We recently designed a group of novel exosite-2-directed sulfated, small, allosteric inhibitors of thrombin. To develop more potent inhibitors, monosulfated benzofuran tri- and tetrameric homologues of the parent designed dimers were synthesized in seven to eight steps and found to exhibit a wide range of potencies. Among these, trimer 9a was found to be nearly 10-fold more potent than the first generation molecules. Michaelis-Menten studies indicated an allosteric mechanism of inhibition. Competitive studies using a hirudin peptide (exosite 1 ligand) and unfractionated heparin, heparin octasaccharide, and γ'-fibrinogen peptide (exosite 2 ligands) demonstrated exosite 2 recognition in a manner different from that of the parent dimers. Alanine scanning mutagenesis of 12 Arg/Lys residues of exosite 2 revealed a defect in 9a potency for Arg233Ala thrombin only confirming the major difference in site of recognition between the two structurally related sulfated benzofurans. The results suggest that multiple avenues are available within exosite 2 for inducing thrombin inhibition.