ZM-306416 (CB-676475) is a novel and potent tyrosine kinase inhibitor of VEGFR1 (vascular endothelial growth factor receptor 1) with potential antineoplastic activity. With an IC50 of less than 10 nM, it inhibits both EGFR and VEGFR1, with the latter being inhibited at 0.33 μM.

Physicochemical Properties

Molecular Formula	C16H13CLFN3O2
Molecular Weight	333.74
Exact Mass	333.068
Elemental Analysis	C, 57.58; H, 3.93; Cl, 10.62; F, 5.69; N, 12.59; O, 9.59
CAS #	690206-97-4
Related CAS #	196603-47-1 (HCl);690206-97-4;
PubChem CID	5329006
Appearance	White to gray solid powder
Density	1.4±0.1 g/cm3
Boiling Point	434.1±45.0 °C at 760 mmHg
Flash Point	216.4±28.7 °C
Vapour Pressure	0.0±1.0 mmHg at 25°C
Index of Refraction	1.653
LogP	4.15
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	4
Heavy Atom Count	23
Complexity	392
Defined Atom Stereocenter Count	0
SMILES	ClC1C([H])=C([H])C(=C(C=1[H])F)N([H])C1C2=C([H])C(=C(C([H])=C2N=C([H])N=1)OC([H])([H])[H])OC([H])([H])[H]
InChi Key	YHUIUSRCUKUUQA-UHFFFAOYSA-N
InChi Code	InChI=1S/C16H13ClFN3O2/c1-22-14-6-10-13(7-15(14)23-2)19-8-20-16(10)21-12-4-3-9(17)5-11(12)18/h3-8H,1-2H3,(H,19,20,21)
Chemical Name	N-(4-chloro-2-fluorophenyl)-6,7-dimethoxyquinazolin-4-amine
Synonyms	ZM 306416; ZM-306416; CB 676475; CB676475; CB-676475; ZM306416
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	VEGFR1 (IC50 = 0.33 μM); Src (IC50 = 0.33 μM); Abl (IC50 = 1.3 μM) ZM 306416 (CB676475) inhibits vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase (IC₅₀ = 25 nM) [1] ZM 306416 (CB676475) shows inhibitory activity against epidermal growth factor receptor (EGFR) tyrosine kinase (IC₅₀ = 38 nM) [2]
ln Vitro	ZM-306416 selective anti-proliferative effect that spares the wild type EGFR cell lines A549 and H2030 (IC50>10 μM) but selectively inhibits the growth of the EGFR-addicted NSCLC cell lines H3255 and HCC4011 (IC50=0.09±0.007 μM and 0.072±0.001 μM, respectively). ZM-306416 is also observed to have a less potent IC50 value of 1.3±0.2 μM toward the ABL kinase, which inhibits the ABL in vitro kinase activity[2]. ZM 306416 (CB676475) dose-dependently blocked the kinase activity of recombinant VEGFR2, with 50% inhibition at 25 nM. It also suppressed VEGF-induced phosphorylation of VEGFR2 in human umbilical vein endothelial cells (HUVECs) by ~70% at 50 nM [1] ZM 306416 (CB676475) inhibited EGF-induced EGFR phosphorylation in A431 cells, with a maximum inhibition of ~80% at 100 nM. It reduced the proliferation of A431 cells with an IC₅₀ of 42 nM and induced G1 phase cell cycle arrest [2]
ln Vivo	Recombinant VEGFR2 kinase domain was incubated with ATP and a specific peptide substrate in the presence of serial dilutions of ZM 306416 (CB676475). The reaction was conducted at 37°C for 60 minutes, and the phosphorylated substrate was detected using a radiometric assay. The inhibition rate was calculated by comparing the radioactivity with the vehicle control, and the IC₅₀ value was derived from the dose-response curve [1] Recombinant EGFR kinase domain was mixed with ATP, a fluorescently labeled substrate, and different concentrations of ZM 306416 (CB676475). The mixture was incubated at 30°C for 45 minutes, and the fluorescence resonance energy transfer (FRET) signal was measured to quantify kinase activity. The IC₅₀ was determined by plotting the inhibition percentage against drug concentration [2]
Enzyme Assay	An assay for luminescence ADP production kinase was utilized to evaluate the activity of confirmed positives in a panel of kinases, which included EGFR, VEGFR1, SRC, and ABL kinase, as previously mentioned. In dose response studies with a 384-well format, the potency of each compound was measured using 12 doubling dilutions in duplicate, with the compound concentrations at 10 μM and 1 μM serving as the upper limit. The PP-384-M Personal Pipettor was used for all reagent transfers. A volume of 1 μL was added to the wells of white 384 well microtiter plates (Corning #3570, Corning, NY) containing the tested compounds or controls. The controls were 30 μM staurosporine in 1% DMSO (v/v) and 1% DMSO (v/v) (high control and low control, respectively). The assay buffer consisted of 25 mM Hepes/NaOH, pH 7.5, 10 mM MgCl2, 2 mM TCEP, 20 mM β-Glycerol Phosphate, and 100 μM Na3VO4. To achieve a final concentration of 50 nM enzyme, 4 μL of kinase dilution in assay buffer was added to each well for each kinase on the panel. It is noteworthy that the concentration of active enzyme in the preparation is unknown due to the unknown specific activity of the panel's kinases. Kinase and the compound were pre-incubated for ten minutes at room temperature following the addition of the enzyme. Afterward, 5 μL of a mixture comprising ATP and Poly(Glu, Tyr) substrates in solution within the assay buffer was introduced into each well, ultimately achieving a 200 μM final concentration. ADP-Glo Reagent (10 μL) was added to each well following a 45-minute reaction at room temperature. 20 microliters of Kinase Detection Reagent were added after 40 minutes of incubation, and another 60 minutes of incubation were required. Using LEADseekerTM, the output of the luminescence signal was measured. The average data from duplicates are represented by the dose response curves, which were plotted using SigmaPlot. The error bars stand for the regression's standard error. Compound IC50 in the assay conditions was calculated with a low limit of 10 nM.
Cell Assay	Confirmed hits' anti-proliferative effect was evaluated using a panel of established cell lines, which included A549-EGFRB, A549, and H2030 cell lines harboring wild type EGFR and H3255 and HCC4011 cell lines harboring the activating L858R EGFR mutation. As previously mentioned, A549-EGFRB cells were cultured. As previously mentioned, H2030, H3255, and HCC4011 cells were cultured. F12K medium (Invitrogen Cat.# 21127-022) supplemented with 10% FBS (PAA Cat.# A15-201) was used to cultivate A549 cells. Employing 12 doubling dilutions in duplicate, with compound concentrations of 10 μM and 1 μM as the upper limit, and the Alamar Blue viability assay as previously described, the anti-proliferative effect of each compound was evaluated in dose response studies in 384-well format. A 1% DMSO (v/v) high control and a 1 μM “killer mix” in 1% DMSO (v/v) low control were the contents of the control wells. It took 120 hours for the compound to fully incubate with the cells. The standard error of the regression is represented by the error bars in dose response curves plotted with SigmaPlot 9.0 (Systat Software, San Jose, CA). The mean data from duplicates is shown. HUVECs were seeded in 6-well plates and cultured overnight. After serum starvation for 12 hours, cells were treated with ZM 306416 (CB676475) (10-100 nM) for 1 hour, then stimulated with VEGF (50 ng/mL) for 15 minutes. Cells were lysed, and Western blot analysis was performed using antibodies against phosphorylated VEGFR2 and total VEGFR2 [1] A431 cells were plated in 96-well plates at 5×10³ cells/well and treated with ZM 306416 (CB676475) (5-200 nM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate the IC₅₀ for proliferation inhibition. For cell cycle analysis, treated cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. Western blot was used to detect phosphorylated EGFR and downstream signaling molecules (ERK1/2) [2]
Animal Protocol
References	[1]. Synthesis of a novel biotin-tagged photoaffinity probe for VEGF receptor tyrosine kinases. Bioorg Med Chem Lett. 2006 Jan 1;16(1):129-33. [2]. A high-content biosensor-based screen identifies cell-permeable activators and inhibitors of EGFR function: implications in drug discovery. J Biomol Screen. 2012 Aug;17(7):885-99.
Additional Infomation	N-(4-chloro-2-fluorophenyl)-6,7-dimethoxy-4-quinazolinamine is a member of quinazolines. ZM 306416 (CB676475) is a small-molecule inhibitor that targets both VEGFR2 and EGFR, making it a potential candidate for the treatment of tumors dependent on angiogenesis and EGFR signaling [1] The inhibitory activity of ZM 306416 (CB676475) against EGFR was identified through a high-content biosensor-based screen, which highlights its utility in drug discovery for EGFR-driven cancers [2]

Solubility Data

Solubility (In Vitro)

DMSO: ~67 mg/mL (~200.8 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.49 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 (7.49 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.9963 mL	14.9817 mL	29.9634 mL
	5 mM	0.5993 mL	2.9963 mL	5.9927 mL
	10 mM	0.2996 mL	1.4982 mL	2.9963 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.