Design and synthesis of phthalazine-based compounds as potent anticancer agents with potential antiangiogenic activity via VEGFR-2 inhibition

Abstract

In the designed compounds, either a biarylamide or biarylurea moiety or an N-substituted piperazine motif was linked to position 1 of the phthalazine core. The anti-proliferative activity of the synthesised compounds revealed that eight compounds (6b, 6e, 7b, 13a, 13c, 16a, 16d and 17a) exhibited excellent broad spectrum cytotoxic activity in NCI 5-log dose assays against the full 60 cell panel with GI₅₀ values ranging from 0.15 to 8.41 µM. Moreover, the enzymatic assessment of the synthesised compounds against VEGFR-2 tyrosine kinase showed the significant inhibitory activities of the biarylureas (12b, 12c and 13c) with IC₅₀s of 4.4, 2.7 and 2.5 μM, respectively, and with 79.83, 72.58 and 71.6% inhibition of HUVEC at 10 μM, respectively. Additionally, compounds (7b, 13c and 16a) were found to induce cell cycle arrest at S phase boundary. Compound 7b triggered a concurrent increase in cleaved caspase-3 expression level, indicating the apoptotic-induced cell death.

Keywords: Substituted phthalazines; VEGFR-2 kinase inhibitors; anti-proliferative; apoptosis.

Figure 1.

Structures of some known VEGFR-2 kinase inhibitors.

Figure 2.

Strategy for the design of the target compounds.

Scheme 1.

(a) Reagents and conditions: (i) dioxane, rt, 1 h. (b) Reagents and conditions: (i) NH₂NH₂ 99%, propanol, 2 h (ii) POCl₃, reflux 70 °C, 2 h (iii) p-phenylenediamine, butanol, 110 °C, 1 h, (iv) benzoyl chlorides, acetonitile, triethylamine, 6 h (v) arylisocyanates, DMF, reflux, 8 h, (vi) 1a–c, Cs₂CO₃, acetonitrile, reflux, 6 h, (vii) piperazines, K₂CO₃, KI, ethanol, reflux, 3 h.

Scheme 2.

Reagents and conditions: (i) NH₂NH₂ acetic, reflux, 3 h (ii) POCl₃, reflux 110 °C, 1 h (iii) p-phenylenediamine, butanol, 110 °C, 1 h, (iv) phenylisocyanates, DMF, reflux, 8–10 h, (v) 1a–c, Cs₂CO₃, acetonitrile, reflux, 6–8 h.

Scheme 3.

Reagents and conditions: (i) aniline derivatives, butanol, K₂CO₃, KI, reflux, 3 h (ii) NBS, dibenzoyl chloride, reflux, 24 h (iii) aniline derivatives, acetone, K₂CO₃, KI, reflux, 6 h (iv) phenol derivatives, NaH, acetone, reflux, 8 h.

Figure 3.

IC₅₀ curves for compounds (6c, 12b, 12c and 13c) against the VEGFR-2 kinase.

Figure 4.

The bar graphs showing the HUVECs growth percentage after treatment with the target compounds.

Figure 5.

Effect of compound 7b on DNA-ploidy flow cytometric analysis of MCF7 cells. MCF-7cells were treated with DMSO 0.01% or compound 7b at its GI₅₀ (0.32 µM), for 24 h and 48 h, and the harvested cells were subjected to Cell-cycle analysis using a FACS Calibur flow cytometer (A,B and C). Bar chart shows percentage of MCF7 cells at each phase of cell cycle in DMSO and compound 7b treated cells.

Figure 6.

Effect of compound 13c on DNA-ploidy flow cytometric analysis of MCF7 cells. MCF-7cells were treated with DMSO 0.01% or compound 13c at its GI₅₀ (0.57 µM), for 24 h and 48 h, and the harvested cells were subjected to cell-cycle analysis using a FACS Calibur flow cytometer (A, B and C). Bar chart shows percentage of MCF7 cells at each phase of cell cycle in DMSO and compound 13c treated cells.

Figure 7.

Effect of compound 16a on DNA-ploidy flow cytometric analysis of HCT-116 cells. HCT-116 cells were treated with DMSO 0.01% or compound 16a at its GI₅₀ (1.20 µM), for 24 h and 48 h, and the harvested cells were subjected to Cell-cycle analysis using a FACS Calibur flow cytometer (A,B and C). Bar chart shows percentage of HCT-116 cells at each phase of cell cycle in DMSO and compound 16a treated cells.

Figure 8.

Effect of compound 7b treatment on induction of apoptosis. MCF-7 cells were treated with DMSO 0.01% or compound 7b at its GI₅₀ (0.32 µM) for 24 or 48h, the harvested cells were stained with Annexin V-FITC apoptosis detection kit and Analyzed on a Flow cytometer.

Figure 9.

Effect of compound 13c treatment on induction of apoptosis. MCF-7 cells were treated with DMSO 0.01% or compound 13c at its GI₅₀(0.57 µM) for 24 or 48h, the harvested cells were stained with Annexin V-FITC apoptosis detection kit and Analyzed on a Flow cytometer.

Figure 10.

Effect of compound 16a treatment on induction of apoptosis. HCT-116 cells were treated with DMSO 0.01% or compound 16a at its GI₅₀(1.20 µM) for 24 or 48h, the harvested cells were stained with Annexin V-FITC apoptosis detection kit and Analyzed on a Flow cytometer.

Figure 11.

Effect of compound 7b on the cellular and nuclear morphology. MCF-7 cells were seeded on cover slips in a 6- well tissue culture plate, the cells then were treated with DMSO 0.01% or compound 7b (0.32 µM) for 24 or 48h, the cells were imaged with the light microscope to examine the cellular morphology (A) or stained with the nuclear stain Dapi to study the nuclear morphology using fluorescence microscope (B).

Figure 12.

Effect of compound 7 b on cleaved caspase-3 protein expression. MCF-7 cells were seeded on cover slips in a 6-well tissue culture plate, the cells then were treated with DMSO 0.01% or compound 7b at its GI₅₀(0.32 µM) for 48h, the cells were then permiabilised, fixed, blocked and stained with the primary and secondary antibodies, the nuclear stain Dapi was used as a counter stain. The image were taken using a fluorescence microscope.

Figure 13.

Overlay of the best-scored docking poses of some relevant compounds on the co-crystallised ligand Sorafenib (cyan sticks) in the binding site of VEGFR2 (PDB: 4ASD):. (A) The docking pose of 6c as magenta sticks, (B) the docking poses of 12b and 12c as yellow and purple sticks, respectively, and (C) the docking pose of 13c as gold sticks. (D) The docking poses of 12c and 13c at a different perspective of the binding site showing the extended hydrophobic contact of the chloro and trifluoromethyl phenyl moiety with the side chains of PHE-918 and LEU-840. The yellow dashed-lines represent the polar contacts (H-bonding interactions). Non-polar hydrogen atoms were omitted for clarity. All presented docking poses are the best-scored by AutoDock Vina.