assay Antiproliferative activity against human A375 cells harboring B-RAF V600E mutant, IC50=0.04μM. 26396681
WM1799 Antiproliferative assay Antiproliferative activity against human WM1799 cells harboring B-RAF V600E mutant, IC50=0.04μM. 26396681
SK-MEL-28 Function assay Inhibition of B-RAF V600E mutant in human SK-MEL-28 cells assessed as phosphorylation of ERK, IC50=0.14μM. 26396681
SK-MEL-28 Antiproliferative assay Antiproliferative activity against human SK-MEL-28 cells harboring B-RAF V600E mutant, IC50=0.16μM. 26396681
A375M Function assay 100 mg/kg fCmin in mouse xenografted with human A375M cells at 100 mg/kg, po q2d, fCmin=0.5μM. 26396681
A375M Function assay 100 mg/kg 48 hrs Inhibition of B-RAF V600E mutant in mouse xenografted with human A375M cells assessed as reduction of phospho-MEK level in tumor at 100 mg/kg, po q24 after 48 hrs by Western blot analysis 26396681
A375M Function assay 100 mg/kg 48 hrs Inhibition of B-RAF V600E mutant in mouse xenografted with human A375M cells assessed as reduction of phospho-MEK level in tumor at 100 mg/kg, po q2d after 48 hrs by Western blot analysis 26396681
A375M Function assay 30 to 100 mg/kg 4 hrs Inhibition of B-RAF V600E mutant in mouse xenografted with human A375M cells assessed as reduction of phospho-MEK level in tumor at 30 to 100 mg/kg, po q2d measured after 4 hrs post-third dose by Western blot analysis 26396681
A375M Antitumor assay 10 to 100 mg/kg 30 days Antitumor activity against human A375M cells xenografted in mouse assessed as tumor regression at 10 to 100 mg/kg, po q2d measured up to 30 days 26396681
TC32 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for TC32 cells 29435139
DAOY qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for DAOY 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
A673 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for A673 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
BT-37 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for BT-37 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
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
Saos-2 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for Saos-2 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
NB1643 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for NB1643 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
BT-12 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for BT-12 cells 29435139
RD qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for RD cells 29435139
NB1643 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Confirmatory screen for NB1643 cells 29435139
SK-N-MC qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Confirmatory screen for SK-N-MC cells 29435139
LAN-5 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Confirmatory screen for LAN-5 cells 29435139
NB-EBc1 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Confirmatory screen for NB-EBc1 cells 29435139
BT-37 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Confirmatory screen for BT-37 cells 29435139
TC32 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Confirmatory screen for TC32 cells 29435139
MG 63 (6-TG R) qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Confirmatory screen for MG 63 (6-TG R) cells 29435139
VERO-E6 Function assay 48 hrs Toxicity CC50 against VERO-E6 cells determined at 48 hours by high content imaging (same conditions as 2_LEY without exposure to 0.01 MOI SARS CoV-2 virus), CC50=9.19μM. ChEMBL
VERO-E6 Function assay 48 hrs Determination of IC50 values for inhibition of SARS-CoV-2 induced cytotoxicity of VERO-E6 cells after 48 hours exposure to 0.01 MOI SARS CoV-2 virus by high content imaging, IC50=14.64μM. ChEMBL
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Solubility

In vitro
Batch:

DMSO : 100 mg/mL (192.89 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Ethanol : 33 mg/mL

Water : Insoluble

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Mass Concentration Volume Molecular Weight
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In vivo
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Chemical Information, Storage & Stability

Molecular Weight 518.41 Formula

C24H16F6N6O

 Storage (From the date of receipt)
CAS No. 927880-90-8 Download SDF Storage of Stock Solutions

Synonyms N/A Smiles CN1C2=C(C=C(C=C2)OC3=CC(=NC=C3)C4=NC=C(N4)C(F)(F)F)N=C1NC5=CC=C(C=C5)C(F)(F)F

Mechanism of Action

Targets/IC50/Ki
VEGFR2
(Cell-free assay)
30 nM(EC50)
B-Raf
(Cell-free assay)
3 nM-60 nM
In vitro
RAF265 (CHIR-265) inhibits C-Raf, wild type B-Raf and mutant (V600E) B-Raf. This compound effectively blocks phosphorylation of Raf's downstream substrates MEK and ERK in cells and also kills melanoma and colorectal cancer cell lines harboring B-Raf mutations independent of PTEN mutation status. Raf kinase inhibition by this agent in mutant B-Raf melanoma cell lines causes cell cycle arrest and induces apoptosis, mimicking the effect of Raf RNAi in these cells. It also potently inhibits the phosphorylation of VEGFR2 and proliferation of VEGF-stimulated hMVEC. In HT29 and MDAMB231 cells, this chemical shows inhibitory activity with IC20 of 1 to 3 μM and IC50 of 5 to 10 μM, respectively. While it leads to a significant decrease in clonogenic survival in all tested cell lines, which means that this compound induces a dominant effect on clonogenic survival. Addition of this agent to RAD001 in HCT116 cells could lead to moderately decreased AKT, S6 protein, and 4EBP1 phosphorylation. It markedly reduces the protein level of Bcl-2 and is greatly inhibitory in CM- and NCI-H727 cells, while having no effect on the TRAIL susceptibility of BON1 and GOT1 cells. Protein kinase D3 (PRKD3) that when knocked down could enhance cell killing by this compound in A2058 melanoma cells, which prevent reactivation of MAPK signaling, induce PARP cleavage, increase caspase activity, interrupt cell-cycle progression, and inhibit colony formation.
Kinase Assay
Assay Protocol
Raf and Mek are combined at 2 × final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2. 0.1 mM EDTA and 1 mM DTT) and dispensed 15 μL per well in polypropylene assay plates. Background levels are determined in wells containing Mek and DMSO without Raf. To the Raf/Mek containing wells is added 3 μL of 10 × of this compound diluted in 100% DMSO. The raf kinase activity reaction is started by the addition of 12 μL per well of 2.5 × 33P-ATP diluted in assay buffer. After 45-60 minutes, the reactions are stopped with the addition of 70 μL of stop reagent (30 mM EDTA). Filtration plates are pre-wetted for 5 min with 70% ethanol, and then rinsed by filtration with wash buffer. Samples (90 μL) from the reaction wells are then transferred to the filtration plates. The filtration plates are washed 6 × with wash buffer using Millipore filtration apparatus. The plates are dried and 100 μL per well of scintillation fluid is added. The CPM is then determined using a Wallac Microbeta 1450 reader.
In vivo
RAF265 (CHIR-265) shows 71% to 72% TVI% (tumor volume inhibition percentage) in HCT116 xenografts at 12 mg/kg. While the combination of this compound and RAD001 shows enhanced antitumor activity with increased T10 (time to achieve a relative tumor volume of 10 times the initial tumor volume) and tumor growth delay. The combination of RAD001 and this chemical also significantly enhances the activation of caspase-3 in HCT116 and MDAMB231 but not in A549 xenografts. This compound inhibits FDG (2-deoxy-2-[18F]fluoro-d-glucose) accumulation and decreases the tumor volumes in A375M xenografts by orally dosed of 100 mg/kg.
References
  • [4] https://pubmed.ncbi.nlm.nih.gov/21527556/
  • [5] https://pubmed.ncbi.nlm.nih.gov/21390189/

Clinical Trial Information

(data from https://clinicaltrials.gov, updated on 2024-05-22)

NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT01352273 Completed
Advanced Solid Tumors
Array Biopharma now a wholly owned subsidiary of Pfizer|Array BioPharma
 June 2011 Phase 1

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