Discovery and characterization of a potent and selective non-amidine inhibitor of human factor Xa

Abstract

Benzothiophene-anthranilamide 1 (3-chloro- N-[2-[[(4-fluorophenyl)amino]carbonyl]-4-methylphenyl]benzo[ b]thiophene-2-carboxamide) was discovered by high throughput screening to be a highly potent and selective non-amidine inhibitor of human factor Xa with a K i of 15±4 nM. Compound 1 is a selective inhibitor of human factor Xa as suggested by the K i (app) determined for nine other human serine proteases and bovine trypsin. The activity of reconstituted human prothrombinase complex was inhibited by compound 1 when assayed in physiological concentrations of the substrate prothrombin. However, 27-fold higher inhibitor concentrations were needed to achieve the same level of inhibition than were required for the inhibition of free factor Xa, due in part to non-specific binding of the inhibitor to phospholipid under the assay conditions. Failure to demonstrate enzymatic cleavage of compound 1 suggests that compound 1 is solely an inhibitor rather than a substrate for factor Xa. The inhibition of factor Xa by compound 1 was reversible upon dilution of the enzyme/inhibitor mixture. Analyses of the inhibition mechanism with Dixon, Cornish–Bowden, and Lineweaver–Burk plots showed that compound 1 is a linear mixed-type inhibitor with 5-fold higher affinity for free factor Xa than the factor Xa/substrate complex. The linear mixed-type inhibition suggests that compound 1 binds to the active site region of factor Xa, but its binding cannot be fully displaced by the substrate S2222 (1:1 mixture of N-benzoyl-Ile-Glu-Gly-Arg- p-nitroanilide and N-benzoyl-Ile-Glu(γ-OMe)-Gly-Arg- p-nitroanilide hydrochloride). Thus, the inhibition mechanism for compound 1 is novel compared to most serine protease inhibitors including amidine-containing factor Xa inhibitors, which rely on binding to the S1 pocket of the enzyme active site. Compound 1 represents an attractive, novel structural template for further development of efficacious, safe, and potentially orally active human factor Xa inhibitors.