Design, synthesis, and evaluation of a novel benzamidine-based inhibitor of VEGF-C binding to Neuropilin-2

Abstract

The Neuropilin (Nrp) family of cell surface receptors have key physiological and pathological functions. Nrp2 is of particular interest due to its involvement in tumor metastasis. Currently, peptide and small molecule inhibitors that target Nrp utilize arginine-based molecules which have limitations due to high inherent flexibility and issues related to stability. Further, there are no known small molecule inhibitors specific for Nrp2. Recent molecular insights identify a key ligand binding region in the b1 domain of Nrp2 responsible for binding the C-terminus of its cognate ligand VEGF-C. Based on this, we report the discovery of a novel benzamidine-based inhibitor that functions through competitive inhibition of VEGF-C binding to Nrp2. Further, we have explored inhibitor functionality and selectivity by defining its structure-activity relationship (SAR) providing valuable insights on this benzamidine-based family of Nrp2 inhibitors. This study provides the basis for further development of a potent and specific small molecule inhibitor that competitively targets pathological Nrp2 function.

Keywords: Angiogenesis; Benzamidine; Lymphatic; Neuropilin; Nrp-2; Receptor; VEGF.

Scheme 1:

Synthesis of Inhibitor 2: Reagents and conditions: (a) EDCI, HOAt, DIEA, DMF, 16 h; (b) 3 N methanolic HCl, 3 h at 0 °C; (c) N-Boc-D-Phenylalanine, EDCI, HOAT, DIEA, DMF, 16 h; (d) Hydroxylamine HCl, DIEA, anhydrous MeOH, 16 h; followed by Ac₂O, AcOH, 30 min then H₂, 10% Pd/C, 12 h; (e) 3 N methanolic HCl, 3 h at 0 °C.

Scheme 2:

Synthesis of Intermediates (3a–3c): Reagents and conditions: (a) EDCI, HOAt, DIEA, DMF, 16 h; (b) 3 N methanolic HCl, 3 h at 0 °C; (c) different N-Boc-D-amino acids, EDCI, HOAt, DIEA, DMF, 16 h; (d) Hydroxylamine HCl, DIEA, anhydrous MeOH, 16 h; followed by Ac₂O, AcOH, 30 min then H₂, 10% Pd/C, 12 h; (e) 3 N methanolic HCl, 3 h at 0 °C.

Scheme 3:

Synthesis of Inhibitors (5–13): Reagents and conditions: (a) EDCI, HOAt, DIEA, DMF, 16 h; (b) 3 N methanolic HCl, 3 h at 0 °C; (c) different N-Boc-D-amino acids, EDCI, HOAt, DIEA, DMF, 16 h; (d) Hydroxylamine HCl, DIEA, anhydrous MeOH, 16 h; followed by Ac₂O, AcOH, 30 min then H₂, 10% Pd/C, 12 h; (e) 3 N methanolic HCl, 3 h at 0 °C.

Figure 1:

Design of benzamidine-based inhibitors of Nrp2. A) CT-Arg binding pocket of Nrp2 highlighting key interaction interfaces B) Model of a benzamidine based inhbitor in the VEGF-C binding pocket of Nrp2.

Figure 2:

Discovery of benzamidine-based inhibitors of Nrp2. A) Initial lead compound with (5) and without (2) carboxylate. B) Inhibitory potency of 5 and 2 in blocking VEGF-C binding to Nrp2. Data points represent the mean value and standard deviation, N=3. Lines represent the fit used for determintation of the IC₅₀.

Figure 3:

Docking of Nrp2 selective inhibitors. Docking of compounds A) 11 and B) 9 supports a ligand-like CT-Arg binding mode via engagement with the Nrp2 coagulation factor loops (labelled L1-L3) and a critical role for the Nrp2 L1 loop residue D301 engagement by the inhibitor R2-functionality (pink circle) in determining Nrp2 selectivitiy.