Angiogenesis is a crucial factor in tumor proliferation and metastasis [1], [2]. Among the various participants involved in different mechanisms of vascular formation, members of the vascular endothelial growth factor (VEGF) family play a dominant role [3]. VEGF is a dimeric glycosylated basic protein, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. VEGF ligands can bind to three different VEGF receptor tyrosine kinases (VEGFR-TK), namely VEGFR-1 (Flt-1), VEGFR-2 (KDR), and VEGFR-3 (Flt-4) [4], [5]. Among them, VEGFR-2 is the most significant angiogenic factor, primarily expressed on the surface of vascular endothelial cells.

VEGFR-2, when activated by VEGF, initiates downstream signaling, resulting in the formation of a vascular network that enhances tumor proliferation and metastasis [6], [7]. Inhibiting the VEGFR-2/VEGF signaling pathway leads to effective anti-angiogenesis and anti-tumor responses [8], [9], [10]. Currently, many small molecule VEGFR-2 inhibitors have been reported, such as sunitinib [11], axitinib [12], lenvatinib [13], tivozanib [14], fruquintinib [15], PAN-90806 [16]. However, these chemotherapic drugs have been associated with many side effects (fatigue, skin abnormalities, diarrhea, hypertension), and resistance mechanisms have emerged [17], [18]. Therefore, it is necessary to discover new anti-angiogenic agents that can reduce side effects and block tumor cell resistance while enriching VEGFR-2 inhibitors [19].

PAN-90806 has been identified as a potent inhibitor of VEGFR-2 kinase (IC50 = 11 nM) [16]. Molecular dynamics simulations were performed on the small molecule PAN-90806, and the crystal structure of VEGFR-2 (4AGC.pdb) was obtained from the RCSB Protein Data Bank (RCSB PDB, http://www.rcsb.org/) [20]. The RMSD value does not show significant fluctuations, indicating the overall stability of the 4AGC-PAN-90806 complex (Fig. S1). According to the bar charts (Fig. 1A) of protein–ligand contacts and contacts throughout trajectory (Fig. 2), the urea (red) and formamide (red) could form stable hydrogen bonds with CYS919 and GLU917, and CYS919 has an interaction fraction exceeding 250 %. The hydrogen bond interactions play important roles in stabilizing the binding mode of PAN-90806 to VEGFR-2, thus maintaining the basic framework of the 5-membered heterocycle (green), formamide (red), and urea (red) fragments. Initially, we utilized the principles of bioisosterism to substitute the isothiazole ring with a thiophene ring. Subsequently, the biological effects of bridging atoms between the aromatic region and the thiophene ring (pink) were focused. In Fig. 1B, it was observed that PAN-90806 did not occupy the entire protein cavity. Extending the linker length allowed the aromatic region to fill the internal cavity (9a-9q). Finally, adhering to conformationally constrained, we directly linked the aromatic region to the thiophene ring (14a-14l).