Fragment deconstruction study and human thrombin-bound crystal structures of new β-d-glucose-containing anticoagulants

We recently synthesized novel glucose-conjugated dual factor Xa (fXa) and thrombin (fIIa) inhibitors 3, bearing 5-chlorothien-2-yl and 1-isopropylpiperidine moieties as binders of the S1 and S3/S4 enzymes’ pockets, respectively, which showed potential for use in the treatment of thrombotic diseases. In particular, the β-d-glucosyl-bearing derivative proved to be a competitive inhibitor with high potency against fXa (Ki = 0.09 nM) and fIIa (Ki = 100 nM), and in vitro/ex vivo micromolar anticoagulant potency.1 Despite the narrower binding site groove of fIIa, the inhibitory potency of the glucosyl derivative, compared to the parent glucosyl-lacking compound 1, increases against fIIa (110-fold) much more than against fXa (7-fold). Experimental deconstruction of the most potent inhibitor molecule into smaller fragments, synthesized and tested, provided us with significant insights into the enzymes’ affinity contributions of the P1 and P3/P4 moieties, and a C3-alkyl-linked -d-glucose fragment (PG). To understand the inhibitors’ binding modes to fIIa, the crystal structures of human thrombin in complex with two glucose-based compounds were solved (pdb codes: 4NZE and 4N3L), and the crystallographic results will be presented and discussed.