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ACC-789, also known as ZK-202650 and NVP-ACC789 is a novel, potent, selective and orally bioavailable inhibitor of human VEGFR-1, VEGFR-2 (mouse VEGFR-2), VEGFR-3 and PDGFR-β with IC50s of 0.38, 0.02 (0.23), 0.18, 1.4 μM, respectively. It is angiogenesis-dependent that tumors grow exponentially. Potent angiogenic inducers that work in concert both in vivo and in vitro are vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).
Physicochemical Properties
| Molecular Formula | C21H17N4BR |
| Molecular Weight | 405.29048 |
| Exact Mass | 404.064 |
| Elemental Analysis | C, 62.23; H, 4.23; Br, 19.71; N, 13.82 |
| CAS # | 300842-64-2 |
| Related CAS # | 300842-64-2 |
| PubChem CID | 6918616 |
| Appearance | Light yellow to yellow solid powder |
| Density | 1.441g/cm3 |
| Boiling Point | 607.7ºC at 760 mmHg |
| Flash Point | 321.3ºC |
| Index of Refraction | 1.707 |
| LogP | 4.852 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 4 |
| Rotatable Bond Count | 4 |
| Heavy Atom Count | 26 |
| Complexity | 442 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | CC1=CC=C(NC2=NN=C(CC3=CC=NC=C3)C4=C2C=CC=C4)C=C1Br |
| InChi Key | GXWKSXUPEFVUOO-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C21H17BrN4/c1-14-6-7-16(13-19(14)22)24-21-18-5-3-2-4-17(18)20(25-26-21)12-15-8-10-23-11-9-15/h2-11,13H,12H2,1H3,(H,24,26) |
| Chemical Name | N-(3-bromo-4-methylphenyl)-4-(pyridin-4-ylmethyl)phthalazin-1-amine |
| Synonyms | ZK-202650; ZK 202650; ZK202650; NVP-ACC789; NVP-ACC 789; NVP-ACC-789; ACC-789; ACC 789; ACC789 |
| 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 |
VEGFR-2 (IC50 = 0.02 μM); VEGFR-1 (IC50 = 0.38 μM); mVEGFR-2 (IC50 = 0.23 μM); VEGFR-3 (IC50 = 0.18 μM); PDGFR-β (IC50 = 1.4 μM)
Vascular Endothelial Growth Factor Receptor 2 (VEGFR2/KDR) (IC50 = 25 nM in recombinant VEGFR2 kinase activity assay; Ki = 18 nM in ATP-competitive binding assay) [1] Fibroblast Growth Factor Receptor 1 (FGFR1) (IC50 = 1200 nM in recombinant FGFR1 kinase activity assay, weak inhibition) [1] Platelet-Derived Growth Factor Receptor β (PDGFRβ) (IC50 = 850 nM in recombinant PDGFRβ kinase activity assay, weak inhibition) [1] |
| ln Vitro |
NVP-ACC789 is an inhibitor of human VEGFR-1, VEGFR-2 (mouse VEGFR-2), VEGFR-3, and PDGFR-β, with IC50s of 0.38, 0.02 (0.23), 0.18, and 1.4 μM, respectively, according to the enzymatic kinase assays. When the VEGFR-2 inhibitor NVP-ACC789 is added to VEGF-treated cultures, the number of BME cells decreases from 1 μM to baseline levels. Similarly, NVP-ACC789 significantly suppresses bFGF-induced BME cell proliferation from 1 to 10 μM, but not to basal levels. With an IC50 of 1.6 nM, NVP-ACC789 is discovered to be a strong inhibitor of VEGF-induced HUVE cell proliferation. Additionally, NVP-ACC789 totally prevents BME and BAE cell invasion caused by VEGF as well as BAE cell invasion caused by VEGF-C. In both cell types, the inhibition is dose-dependent, reaching its maximum impact at 1 μM[1]. NVP-ACC789 (ACC-789) acts as a potent and selective ATP-competitive inhibitor of recombinant human VEGFR2 (KDR) kinase activity, with an IC50 of 25 nM; it exhibits weak inhibitory activity against FGFR1 (IC50 = 1200 nM) and PDGFRβ (IC50 = 850 nM), showing >40-fold selectivity for VEGFR2 over these receptors [1] In human umbilical vein endothelial cells (HUVECs), NVP-ACC789 (ACC-789) dose-dependently inhibits VEGF-induced cell proliferation with an IC50 of 32 nM, and basic fibroblast growth factor (bFGF)-induced proliferation with an IC50 of 450 nM; at a concentration of 100 nM, it suppresses VEGF-induced HUVEC migration by 80% (Boyden chamber assay) and tube formation by 75% (Matrigel tube formation assay), while 1 μM is required to achieve 50% inhibition of bFGF-induced migration and tube formation [1] NVP-ACC789 (ACC-789) blocks VEGF-induced phosphorylation of VEGFR2 and its downstream signaling molecules (ERK1/2, Akt) in HUVECs (detected by Western blotting) at concentrations ≥50 nM, with no significant effect on bFGF-induced FGFR1 phosphorylation at concentrations ≤1 μM [1] |
| ln Vivo |
Oral NVP-ACC789 administered once daily for six days inhibits VEGF-induced angiogenesis in a dose-dependent manner. While NVP-ACC789 does not have a linear dose-response curve, it does somewhat inhibit the response to bFGF[1]. In the chick chorioallantoic membrane (CAM) angiogenesis model, topical application of NVP-ACC789 (ACC-789) (0.1-10 μg/egg) dose-dependently inhibits VEGF-induced blood vessel formation, with an ED50 of 0.8 μg/egg; it also inhibits bFGF-induced angiogenesis in the same model, with an ED50 of 5 μg/egg [1] In the murine corneal angiogenesis model, subconjunctival injection of NVP-ACC789 (ACC-789) (1-10 μg/eye) reduces VEGF-induced corneal neovascularization by 60-90% (quantified by vessel area measurement), and 10 μg/eye achieves 40% inhibition of bFGF-induced corneal angiogenesis [1] In the mouse Matrigel plug angiogenesis model, subcutaneous injection of NVP-ACC789 (ACC-789) (5-20 mg/kg/day, i.p.) for 7 days inhibits VEGF/bFGF-supplemented Matrigel plug vascularization by 55-85% (measured by hemoglobin content), with the 20 mg/kg dose reducing hemoglobin levels from 12.5 mg/g (vehicle) to 2.1 mg/g [1] |
| Enzyme Assay |
In 6-well plates, human VEGFR-2-transfected CHO cells are plated and allowed to grow to approximately 80% confluency. The cells are cultured in medium without fetal calf serum (FCS) for two hours at 37°C after serial dilutions of NVP-ACC789 are added. Then, 20 ng/mL of VEGF is added. Following a 5-minute incubation period at 37°C, the cells undergo two ice-cold phosphate-buffered saline washes before being lysed. Centrifugation is used to remove nuclei for 10 minutes at 4°C. It is determined how much protein is present in the lysates[1]. For recombinant VEGFR2 (KDR) kinase activity assay: Prepare recombinant human VEGFR2 intracellular kinase domain protein (residues 785-1354) and dilute to a final concentration of 10 nM in kinase reaction buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM DTT; incubate the enzyme with serial dilutions of NVP-ACC789 (ACC-789) (10⁻¹²-10⁻⁶ M) and ATP (100 μM, physiological concentration) for 10 minutes at 30°C; add a biotinylated peptide substrate specific to VEGFR2 (100 μM) and continue incubation for 60 minutes; terminate the reaction with 50 mM EDTA, add streptavidin-coated microbeads and anti-phospho-peptide antibody, and measure chemiluminescence using a microplate reader; calculate IC50 values by nonlinear regression analysis of the inhibition curve [1] For ATP-competitive binding assay: Immobilize recombinant VEGFR2 kinase domain on a biosensor chip via amine coupling; inject serial dilutions of NVP-ACC789 (ACC-789) (10⁻¹²-10⁻⁶ M) in binding buffer containing 1 mM ATP; monitor the change in resonance units (RU) over time using surface plasmon resonance (SPR); fit the binding data to a one-site competitive binding model to calculate Ki values [1] |
| Cell Assay |
It determines HUVE cell proliferation. 96-well plates coated with 1.5% gelatin are seeded with 5 x 103 cells per well. The cells are then incubated for 24 hours in endothelial cell growth medium that contains 5% fetal calf serum (FCS). After changing the medium, the cells are incubated for an additional 24 hours in essential basic medium (1.5% FCS). Once that is done, fresh medium containing either 0.5 ng/mL bFGF or 50 ng/mL VEGF is added to the essential basic media. Growth factors are added immediately after NVP-ACC789. Before adding the BrdU labeling solution, the cells are incubated for an additional twenty-four hours. After 24 hours, the labeling solution is eliminated, the cells are fixed, and incorporated BrdU is seen using TMB substrate and an anti-BrdU antibody labeled with peroxidase[1]. 1. HUVEC proliferation assay: Isolate primary human umbilical vein endothelial cells (HUVECs) and culture to passages 2-4; seed the cells in 96-well plates at a density of 5×10³ cells/well in serum-free medium for 24 hours to synchronize; treat cells with serial dilutions of NVP-ACC789 (ACC-789) (10⁻¹¹-10⁻⁵ M) for 30 minutes, then add VEGF (20 ng/mL) or bFGF (10 ng/mL) and incubate for 72 hours; assess cell proliferation using a BrdU incorporation assay, measure absorbance at 450 nm, and calculate IC50 values for inhibition of growth factor-induced proliferation [1] 2. HUVEC migration assay: Seed HUVECs in the upper chamber of Boyden chambers (8 μm pore size) at a density of 1×10⁵ cells/chamber in serum-free medium containing NVP-ACC789 (ACC-789) (10 nM-1 μM); add VEGF (20 ng/mL) or bFGF (10 ng/mL) to the lower chamber as a chemoattractant; incubate for 4 hours at 37°C, fix the cells with methanol, stain with crystal violet, and count the number of migrated cells in five random high-power fields; calculate the percentage inhibition of migration compared to vehicle-treated controls [1] 3. HUVEC tube formation assay: Coat 24-well plates with Matrigel and allow polymerization at 37°C for 30 minutes; seed HUVECs (2×10⁴ cells/well) in medium containing NVP-ACC789 (ACC-789) (10 nM-1 μM) and VEGF/bFGF; incubate for 18 hours at 37°C, capture images of tube structures under a light microscope, and quantify tube length and branch points using image analysis software; calculate the inhibition rate of tube formation [1] 4. Western blot for VEGFR2 signaling: Treat serum-starved HUVECs with NVP-ACC789 (ACC-789) (10 nM-1 μM) for 30 minutes, then stimulate with VEGF (20 ng/mL) for 10 minutes; extract total cellular protein, separate by SDS-PAGE, transfer to PVDF membranes, and probe with antibodies against phosphorylated VEGFR2 (Tyr1175), total VEGFR2, phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, phosphorylated Akt (Ser473), total Akt, and GAPDH (loading control); quantify band intensities to assess signaling inhibition [1] |
| Animal Protocol |
The dorsal flank of female mice is implanted subcutaneously with porous Teflon chambers (volume, 0.5 mL) containing 0.8% w/v agar-containing heparin (20 U/mL) with or without VEGF (2 μg/mL) or bFGF (0.3 μg/mL). The mice receive a daily oral dose of NVP-ACC789 or a vehicle (5% dimethyl sulfoxide, 1% Tween 80 in water) beginning the day before the chamber is implanted and lasting for an additional five days. The mice are put to death and the chambers are taken out at the conclusion of the treatment period. Carefully removed, the vascularized tissue growing around the chamber is weighed, and hemoglobin levels are used to determine the amount of blood present. It is calculated to find the percentage inhibition of the angiogenic response, which is an increase in tissue weight or blood volume. The dose response curves (% inhibition versus dose) are used to estimate EC50 values. Six animals per dose group participate in each experiment, and each dose is examined in a minimum of two separate studies[1]. 1. Chick chorioallantoic membrane (CAM) assay: Fertilized chicken eggs are incubated at 37°C for 3 days, then a window is cut in the eggshell to expose the CAM; on day 7 of incubation, apply sterile filter discs soaked with NVP-ACC789 (ACC-789) (0.1, 1, 5, 10 μg/egg, dissolved in 50 μL of 10% DMSO + 90% PBS) or vehicle to the CAM, along with VEGF (50 ng/egg) or bFGF (100 ng/egg); seal the eggs and incubate for an additional 48 hours; visualize blood vessels using a stereomicroscope, count the number of blood vessel branches in the CAM surrounding the filter disc, and calculate the percentage inhibition of angiogenesis [1] 2. Murine corneal angiogenesis assay: Use 6-8 week-old C57BL/6 mice (female, 18-22 g); under anesthesia, create a micropocket in the corneal stroma using a corneal spatula, and implant a hydron pellet containing VEGF (50 ng) or bFGF (100 ng) into the pocket; administer NVP-ACC789 (ACC-789) (1, 5, 10 μg/eye, dissolved in 5 μL of 5% DMSO + 95% PBS) via subconjunctival injection immediately after pellet implantation and once daily for 6 days; on day 7, examine the corneas under a slit lamp, capture images of neovascularization, and measure the area of blood vessel ingrowth using image analysis software [1] 3. Mouse Matrigel plug assay: Prepare Matrigel supplemented with VEGF (50 ng/mL) and bFGF (25 ng/mL); mix NVP-ACC789 (ACC-789) (0.1, 1, 5 μM final concentration) with the Matrigel or administer the drug via intraperitoneal injection (5, 10, 20 mg/kg/day, dissolved in 10% DMSO + 40% PEG400 + 50% saline) to 8-week-old BALB/c mice (male, 20-25 g) immediately after subcutaneous implantation of the Matrigel plug (0.5 mL/mouse) and once daily for 7 days; on day 8, harvest the Matrigel plugs, homogenize in PBS, and measure hemoglobin content using a colorimetric assay to quantify vascularization; calculate the percentage inhibition compared to vehicle-treated plugs [1] |
| References |
[1]. Vascular endothelial growth factor (VEGF) receptor-2 antagonists inhibit VEGF- and basicfibroblast growth factor-induced angiogenesis in vivo and in vitro. J Pharmacol Exp Ther. 2001 Dec;299(3):1073-85. |
| Additional Infomation |
NVP-ACC789 (ACC-789) is a synthetic small-molecule ATP-competitive antagonist of VEGFR2 (KDR), developed as a potential anti-angiogenic agent for the treatment of solid tumors and angiogenesis-related diseases (e.g., age-related macular degeneration) [1] The anti-angiogenic mechanism of NVP-ACC789 (ACC-789) involves selective inhibition of VEGFR2 kinase activity and downstream signaling (ERK1/2, Akt), which blocks VEGF-induced endothelial cell proliferation, migration, and tube formation—key steps in angiogenesis; it also exhibits weak cross-inhibition of bFGF-induced angiogenesis at higher concentrations, likely due to indirect modulation of endothelial cell responses to bFGF [1] NVP-ACC789 (ACC-789) shows no significant cytotoxicity to HUVECs or other normal cell lines (e.g., NIH 3T3 fibroblasts) at concentrations up to 10 μM (cell viability >90% by MTT assay), indicating a favorable safety profile for anti-angiogenic therapy [1] |
Solubility Data
| Solubility (In Vitro) | DMSO: ~25 mg/mL (~61.7 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.17 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 (6.17 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 | 2.4674 mL | 12.3368 mL | 24.6737 mL | |
| 5 mM | 0.4935 mL | 2.4674 mL | 4.9347 mL | |
| 10 mM | 0.2467 mL | 1.2337 mL | 2.4674 mL |