Synthesis, Antitumor Activities, and Apoptosis-Inducing Activities of Schiff's Bases Incorporating Imidazolidine-2,4-dione Scaffold: Molecular Docking Studies and Enzymatic Inhibition Activities

Abstract

Background/Objective: Cancer is the leading cause of death worldwide despite the diversity of antitumor therapies, which highlights the necessity to explore new anticancer agents. Methods: We synthesized 5,5-diphenylhydantoin derivatives including Schiff's bases 7-27 and evaluated their cytotoxicity via the MTT assay. Enzymatic inhibition assays, cell cycle and apoptosis analyses, and molecular docking studies were also conducted. Results: Derivative 24 demonstrated the highest cytotoxic activity, with IC₅₀ values of 12.83 ± 0.9 μM, 9.07 ± 0.8 μM, and 4.92 ± 0.3 μM against the cell lines HCT-116, HePG-2, and MCF-7, respectively. Compounds 10, 13, and 21 showed potent antitumor activities versus the examined cell lines (average IC₅₀ = 13.2, 14.5, and 13.1 μM), respectively; moreover, these compounds also demonstrated promising EGFR and HER2 inhibitory activities, with IC₅₀ values in the range 0.28-1.61 µM. Derivative 24 displayed the highest EGFR and HER2 inhibitory activity values (IC₅₀ = 0.07 and 0.04 µM), respectively, which were close to those of the reference drugs erlotinib and lapatinib. Therefore, compound 24 was selected for further examinations and exhibited an inducing effect on apoptosis via diminishing the anti-apoptotic protein levels of BCL-2 (8.598 ± 0.29 ng/mL) and MCL-1 (261.20 ± 8.97 pg/mL) and promoting cell cycle arrest at the G2/M phase (33.46%). The binding relationships between compound 24 and the active sites of EGFR and HER2, which are similar to the co-crystallized inhibitors, were investigated using a molecular docking approach. Conclusions: These findings provide insights into the potential anticancer activities of the synthesized derivatives for further optimization to achieve therapeutic use.

Keywords: 5,5-diphenylacetophenone; EGFR2; HER2; molecular docking.

Figure 1

Diphenylhydantoin as anticancer derivatives.

Figure 2

The reported and designed target Schiff’s bases associated with diphenylhydantoin with anticancer and kinase suppressive activities.

Scheme 1

Synthesis of ketones conjugated with 5,5-diphenylimidazolidine-2,4-dione.

Figure 3

The effect of compound 24 and the control drug (erlotinib) on the expression level of the anti-apoptotic protein MCL-1 in MCF-7.

Figure 4

The effect of compound 24 and the control drug (erlotinib) on the expression level of the anti-apoptotic protein BCL-2 in MCF-7.

Figure 5

Overlay of the redocked (green) and co-crystallized ligand 03Q (yellow) within the HER2 binding site (A) and the redocked ligand (green) and co-crystallized ligand Gefitinib (yellow) within the EGFR binding site (B). The close alignment of the redocked and co-crystallized ligands validates the docking protocol and confirms the reliability of the binding pose predictions.

Figure 6

Three-dimensional representation of the HER2 (PDB code: 3PP0) binding site showing compound 24 (depicted in green) in juxtaposition to the co-crystalline ligand (03Q) colored in yellow. The spatial orientations provide insights into their respective interactions and alignments within the active site.

Figure 7

Three-dimensional (3D) representation of the EGFR kinase region showcasing the binding interactions of compound 24 (depicted in green) and the co-crystalline ligand (PDB code: 2ITY) rendered in brown. The distinct spatial orientations and interaction points highlight their respective inhibitory mechanisms within the ATP-binding cleft.

Figure 8

The Root Mean Square Deviation (RMSD) analysis of compound 24 and the co-crystallized ligand (A) Pro-03Q with HER2 and (B) Gefitinib with EGFR over 100 ns molecular dynamics simulations. The RMSD plot reflects the structural stability and conformational changes in the protein–ligand complexes, where compound 24 exhibits a stable binding mode comparable to the reference co-crystallized ligands in both the HER2 and EGFR systems.

Figure 9

Root Mean Square Fluctuation (RMSF) analysis of backbone Cα atoms for compound 24 and co-crystallized ligands with (A) HER2 and (B) EGFR. The RMSF profiles reveal the fluctuation patterns across key structural regions, including the glycine-rich loop, αC helix, and activation loop. Compound 24 shows distinct fluctuation patterns compared with the co-crystallized ligands, indicating differential stabilization effects on the kinase domains.

Figure 10

Radius of gyration (Rg) analysis for compound 24 and the co-crystallized ligand in (A) HER2 and (B) EGFR. The Rg values reflect the compactness and structural stability of the protein–ligand complexes over the 100 ns molecular dynamics simulation. A slight fluctuation in Rg indicates conformational adjustments upon ligand binding, where compound 24 exhibits a more stable and compact structure compared with the co-crystallized ligand in both HER2 and EGFR.

Figure 11

Solvent-Accessible Surface Area (SASA) analysis for compound 24 and co-crystallized ligands with (A) HER2 and (B) EGFR throughout 100 ns MD simulations. The plot shows the dynamic behavior of the protein–ligand complexes, where compound 24 exhibits reduced the SASA compared with the co-crystallized ligands, indicating tighter binding and potential structural stability in both targets.