Enantiomerically Pure Thrombin Inhibitors for Exploring the Molecular‐Recognition Features of the Oxyanion Hole

A new route via intermediate pseudoenantiomers was developed to synthesize racemic and enantiomerically pure new non‐peptidic inhibitors of thrombin, a key serine protease in the blood‐coagulation cascade. These ligands feature a conformationally rigid tricyclic core and are decorated with substituents to fill the major binding pockets (distal (D), proximal (P), selectivity (S1), and oxyanion hole) at the thrombin active site (Fig. 1). The key step in the preparation of the new inhibitors is the 1,3‐dipolar cycloaddition between an optically active azomethine ylide, prepared in situ from L ‐(4R)‐hydroxyproline and 4‐bromobenzaldehyde, and N‐piperonylmaleimide (Scheme 1). According to this protocol, tricyclic imide (compounds (±)‐ 15 ‐(±)‐ 18 and (+)‐ 21 ) and lactam (compound (+)‐ 2 ) inhibitors with OH or ether substituents at C(7) in the proline‐derived pyrrolidine ring were synthesized to specifically explore the binding features of the oxyanion hole (Schemes 2–4). Biological assays (Table) showed that the polar oxyanion hole in thrombin is not suitable for the accommodation of bulky substituents of low polarity, thereby confirming previous findings. In contrast, tricyclic lactam (+)‐ 2 (K_i=9 nM , K_i(trypsin)/K_i(thrombin)=1055) and tricyclic imide (+)‐ 21 (K_i=36 nM , K_i(trypsin)/K_i(thrombin)=50) with OH‐substituents at the (R)‐configured C(7)‐atom are among the most‐potent and most‐selective thrombin inhibitors in their respective classes, prepared today. While initial modeling predicted H‐bonding between the OH group at C(7) in (+)‐ 2 and (+)‐ 21 with the H₂O molecule bound in the oxyanion hole (Fig. 2), the X‐ray crystal structure of the complex of (+)‐ 21 (Fig. 7, b) revealed a different interaction for this group. The propionate side chain of Glu192 undergoes a conformational change, thereby re‐orienting towards the OH group at C(7) under formation of a very short ionic H‐bond (O? H???^?OOC; d(O???O)=2.4 Å). The energetic contribution of this H‐bond, however, is negligible, due to its location on the surface of the protein and the unfavorable conformation of the H‐bonded propionate side chain.