In our molecular recognition studies, aimed at quantifying the energetics of individual protein-ligand interactions, [1] we became interested in exploring cation-p interactions [2,3] in the D-pocket of thrombin, a central serine protease in the blood coagulation cascade. The bottom of this hydrophobic pocket is lined by the indole residue of Trp 215 (Figure 1), an aromatic amino acid side chain frequently involved in cationp interactions in biological systems.[4] To probe this interaction, we prepared the tricyclic inhibitors [5] (AE )-1 and (AE )-2, predicted by computer modeling [6] to position a quaternary ammonium ion and an uncharged tert-butyl group above the indole ring of Trp 215.The synthesis of (AE )-1 started with the 1,3-dipolar cycloaddition between maleimide 3, aldehyde 4, and l-proline (5) to give (AE )-6, which was transformed into amidinium salt (AE )-7 using a Pinner-reaction (Scheme 1, for full experimen-

A series of 16 tricyclic thrombin inhibitors was prepared by using the 1,3-dipolar cycloaddition of azomethine ylides derived from 3- or 4-hydroxyproline and 4-bromobenzaldehyde, with N-(4-fluorobenzyl)maleimide as the key step. The terminal pyrrolidine ring of the inhibitors was systematically substituted to explore the potential bioisosteric behavior of C-F, C-OH, and C-OMe residues pointing into the environment of the catalytic center of a serine protease. X-ray crystal structure analyses revealed a distinct puckering preference of this ring. Substitution by F, HO, and MeO has a strong effect on the basicity of the adjacent pyrrolidine nitrogen center which originates from two sigma-inductive pathways between this center and the electronegative O and F atoms. gem-Difluorination decreases the pKa value of this tertiary amine center to <2, making the conjugated ammonium ion a moderately strong acid. Unexpectedly, F substitution next to the nitrogen center reduced the lipophilicity of the ligands, as revealed by measurements of the logarithmic partition coefficient log D. The biological assays showed that all compounds are thrombin inhibitors with activities between Ki=0.08 and 2.17 microM. Bioisosteric behavior of F, HO, and MeO substituents was observed. Their electronegative F and O atoms undergo energetically similar polar interactions with positively polarized centers, such as the N atom of His 57 which is hydrogen bonded to the catalytic Ser 195. However, for energetically similar polar interactions of C-F, C-OH, and C-OMe to occur, sufficient space is necessary for the accommodation of the Me group of the C-OMe residue, and a H-bond acceptor must be present to prevent unfavorable desolvation of the C-OH residue.

Im Verlauf unserer Studien zur molekularen Erkennung mit dem Ziel einer Quantifizierung des energetischen Beitrages einzelner Protein-Ligand-Wechselwirkungen [1] richtete sich unser Interesse auf die Erforschung von Kation-p-Wechselwirkungen [2,3] in der D-Tasche von Thrombin, einer zentralen Serin-Protease in der Blutgerinnungskaskade. Den Boden dieser hydrophoben Tasche bildet der Indol-Rest von Trp 215 (Abbildung 1), einer aromatischen Aminosäure-Seitenkette, die in biologischen Systemen häufig Kation-p-Wechselwirkungen eingeht.[4] Zur Erkundung dieser Wechselwirkung wurden die tricyclischen Inhibitoren (AE )-1 und (AE )-2 synthetisiert, [5] die entsprechend der Voraussage eines Computer-Modells [6] beim Binden im aktiven Zentrum von Thrombin ein quartäres Ammonium-Ion bzw. einen ungeladenen tert-Butyl-Rest oberhalb des Indol-Ringes von Trp 215 positionieren.

The heterocyclic compound specified in the title (and readily prepared from commercial precursors) has a sterically protected C==O group, so that direct lithiation by BuLi at the exocyclic CH(2) group is possible (3 --> Li-3). The lithiated N,S-acetal derivative (Li-3) adds diastereoselectively to aldehydes (Table 2), unsymmetrical ketones (Table 3), chalcone (1,4-addition, Scheme 2), and N-phosphinoyl- and N-sulfonylimines (Table 4). Protection of the newly formed OH groups (Scheme 3) and/or MeS/OH displacement by Hg(O(2)CCF(3))(2) in aqueous THF/acetonitrile converts the N,S-acetals into hemiaminals (--> 20) which, in turn, are readily cleaved to aldehydes, with recovery of the chiral auxiliary (1, Scheme 4). The aldehydes (especially those lacking alpha-carbonyl hydrogens) may be isolated, or they are trapped in situ by reduction to (selectively protected) diols or amino alcohols, by addition of Grignard or Li reagents, which provides diols with two stereogenic centers, by oxidation to give 2-hydroxy esters, or by olefination to provide 4-hydroxy-2-alkenoates (Scheme 5). The scope and limitations of the new, overall enantioselective transformation are determined, and the readily recovered chiral auxiliary used is compared with oxazolidinones of other substitution patterns (Scheme 7). The configuration of a number of products has been assigned by single-crystal X-ray diffraction (cf. Figure 5). These structures and similarities of NMR data led to configurational assignment of the other products (see formulas in the schemes and tables) by analogy. A simple mechanistic model for the stereochemical course of the addition of Li-3 to aldehydes and ketones is presented (Figure 6).

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.