Exploring a unique pocket in Galectin-8N by the synthesis of D-galactoside-based benzimidazole inhibitors

Abstract: Galectins are a family of glycan-binding proteins where galectin-8 is a part of the subgroup known as tandem-repeat type galectins. Galectin-8 is composed of two homologous CRDs, one N- and one C-terminal CRD. As it stands now, galectin-8N has been found to bind to a larger number of glycans than galectin-8C. Thus, it is currently of a higher therapeutical interest to focus research on the N-terminal CRD of galectin-8 than on the C-terminal. Studies on galectin- 8N inhibitors have revealed that benzimidazole-galactoside derivates show a greater affinity and selectivity for galectin-8N by binding to unique amino acids, namely Arg45. However, recent research has shown that there are two more unique amino acids, Arg59 and Tyr141, existing in a pocket of galectin-8N. It was found that this pocket and its unique amino acids can be exploited by placing substituents on the C-4 position of the benzimidazole leading to a galectin-8N inhibitor with a higher affinity and selectivity, which was the aim of this thesis. Ten inhibitors were synthesized in six synthesis steps starting with an equatorial chlorination at C-1 of β-D-galactose pentaacetate, followed by an SN2 reaction yielding an α-thioglycoside. A Zemplén deacetylation was then performed following a regioselective 3-O-monoalkylation with methyl bromoacetate as the reagent, which was then cyclized into a benzimidazole. For the final step, Suzuki-couplings were executed at C-4 of the benzimidazole with a p- carboxyphenyl substituent having the highest affinity for galectin-8N with Kd of 11 ± 1 μM of the ten synthesized inhibitors. Whilst none of the synthesized compounds managed to surpass current more potent galectin-8N inhibitors, it highlighted the importance of a substituent at the C-6 position of the benzimidazole, which was omitted in this project. However, the importance of the substituent at C-4 should not be ignored considering the synthesized compounds differ by a factor of a 100 with the p-carboxyphenyl substituent revealing promising results for further exploration of the pocket.