New nicotinamide-thiadiazol hybrids as VEGFR-2 inhibitors for breast cancer therapy: design, synthesis and in silico and in vitro evaluation

Abstract

Vascular endothelial growth factor receptor-2 (VEGFR-2) is a key regulator of tumor angiogenesis and has become an important target in anticancer drug development. In this study, novel nicotinamide-thiadiazol hybrids were synthesized and evaluated for their anti-breast cancer potential through VEGFR-2 inhibition. The compounds were assessed in vitro for their cytotoxicity against MDA-MB-231 and MCF-7 cell lines. Among the nicotinamide-thiadiazol hybrids, 7a exhibited the most potent anticancer activity, with IC₅₀ values of 4.64 ± 0.3 μM in MDA-MB-231 and 7.09 ± 0.5 μM in MCF-7, showing comparable efficacy to sorafenib. VEGFR-2 inhibition assays confirmed strong inhibitory potential with an IC₅₀ of 0.095 ± 0.05 μM. In vitro cell cycle analysis indicated that 7a induced S-phase arrest, while apoptosis assays demonstrated a substantial increase in late apoptotic cells (44.01%). Other in vitro mechanistic studies further confirmed the activation of the intrinsic apoptotic pathway, as evidenced by caspase-3 activation (8.2-fold), Bax upregulation (6.9-fold), and Bcl-2 downregulation (3.68-fold). Computational studies, including molecular docking and 200 ns molecular dynamics (MD) simulations, confirmed the stable interaction of 7a with VEGFR-2, showing binding affinities comparable to sorafenib. Further validation through MM-GBSA, ProLIF, PCAT, and FEL analyses reinforced its strong binding capability. Additionally, ADMET predictions suggested favorable pharmacokinetic properties, including good absorption, high plasma protein binding, and non-CYP2D6 inhibition. Moreover, toxicity analysis classified 7a as non-mutagenic and non-carcinogenic, with a lower predicted toxicity than sorafenib. Finally, density functional theory (DFT) calculations highlighted the structural stability and reactivity of 7a, further supporting its potential as a VEGFR-2 inhibitor. These findings suggest that 7a is a promising VEGFR-2 inhibitor with significant anticancer potential, favorable pharmacokinetics, and an improved safety profile. Further preclinical studies and structural modifications are warranted to optimize its therapeutic potential.

Fig. 1. Rational design of the new nicotinamide–thiadiazol hybrids as VEGFR-2 inhibitors. Sorafenib and sunitinib as examples of FDA-approved VEGFR-2 inhibitors showing the essential pharmacophoric features. Compounds III and IV as reported highly effective VEGFR-2 inhibitors. The designed compounds have the main pharmacophoric features of VEGFR-2 inhibitors.

Scheme 1. The synthetic methodology exploited for the synthesis of nicotinamide derivatives 7a–g.

Scheme 2. Anticipated mechanism for the synthesis of the target nicotinamide derivatives 7a–g.

Fig. 2. Structure–activity relationship (SAR) of the synthesized compounds based on the cytotoxic evaluation against MDA-MB-231 and MCF-7 cell lines.

Fig. 3. The effect of comp. 7a on different phases of MDA-MB-231 cell cycle.

Fig. 4. Apoptosis and necrosis rates of MDA-MB-231 cells treated with compound 7a compared to control.

Fig. 5. Validation within VEGFR-2 active pocket through the superimposition of the docked ligand (purple) upon the original ligand (turquoise).

Fig. 6. MS, 3D, and 2D images of the most promising nicotinamide derivative 7a interacted with the VEGFR-2 active site.

Fig. 7. (A) RMSD values from the trajectory for the VEGFR-2, 7a RMSD values, RoG for the VEFR-2 protein in the VEGFR-2_7a complex, SASA for the VEGFR-2 protein in the VEGFR-2_7a complex, change in the number of hydrogen bonds and distance from the center of mass of 7a compound and VEGFR-2 protein. (B) RMSF for the VEFR-2 protein in VEGFR-2_ 7a complex.

Fig. 8. Energetic components and their values for the VEGFR-2_7a complex.

Fig. 9. Binding free energy decomposition of the VEGFR-2_7a complex.

Fig. 10. Amino acids, interactions types of VEGFER-2 and 7a.

Fig. 11. Eigenvalues' changes showing an increase in eigenvectors (blue) and retained cumulative variance in the eigenvectors (red).

Fig. 12. The initial ten eigenvectors' distribution.

Fig. 13. Cosine content values of the initial ten eigenvectors for the trajectory.

Fig. 14. 2D and 3D projection of the VEGFR-2_7a trajectory's FEL on (A) first two, (B) first and third and (C) second and third eigenvectors.

Fig. 15. The optimized structure (A), the Mulliken charge distribution color scale (B), the HOMO/LUMO distribution functions and energy gap (C), the TDOS analysis (D), and the molecular electrostatic potential map (E) at B3LYP/6-31G(d,p) level for 7a.