AZ 628

Catalog No.S2746 Batch:S274601

Technical Data

Formula	C₂₇H₂₅N₅O₂
Molecular Weight	451.52	CAS No.	878739-06-1
Solubility (25°C)*	In vitro	DMSO	90 mg/mL (199.32 mM)
		Water	Insoluble
		Ethanol	Insoluble
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description

AZ628 is a new pan-Raf inhibitor for BRAF, BRAFV600E, and c-Raf-1 with IC50 of 105 nM, 34 nM and 29 nM in cell-free assays, also inhibits VEGFR2, DDR2, Lyn, Flt1, FMS, etc. AZ628 induces apoptosis.

Targets

C-Raf-1 ^[1] (Cell-free assay)	B-Raf (V600E) ^[1] (Cell-free assay)	B-Raf ^[1] (Cell-free assay)
29 nM	34 nM	105 nM

In vitro

AZ628 prevents activation of number of tyrosine protein kinases including VEGFR2, DDR2, Lyn, Flt1, FMS and others. AZ628 suppresses anchorage-dependent and -independent growth, gives rise to cell cycle arrest, and induces apoptosis in colon and melanoma cell lines harboring B-RafV600E mutation. The profile of AZ628 cross-reactivity suggests that similar to sorafenib, AZ628 may be antiangiogenic based on prevention of VEGFR2. ^[1] AZ628-resistant clones are approximately 100-fold more resistant to AZ628 than the parental cell line, exhibiting IC50 of approximately 10 μM, compared with 0.1 μM for the parental cell line. Effective suppression of p-ERK1/2 levels is observed in the M14 parental cell line following treatment with increasing concentrations of AZ628. AZ628-resistant clones express elevated CRAF. Elevated CRAF expression is a potential mechanism of acquired resistance to continuous AZ628 exposure, resulting in sustained activation of ERK1/2. p-ERK1/2 activity is not significantly inhibited by exposure to AZ628 in one of these three AZ628-insensitive cell lines (Wm1552C). Unlike in the AZ628-resistant M14 cells in which AZ628 fails to suppress the activation of ERK, AZ628 treatment efficiently attenuates ERK activation in the NRAS mutant melanoma cells.^[2]

Protocol (from reference)

Cell Assay:^{^[1]}	Cell lines M14 expressing the V600E BRAF mutation Concentrations 0.01-10 μM Incubation Time Overnight Method Approximately 0.5-2.5 × 10⁵ M14 cells are seeded in 12 or 24 - well plates, respectively, in medium supplemented with 5% FBS. After overnight incubation, the cells are treated with various concentrations of AZ628. Fresh medium and drug is replaced every 2 days until the untreated control wells reached confluence. At this time-point, the media is removed and the cells are fixed in 4% formaldehyde in PBS for 20 minutes at room temperature. Cells are then washed twice with PBS and stained with a 1:5000 solution of the fluorescent nucleic acid stain Syto60. Quantitation of fluorescent signal intensity is carried out at 700 nm, using an Odyssey Infrared Imager. Each experiment is performed in quadruplicate and the results shown represent the average of the four values compared to untreated wells. Error bars represent standard deviation of the 4 values from the mean. High-throughput cell growth/viability assays are performed.

Cell Assay:^{^[1]}

Cell lines
M14 expressing the V600E BRAF mutation
Concentrations
0.01-10 μM
Incubation Time
Overnight
Method
Approximately 0.5-2.5 × 10⁵ M14 cells are seeded in 12 or 24 - well plates, respectively, in medium supplemented with 5% FBS. After overnight incubation, the cells are treated with various concentrations of AZ628. Fresh medium and drug is replaced every 2 days until the untreated control wells reached confluence. At this time-point, the media is removed and the cells are fixed in 4% formaldehyde in PBS for 20 minutes at room temperature. Cells are then washed twice with PBS and stained with a 1:5000 solution of the fluorescent nucleic acid stain Syto60. Quantitation of fluorescent signal intensity is carried out at 700 nm, using an Odyssey Infrared Imager. Each experiment is performed in quadruplicate and the results shown represent the average of the four values compared to untreated wells. Error bars represent standard deviation of the 4 values from the mean. High-throughput cell growth/viability assays are performed.

References

Customer Product Validation

: Data from [Data independently produced by Cell Death Dis, 2014, 5, e1278]

: Data from [Data independently produced by Cancer Discov, 2013, 3(3), 350-62]

Selleck's AZ 628 has been cited by 43 publications

Integrated drug response prediction models pinpoint repurposed drugs with effectiveness against rhabdomyosarcoma [ PLoS One, 2024, 19(1):e0295629]	PubMed: 38277404
Glutamine deficiency in solid tumor cells confers resistance to ribosomal RNA synthesis inhibitors [ Nat Commun, 2022, 13(1):3706]	PubMed: 35764642
Classical RAS proteins are not essential for paradoxical ERK activation induced by RAF inhibitors [ Proc Natl Acad Sci U S A, 2022, 119(5)e2113491119]	PubMed: 35091470
EGFR/MET promotes hepatocellular carcinoma metastasis by stabilizing tumor cells and resisting to RTKs inhibitors in circulating tumor microemboli [ Cell Death Dis, 2022, 13(4):351]	PubMed: 35428350
Glutathione Peroxidase 4 as a Therapeutic Target for Anti-Colorectal Cancer Drug-Tolerant Persister Cells [ Front Oncol, 2022, 12:913669]	PubMed: 35719967
B-Raf inhibitor vemurafenib counteracts sulfur mustard-induced epidermal impairment through MAPK/ERK signaling [ Drug Chem Toxicol, 2022, 1-10]	PubMed: 34986718
Intra-heterogeneity in transcription and chemoresistant property of leukemia-initiating cells in murine Setd2-/- acute myeloid leukemia [ Cancer Commun (Lond), 2021, 10.1002/cac2.12189]	PubMed: 34196511
Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening [ Cell Rep, 2021, 35(11):109233]	PubMed: 34133938
Genome-scale CRISPR-Cas9 knockout screening in hepatocellular carcinoma with lenvatinib resistance [ Cell Death Discov, 2021, 7(1):359]	PubMed: 34795217
Inhibitors of BRAF dimers using an allosteric site [ Nat Commun, 2020, 11(1):4370]	PubMed: 32873792

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