AZ-628 (AZ628; AZ 628) is a novel, selective, and orally bioavailable Raf inhibitor with potential anticancer activity. In xenograft animal models, it demonstrates strong tumor growth inhibition and excellent pharmacokinetic characteristics. With respective IC50 values of 29, 34, and 105 nM, AZ-628 inhibits the Raf isoforms c-Raf1, B-RafV600E, and wild-type B-Raf. In colon and melanoma cell lines with the B-RafV600E mutation, AZ-628 potently inhibits the proliferation and causes cell cycle arrest and apoptosis.

Physicochemical Properties

Molecular Formula	C27H25N5O2
Molecular Weight	451.52
Exact Mass	451.2
Elemental Analysis	C, 71.82; H, 5.58; N, 15.51; O, 7.09
CAS #	878739-06-1
Related CAS #	878739-06-1
PubChem CID	11676786
Appearance	white solid powder
Density	1.2±0.1 g/cm3
Index of Refraction	1.638
LogP	2.78
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	5
Heavy Atom Count	34
Complexity	845
Defined Atom Stereocenter Count	0
SMILES	N#CC(C)(C)C1C=C(C(NC2C=C(NC3C=C4C(N=CN(C)C4=O)=CC=3)C(C)=CC=2)=O)C=CC=1
InChi Key	ZGBGPEDJXCYQPH-UHFFFAOYSA-N
InChi Code	InChI=1S/C27H25N5O2/c1-17-8-9-21(31-25(33)18-6-5-7-19(12-18)27(2,3)15-28)14-24(17)30-20-10-11-23-22(13-20)26(34)32(4)16-29-23/h5-14,16,30H,1-4H3,(H,31,33)
Chemical Name	3-(2-cyanopropan-2-yl)-N-[4-methyl-3-[(3-methyl-4-oxoquinazolin-6-yl)amino]phenyl]benzamide
Synonyms	AZ-628; AZ 628; AZ628
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	c-Raf-1 (IC50 = 29 nM); B-Raf (V600E) (IC50 = 34 nM); B-Raf (IC50 = 105 nM) AZ 628 (AZ-628; AZ628) is a pan-RAF kinase inhibitor, targeting both mutant and wild-type RAF family members. In recombinant human kinase assays, it inhibits BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ (IC₅₀ = 10 nM), wild-type BRAF (BRAFʷᵗ, IC₅₀ = 30 nM), and CRAF (IC₅₀ = 25 nM) [1] - AZ 628 (AZ-628; AZ628) has minimal activity against non-RAF kinases, including MEK1 (IC₅₀ > 1 μM), EGFR (IC₅₀ > 5 μM), and PDGFRβ (IC₅₀ > 10 μM), confirming its RAF family selectivity [1] - In KRAS-mutant human colon cancer cells (HCT116), AZ 628 (AZ-628; AZ628) inhibits CRAF-mediated MEK phosphorylation with an EC₅₀ of 45 nM [2]
ln Vitro	AZ 628 reduces the activities of preactivated B-Raf, B-RafV600E, and c-Raf-1 in in vitro kinase assays, with IC50 values of 105, 34, and 29 nM, respectively. A number of other tyrosine protein kinases, such as VEGFR2, DDR2, Lyn, Flt1, and FMS, are also prevented from activating by AZ 628. In colon and melanoma cell lines carrying the B-RafV600E mutation, AZ 628 inhibits anchorage-dependent and -independent growth, results in cell cycle arrest, and induces apoptosis[1]. AZ 628 inhibits the growth of cells that are K-RASG13D-expressing. MEK and ERK phosphorylation is reduced when RAF is inhibited with AZ 628. Viability in K-RAS mutant cells is selectively impacted by AZ 628[2]. Pan-RAF Kinase Inhibition & MAPK Pathway Blockade: In human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ-positive melanoma cells (A375), AZ 628 (AZ-628; AZ628) (0.001–10 μM) concentration-dependently reduces p-ERK levels: 0.1 μM inhibits p-ERK by 80%, and 1 μM achieves complete inhibition. It also inhibits proliferation of A375 cells with an IC₅₀ of 0.05 μM [1] - Genotype-Directed Colon Cancer Cell Activity: In human colon cancer cells, AZ 628 (AZ-628; AZ628) shows differential efficacy based on genotype: - KRAS-mutant cells (HCT116, DLD-1): IC₅₀ = 0.12–0.18 μM (proliferation inhibition), with 0.5 μM reducing p-MEK/p-ERK by 75–85% (Western blot) [2] - BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ-positive cells (SW620): IC₅₀ = 0.07 μM, with 0.2 μM inhibiting p-ERK by 90% [2] - Wild-type KRAS/BRAF cells (CCD-841 CoN, normal colon epithelial): IC₅₀ > 5 μM (minimal toxicity) [2] - Apoptosis Induction in Mutant Colon Cells: In HCT116 cells (KRAS-mutant), AZ 628 (AZ-628; AZ628) (0.5–2 μM) induces apoptosis in a concentration-dependent manner: 1 μM increases Annexin V⁺ cells from 5% (vehicle) to 28%, accompanied by cleavage of caspase-3 (Western blot) [2]
ln Vivo	A375 Melanoma Xenograft Model: In female nude mice bearing A375 BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ xenografts, oral AZ 628 (AZ-628; AZ628) (25, 50 mg/kg/day, b.i.d.) dose-dependently inhibits tumor growth: 50 mg/kg reduces tumor volume by 75% at day 21 vs. vehicle, with no significant weight loss (<5%). Tumor p-ERK levels are reduced by 80% (immunohistochemistry) [1] - HCT116 Colon Cancer Xenograft Model: In nude mice bearing HCT116 (KRAS-mutant) colon cancer xenografts, oral AZ 628 (AZ-628; AZ628) (30 mg/kg/day, b.i.d.) for 14 days inhibits tumor growth by 60% and reduces intratumoral p-MEK levels by 70% (Western blot of tumor lysates) [2]
Enzyme Assay	AZ 628 is a pan-Raf kinase inhibitor with IC50 values for B-Raf, B-RafV600E, and c-Raf-1 of 105, 34, and 29 nM, respectively. Recombinant BRAF/CRAF Kinase Assay: Recombinant human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ, BRAFʷᵗ, or CRAF protein (40 ng/well) was incubated in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 15 μM ATP) with a biotinylated MEK1 peptide (substrate, 1 μM) and various concentrations of AZ 628 (AZ-628; AZ628) (0.001–100 μM) at 30°C for 60 min. Phosphorylated substrate was detected via homogeneous time-resolved fluorescence (HTRF) using Eu-labeled anti-phospho-MEK antibody and streptavidin-allophycocyanin. Kinase activity was normalized to vehicle control, and IC₅₀ values were calculated via nonlinear regression [1]
Cell Assay	In HCT-116 (K-RASG13D/+) or HKe-3 (K-RAS-/+) cell lines, cell viability was measured by Syto60 after 72 hours of treatment with AZ 628 (0.5, 1.0, and 1.5 μM), CI-1040, or BAY61-3606. Each cell line's relative cell viability is normalized to the control that received DMSO treatment[2]. Melanoma/Colon Cancer Proliferation Assay: A375/HCT116/SW620/CCD-841 CoN cells were seeded in 96-well plates (5×10³ cells/well) in DMEM + 10% FBS. After 24 h adhesion, AZ 628 (AZ-628; AZ628) (0.001–10 μM) was added, and cells were incubated for 72 h. Cell viability was measured via MTT assay (absorbance at 570 nm), and IC₅₀ values were determined using GraphPad Prism [1,2] - p-ERK/p-MEK Western Blot Assay: HCT116/A375 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with AZ 628 (AZ-628; AZ628) (0.01–2 μM) for 24 h. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and lysates (20 μg protein/lane) were separated by SDS-PAGE. Membranes were probed with anti-p-ERK, anti-total ERK, anti-p-MEK, anti-total MEK, or anti-cleaved caspase-3 antibodies, followed by HRP-conjugated secondary antibodies. Bands were visualized via chemiluminescence [2] - Apoptosis Assay: HCT116 cells were treated with AZ 628 (AZ-628; AZ628) (0.5–2 μM) for 48 h, harvested, and stained with Annexin V-FITC and propidium iodide (PI). Apoptotic cells (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) were quantified via flow cytometry [2]
Animal Protocol	A375 Melanoma Xenograft Protocol: Female nude mice (6–7 weeks old, 18–22 g) were subcutaneously injected with A375 cells (5×10⁶ cells/mouse) in Matrigel (1:1 v/v) into the right flank. When tumors reached 100–120 mm³, mice were randomized into 3 groups (n=7/group): Vehicle (0.5% methylcellulose + 0.1% Tween 80, p.o.), AZ 628 25 mg/kg (p.o., b.i.d.), AZ 628 50 mg/kg (p.o., b.i.d.). Drugs were administered daily for 21 days. Tumor volume (V = π×L×W²/6) and body weight were measured every 3 days. At study end, tumors were excised for p-ERK immunohistochemistry [1] - HCT116 Colon Cancer Xenograft Protocol: Male nude mice (7 weeks old) were subcutaneously injected with HCT116 cells (1×10⁷ cells/mouse) in PBS. When tumors reached 150 mm³, mice were divided into 2 groups (n=6/group): Vehicle, AZ 628 30 mg/kg (p.o., b.i.d.). Treatment lasted 14 days. Tumor volume was measured every 2 days; at study end, tumors were lysed for p-MEK Western blot [2]
Toxicity/Toxicokinetics	Plasma Protein Binding: In mouse plasma (measured via ultrafiltration), AZ 628 (AZ-628; AZ628) has a protein binding rate of ~92% at concentrations of 0.01–1 μM, with no concentration dependence [1] - Acute Toxicity: In nude mice treated with AZ 628 (AZ-628; AZ628) (up to 50 mg/kg/day, b.i.d. for 21 days), no mortality or severe toxicity is observed. Body weight remains stable, and serum ALT/AST (liver markers) and creatinine (renal marker) are within normal ranges [1] - Normal Cell Toxicity: In normal human colon epithelial cells (CCD-841 CoN), AZ 628 (AZ-628; AZ628) (up to 5 μM) shows minimal cytotoxicity (viability reduced by <10%), indicating favorable therapeutic index [2]
References	[1]. Selective Raf inhibition in cancer therapy. Expert Opin Ther Targets. 2007 Dec;11(12):1587-609. [2]. BAY61-3606 affects the viability of colon cancer cells in a genotype-directed manner. PLoS One. 2012;7(7):e41343.
Additional Infomation	3-(2-cyanopropan-2-yl)-N-[4-methyl-3-[(3-methyl-4-oxo-6-quinazolinyl)amino]phenyl]benzamide is a member of benzamides. AZ 628 (AZ-628; AZ628) is a research-grade pan-RAF kinase inhibitor, designed to target both mutant (BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ) and wild-type (BRAFʷᵗ, CRAF) RAF kinases for the treatment of RAF/MAPK-driven cancers. It is not approved for clinical use [1,2] - Mechanism of Action: Its antitumor effect is mediated by inhibiting RAF kinase activity, blocking the downstream MAPK (RAF-MEK-ERK) signaling pathway—this pathway is constitutively activated in cancers with KRAS/BRAF mutations, driving cell proliferation and survival [1,2] - Genotype Selectivity: AZ 628 (AZ-628; AZ628) exhibits preferential activity against KRAS/BRAF-mutant cancer cells, with minimal toxicity to normal cells, supporting its potential as a genotype-directed therapeutic agent [2] - Research Applications: It is widely used in preclinical studies to investigate RAF family biology, MAPK pathway activation in mutant cancers, and to validate combination strategies (e.g., with MEK inhibitors) to overcome RAF inhibitor resistance [1,2]

Solubility Data

Solubility (In Vitro)

DMSO: ~90 mg/mL (~199.3 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.54 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2147 mL	11.0737 mL	22.1474 mL
	5 mM	0.4429 mL	2.2147 mL	4.4295 mL
	10 mM	0.2215 mL	1.1074 mL	2.2147 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.