Aromatic heterocycle galectin-1 interactions for selective single-digit nM affinity ligands

Abstract

A series of 3-triazole-thiogalactosides and 3,3'-triazole-thiodigalactosides substituted with different five-membered heterocycles at the C-4 triazole position were found to have high selectivity for galectin-1. Initial studies on the 3-triazole-thiogalactosides indicated that five membered heterocycles in general gave increased affinity for galectin-1 and improved selectivity over galectin-3. The selectivity profile was similar for thiodigalactosides exemplified by 3,3' substituted thien-3-yltriazole and thiazol-2-yltriazole, both having single-digit nM galectin-1 affinity and almost 10-fold galectin-1 selectivity. The binding interactions of a thiodigalactoside based galectin-1 inhibitor with two thien-3-yltriazole moieties were studied with X-ray crystallography. One of the thiophene moieties was positioned deeper into the pocket than previously reported phenyltriazoles and formed close contacts with Val31, Ser29, Gly124, and Asp123. The affinity and structural analysis thus revealed that steric and electronic optimization of five-membered aromatic heterocycle binding in a narrow galectin-1 subsite confers high affinity and selectivity.

Fig. 1. Thien-3-yl thiodigalactoside derivative 1 with high affinity for galectin-1.

Scheme 1. Synthesis of triazoles 4–9 and 12–15. Reagents and conditions: (a) NaOMe, MeOH, rt; (b) alkyne, CuI, DIPEA, MeCN, 50 °C; (c) (i). 1-{[2-(Trimethylsilyl)ethoxy]methyl}-4-[2-(trimethylsilyl)ethynyl]-1H-imidazole, CuI, DIPEA, MeCN, 50 °C; (ii). BF₃OEt₂, CH₂Cl₂, rt; (d) methyl thioglycolate, BF₃ OEt₂, CH₂Cl₂, rt; (e) (i). Alkyne, CuI, DIPEA, MeCN, 50 °C; (ii). NaOMe, MeOH, rt; (iii). LiOH, THF : H₂O (9 : 1), rt. Tol = p-methylphenyl.

Scheme 2. Synthesis of thiodigalactosides 19–20 and 22–23. Reagents and conditions: (a) (i). alkyne (3 equiv.), CuI, DIPEA, DMF, 50 °C; (ii). NaOMe, MeOH, rt; (b) (i). Thiazol-2-ylethynyltrimethylsilane (1.5 equiv.), CuI, DIPEA, DMF, 50 °C; (ii). NaOMe, MeOH, rt; (c) alkyne, CuI, DIPEA, DMF, 50 °C.

Scheme 3. Synthesis of fluorescent probe 25. Reagents and conditions: (a) 1,3-propanedithiol, Et₃N, MeOH, rt; (b) 5-FAM-NHS, DIPEA, DMSO, rt.

Fig. 2. Difference electron density of 1 in the galectin-1 binding site. Difference electron density calculated from refinement with the ligand (stick representation) omitted from the model (|F_o| − |F_c| α_calc; grey mesh, contoured at 3σ) and the protein represented by a grey solvent-accessible surface. The narrow pocket accommodating one of the thiophene rings is indicated with a yellow arrow.

Fig. 3. Galectin-1 binding site interactions with 1. H-bond interactions between ligand (carbons white) and protein/water (carbons green) are shown as dashed lines.

Fig. 4. Comparison of the binding conformation of 1 when bound to galectin-1 (carbons pink) and galectin-3  (carbons green).