New Series of VEGFR-2 Inhibitors and Apoptosis Enhancers: Design, Synthesis and Biological Evaluation

Figures

• 

  Figure 1. VEGFR-2 inhibitors (type II) pharmacophoric features as presented in sorafenib, AZD-2932, and the new derivatives and their corresponding binding regions as well.

• 

  Scheme 1. General procedures for preparation of intermediates 3a-g and 5; conditions: (i) DMF, NaHCO3, ice bath, stirring, 1h.

• 

  Scheme 2. General procedures for preparation of final compounds 10a-g and 11; Reagents and conditions: (i) oxalic acid, HCl, H2O, reflux. (ii) POCl3, DMF, reflux, 3h. (iii) piperazine, Et3N, absolute ethanol, ice bath. (iv) acetonitrile, NaHCO3, reflux, 2h.

• 

  Scheme 3. General procedures for synthesis of final compounds 13a-g and 14; Reagents and conditions: (i) NaOH, H2O, stirring, 50 °C. (ii) acetonitrile, NaHCO3, reflux, 2h.

• 

  Figure 2. Concentration dependent kinase inhibition plot of compound 11. The percent VEGFR-2 inhibition was determined for compound 11 relative to control, and the data were fitted to the dose-response curve to obtain IC50 values. Error bars represent SEM of three replicates.

• 

  Figure 3. Concentration dependent kinase inhibition plot of sorafenib. The percent VEGFR-2 inhibition was determined for sorafenib relative to control, and the data were fitted to the dose-response curve to obtain IC50 values. Error bars represent SEM of three replicates.

• 

  Figure 4. Flow cytometric analysis of HepG-2 cell cycle phases after 48 h treatment with 9.52 µM (IC50 value) of compound 11. After treatment with 11, HepG-2 cells were harvested, stained and then cell cycle phases were analyzed using Flowing software. (A) Illustrating histograms show the cell cycle distribution of both HepG-2 test cells and control. (B) Column diagram represents percentage of cells accumulation in the different phases of cell cycle. Results have been calculated as mean ± SEM of 3 experiments. *p < 0.05, ***p < 0.001 statistically indicate the significance of the obtained results compared to control (HepG-2).

• 

  Figure 5. Apoptosis rate in HepG-2 cells treated with (9.52 µM) of compound 11 for 48 h in comparison to control HepG-2 cells. (A) The obtained flow cytometric figures for both test and control HepG-2 cells. (B) Data from quantification of apoptosis are presented as mean ± SEM of 3 experiments. ***p < 0.001 statistically indicate the significance of the obtained results in comparison to control (HepG-2).

• 

  Figure 6. The immunoblotting of the expression of BAX, Bcl-2, Caspase-3, and p53 (Normalized to β-actin). (A) A given Western blot illustrations display the impact of 9.52 µM (IC50 value) of compound 11 on the tested proteins expression in HepG-2 cells treated for 48h. (B) The evaluated proteins quantity in the cell lysates. The reference HepG-2 cells were set to “1”, and all results from 3 experiments are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate the importance of the obtained results statistically.

• 

  Figure 7. Binding mode of compound 11.

• 

  Figure 8. Overlay of compound 11 (gray colored) and the ligand (green colored).