Inhibitors of human neuraminidase enzymes (NEU) are recognized as important tools for the study of the biological functions of NEU and will be potent tools for elucidating the role of these enzymes in regulating the repertoire of cellular glycans. Here we report the discovery of selective inhibitors of the human neuraminidase 1 (NEU1) and neuraminidase 2 (NEU2) enzymes with exceptional potency. A library of modified 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) analogues, with variability in the C5- or C9-position, were synthesized and evaluated against four human neuraminidase isoenyzmes (NEU1–4). Hydrophobic groups with an amide linker at the C5 and C9 positions were well accommodated by NEU1, and a hexanamido group was found to give the best potency at both positions. While the C5-hexanamido-C9-hexanamido-DANA analogue did not show synergistic improvements for combined modification, an extended alkylamide at an individual position combined with a smaller group at the second gave increased potency. The best NEU1 inhibitor identified was a C5-hexanamido-C9-acetamido-DANA that had a K i of 53 ± 5 nM and 340-fold selectivity over other isoenzymes. Additionally, we demonstrated that C5-modifications combined with a C4-guandino group provided the most potent NEU2 inhibitor reported, with a K i of 1.3 ± 0.2 μM and 7-fold selectivity over other NEU isoenzymes.

Human neuraminidases (NEU) are associated with human diseases including cancer, atherosclerosis, and diabetes. To obtain small molecule inhibitors as research tools for the study of their biological functions, we designed a library of 2-deoxy-2,3-didehydro- N-acetylneuraminic acid (DANA) analogues with modifications at C4 and C9 positions. This library allowed us to discover selective inhibitors targeting the human NEU3 isoenzyme. Our most selective inhibitor for NEU3 has a K of 320 ± 40 nM and a 15-fold selectivity over other human neuraminidase isoenzymes. This inhibitor blocks glycolipid processing by NEU3 in vitro. To improve their pharmacokinetic properties, various esters of the best inhibitors were synthesized and evaluated. Finally, we confirmed that our best compounds exhibited selective inhibition of NEU orthologues from murine brain.

Bruton's tyrosine kinase (Btk) is an attractive drug target for treating several B-cell lineage cancers. Ibrutinib is a first-in-class covalent irreversible Btk inhibitor and has demonstrated impressive effects in multiple clinical trials. Herein, we present a series of novel 2,5-diaminopyrimidine covalent irreversible inhibitors of Btk. Compared with ibrutinib, these inhibitors exhibited a different selectivity profile for the analyzed kinases as well as a dual-action mode of inhibition of both Btk activation and catalytic activity, which counteracts a negative regulation loop for Btk. Two compounds from this series, 31 and 38, showed potent antiproliferative activities toward multiple B-cell lymphoma cell lines, including germinal center B-cell-like diffuse large B cell lymphoma (GCB-DLBCL) cells. In addition, compound 31 significantly prevented tumor growth in a mouse xenograft model.