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

Abstract

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 (Ki=9 nM, Ki(trypsin)/Ki(thrombin)=1055) and tricyclic imide (+)‐21 (Ki=36 nM, Ki(trypsin)/Ki(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.

Rational Design and Characterization of D-Phe-Pro-D-Arg-Derived Direct Thrombin Inhibitors

“…The structure of unliganded human α-thrombin was solved by molecular replacement with Phaser [27] using the coordinates from PDB entry 1VZQ [28] as search model. The refined model of unliganded human α-thrombin was subsequently used as search model in the structural determination of the fPrI, fPrC and fPrt thrombin complexes.…”

Development of Orally Active Thrombin Inhibitors for the Treatment of Thrombotic Disorder Diseases

“…Structurally, thrombin’s active site [12] (Figure 2) is composed of a catalytic triad (Ser195, His57, and Asp102), a basic-recognition S1 pocket (Asp189 is the most important residue), a hydrophobic proximal S2 pocket (Tyr60A and Trp60D are the main residues) and a bigger lipohilic distal S3 pocket (Leu 99, Ile174, and Trp215) [10]. Additionally, subsites of S1′, S2′, S3′ and the oxyanion hole have also been considered in the design of stronger and more selective thrombin inhibitors [13].…”

Crystallization and preliminary crystallographic characterization of three peptidic inhibitors in complex with α-thrombin

“…The molecular coordinates of the light chain and heavy chain of human -thrombin from PDB entry 1vzq (Schä rer et al, 2004) were used as a search model to solve the structure of the unliganded protease by molecular replacement. The model was subjected to alternating cycles of manual building with Coot (Emsley et al, 2010) and refinement with PHENIX (Adams et al, 2010) until an R factor of approximately 0.20 was reached.…”