Design and synthesis of sorafenib-inspired benzofuran hybrids as VEGFR-2 inhibitors: antiproliferative evaluation, mechanistic insights, and docking studies in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide owing to its high metastatic potential. Vascular endothelial growth factor receptor-2 (VEGFR-2), a key regulator of angiogenesis and cell proliferation, is a critical therapeutic target in cancer, particularly in HCC. However, the clinical use of various VEGFR-2 inhibitors is limited by adverse effects, poor selectivity and drug resistance. In this study, two novel series of sorafenib-inspired benzofuran hybrids, azines (6a-k) and thiosemicarbazones (7a-g), were synthesized and evaluated for their cytotoxicity against HepG-2 cell lines. Compounds 6b, 6c, 7a, 7b, 7e, 7f, and 7g exhibited potent activity with IC₅₀ values of 7.21-18.01 μM. Among them, 7a, 7b, 7f, and 7g demonstrated high selectivity (SI = 5.7-11.2) toward HepG-2 cells over normal WI-38 cells. Enzyme assays confirmed significant VEGFR-2 inhibition, led by 7g (IC₅₀ = 0.072 μM), comparable to sorafenib (IC₅₀ = 0.069 μM). In HepG-2 cells, 7g induced G0/G1 arrest and apoptosis, upregulating Bax, caspase-8 and -9, and downregulating Bcl-2. Molecular docking confirmed the strong binding affinity of 7g within the VEGFR-2 active site through key interactions with Glu885, Asp1046, and Cys1045, mirroring sorafenib. A 100 ns molecular dynamics simulation further demonstrated that compound 7g retains a highly stable binding mode within VEGFR-2, supported by low RMSD fluctuations and persistent key hydrogen-bond and hydrophobic interactions. These findings nominate 7g as a promising VEGFR-2-targeted lead for HCC upon further optimization.

Fig. 1. Structure of sorafenib and representative benzofuran-based compounds previously reported as VEGFR-2 inhibitors.

Fig. 2. Pharmacophoric model of sorafenib as a VEGFR-2 type II inhibitor, highlighting four key structural features: (A) hinge-binding region (rose), (B) aromatic linker (pink), (C) hydrogen-bonding moiety (green), and (D) hydrophobic tail (blue). Alternative pharmacophoric moieties were illustrated for each key structural feature. Each moiety is known for its potential VEGFR-2 inhibition and cytotoxic effects.

Fig. 3. Reported anticancer and VEGFR-2 inhibitory compounds featuring alternative pharmacophoric incorporations.

Fig. 4. Newly designed series 6a–k and 7a–g obtained through structural modification of the sorafenib scaffold via molecular hybridization, illustrating alterations in each key pharmacophoric region.

Fig. 5. Visual summarization of SAR study for the anti-cancer activity of the novel compounds (6a–k and 7a–g).

Scheme 1. Synthesis of the targeted VEGFR-2 inhibitors (6a–k and 7a–g), reagents and conditions: (a) HCl, NaNO₂, H₂O/NaOH, Na₂CO₃, stirring, 0 °C, 4 h; (b) chloroacetone, K₂CO₃, acetone, reflux, 6 h; (c) hydrazine hydrate, ethanol, reflux, 4 h; (d) ethanol, 3–5 dps of glacial acetic acid, reflux, 3 h; (e) ethanol, reflux, 4 h.

Fig. 6. Cell cycle distribution of HepG-2 cells treated with compound 7g (left side) and control (right side).

Fig. 7. Necrotic and apoptotic cell death of HepG-2 cells treated with compound 7g on the left and control HepG-2 on the right.

Fig. 8. Effect of compound 7g on the expression levels of apoptotic genes (Bax, Bcl-2, caspase-8, and caspase-9) in HepG-2 cells compared to the control.

Fig. 9. The re-docked pose of the ligand (green) superimposition onto the co-crystallized sorafenib ligand (orange) with RMSD of 1.47 Å in the active site of VEGFR-2 (PDB ID: 3WZE) for the validation step.

Fig. 10. 2D interaction diagram of sorafenib with VEGFR-2 active site (upper) and its 3D binding mode (lower).

Fig. 11. 2D interaction diagrams of compounds (A) 7g, (B) 7b, (C) 7f, and (D) 7a with VEGFR-2 active site (left), and their corresponding 3D binding modes (right).

Fig. 12. The 100 ns simulation RMSD values for the proteins VEGFR-2 Cα-atoms (blue) and compound 7g heavy atoms (red).

Fig. 13. 100 ns simulation interaction diagram panel for compound 7g and VEGFR-2; (A) fractions of interaction between compound 7g and VEGFR-2 active site residues, (B) 2D-interaction diagram of compound 7g within the VEGFR-2 active site.

Fig. 14. Frontier Kohn–Sham molecular orbitals of compounds 7g; (A) optimized structure, (B) LUMO, (C) HOMO.