KI8751 is a potent and selective inhibitor of VEGFR2 with IC50 of 0.9 nM.

VEGFR2:0.9 nM

在LC-6细胞的裸鼠异种移植模型中,Ki8751（5 mg/kg）在不影响体重的情况下完全抑制肿瘤生长.在携带人类肿瘤异种移植物GL07,St-4,LC6,DLD-1和A375细胞的裸鼠中,Ki8751（20 mg/kg）抑制肿瘤生长.

在人脐静脉内皮细胞（HUVECs）中,Ki8751（1 nM-100 nM）有效降低VEGF刺激的细胞增殖和血管通透性。在转移性结直肠癌（CRC）细胞MIP,RKO,SW620和SW480中,Ki8751（10 nM）增加细胞衰老,但在HCT116中无此效果。Ki8751有效且选择性抑制VEGFR-2,IC50为0.9 nM。Ki8751也抑制PDGFRα,c-Kit和 FGFR-2,具有更高的IC50值为40 nM–170 nM。

Cellular Kinase Assays: NIH3T3 cells prepared by transfection of human KDR. The cells are cultured in a collagen type I coated 96-well plate in an amount of 1.5 × 104 per well. The medium is then replaced by a DMEM medium containing 0.1% FCS. Ki8751 diluted in DMSO is added to each well and cultured. rhVEGF is added to a final concentration of 100 ng/mL, and the stimulation of cells is carried out at 37 °C. The cells are washed with PBS (pH 7.4), 50 μL of a solubilization buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 0.2% Triton X-100, 10% glycerol, 5 mM Na3VO4, 5 mM disodium ethylenediamine tetraacetate, and 2 mM Na4P2O7) is then added and a cell extract is prepared. Separately, PBS (50 μL, pH 7.4) containing 5 μg/mL of antiphosphotyrosine antibody (PY20) is added to a microplate for ELISA. After washing of the plate, 300 μL of a blocking solution is added. The cell extract is transferred to the plate. An anti-VEGFR2 antibody and a peroxidase-labeled anti-rabbit Ig antibody are added. Next, a chromophoric substrate for peroxidase is added, and the absorbance at 450 nm is measured with microplate reader. The VEGFR2 phosphorylation activity for each well is determined by presuming the absorbance with the addition of VEGF and without the addition of the test sample to be 100% VEGFR2 phosphorylation activity and VEGF to be 0% VEGFR2 phosphorylation activity. The concentration of the inhibition (%) of VEGFR2 Phosphorylation is determined for each case, and IC50 value is calculated.

To evaluate the inhibition of VEGF-Stimulated HUVEC proliferation by Ki8751, HUVECs are plated at a density of 4000 cells/200 μL/well in a type I collagen pre-coated 96-well plates. After 24 hours, the cells are incubated for 1 hour with Ki8751 and then stimulated with 20 ng/mL rhVEGF. The cultures are incubated at 37 °C for 72 hours, then pulsed with 1 μCi/well [3H]thymidine and re-incubated for 14 hours. Cells are assayed for the incorporation of tritium using a beta counter. (Only for Reference)