Discovery of multi-target receptor tyrosine kinase inhibitors as novel anti-angiogenesis agents

Abstract

Recently, we have identified a biphenyl-aryl urea incorporated with salicylaldoxime (BPS-7) as an anti-angiogenesis agent. Herein, we disclosed a series of novel anti-angiogenesis agents with BPS-7 as lead compound through combining diarylureas with N-pyridin-2-ylcyclopropane carboxamide. Several title compounds exhibited simultaneous inhibition effects against three pro-angiogenic RTKs (VEGFR-2, TIE-2 and EphB4). Some of them displayed potent anti-proliferative activity against human vascular endothelial cell (EA.hy926). In particular, two potent compounds (CDAU-1 and CDAU-2) could be considered as promising anti-angiogenesis agents with triplet inhibition profile. The biological evaluation and molecular docking results indicate that N-pyridin-2-ylcyclopropane carboxamide could serve as a hinge-binding group (HBG) for the discovery of multi-target anti-angiogenesis agents. CDAU-2 also exhibited promising anti-angiogenic potency in a tissue model for angiogenesis.

Figure 1. Sequence alignment of ATP-binding pocket of the three RTKs with PDB code and name (VEGFR-2, Tie-2, and EphB4; Conserved residues are colored blue).

Figure 2. Protein structure alignment and superposition of three angiogenic RTKs. VEGFR-2 (Red), EphB4 (Cyan), and TIE-2 (Green).

Figure 3. Structural optimization of taspine and identification of BPS-7.

Figure 4

Exploration structural diversity of hinge-binding group (HBG) and structures of title compounds.

Figure 5. Synthetic route of the title compounds (CDAU-1~CDAU-11).

Reagents and conditions: (a) Pinacol, MgSO₄, Et₃N, THF; (b) R-NH₂, BTC, Et₃N, DCM, 0 °C to rt; (c) Cyclopropanecarbonyl chloride, Et₃N, DCM, 0 °C to rt; (d) Pd(PPh₃)₄, K₂CO₃, H₂O, dioxane, reflux, 100 °C.

Figure 6. Synthetic route of the title compounds (CDAU-12~CDAU-25).

Reagents and conditions: (a) R-NH₂, BTC, Et₃N, DCM, 0 °C to rt; (b) Pd(PPh₃)₄, K₂CO₃, H₂O, CH₃CN, reflux, 100 °C; (c) Cyclopropanecarbonyl chloride, Et₃N, THF, 0 °C to rt.

Figure 7. Low-energy docking model of the RTK/inhibitor complexes. Hydrogen bonds interactions are shown as dark-yellow dotted lines.

(A)VEGFR-2 (PDB ID: 4ASD); (B) TIE-2 (PDB ID: 2P4I); (C) EphB4 (PDB ID: 4BB4).

Figure 8. Anti-angiogenesis potency of compound CDAU-2 in tissue model for angiogenesis (TMA).

(A–F) the representative images of lung tissue blood vessels in the TMA on the 5^th day; (A) the untreated control group; (B–F) lung tissue vessels in the CDAU-2 treated group; (B) 1.94 μM; (C) 3.87 μM; (D) 7.75 μM; (E) 15.6 μM; (F) 62.5 μM. Vessels grew normally in control group; vessels in the CDAU-2 treated group exhibited the slow increase compared with the control group.

Figure 9. Design strategy and potential action mechanism of multi-target anti-angiogenesis agents with VEGFR-2/TIE-2/EphB4 as targets.

Simultaneous blockade of VEGFR-2/TIE-2/EphB4 signaling pathways leads to inhibition of EC survival, vascular permeability, migration, and proliferation within angiogenesis.