Synthesis of Novel Nonpeptidic Thrombin Inhibitors

Abstract

A novel class of nonpeptidic, active, and selective thrombin inhibitors has resulted from X‐ray‐structure‐based design and subsequent improvement of the initial lead molecules. These inhibitors possess a bi‐ or tricyclic central core structure with attached side chains to reach the three binding pockets (selectivity S1 pocket, distal D pocket, and proximal P pocket) present in the active site of the enzyme. The key step in the preparation of these compounds is the 1,3‐dipolar cycloaddition between an azomethine ylide, prepared in situ by the decarboxylative method from an aromatic aldehyde and an α‐amino acid, with an N‐substituted maleimide (e.g., see Schemes 1 and 2). All potent inhibitors contain an amidinium residue in the side chain for incorporation into the S1 pocket, which was introduced in the last step of the synthesis by a Pinner reaction. The compounds were tested in biological assays for activity against thrombin and the related serine protease trypsin. The first‐generation lead compounds (±)‐11 and (±)‐19 (Scheme 1) with a bicyclic central scaffold showed Ki values for thrombin inhibition of 18 μM and 0.67 μM, respectively. Conformationally more restricted second‐generation analogs (Scheme 2) were more active ((±)‐22i: Ki=90 nM (Table 1)); yet the selectivity for thrombin over trypsin remained weak. In the third‐generation compounds, a small lipophilic side chain for incorporation into the hydrophobic P pocket was introduced (Schemes 7 and 8). Since this pocket is present in thrombin but not in trypsin, an increase in binding affinity was accompanied by an increase in selectivity for thrombin over trypsin. The most selective inhibitor (Ki=13 nM, 760‐fold selectivity for thrombin over trypsin; Table 2) was (±)‐1 with an i‐Pr group for incorporation into the P pocket. Optical resolution of (±)‐1 (Scheme 9) provided (+)‐1 with a Ki value of 7 nM and a 740‐fold selectivity, whereas (−)‐1 was 800‐fold less active (Ki=5.6 μM, 21‐fold selectivity). The absolute configuration of the stronger‐binding enantiomer was assigned based on the X‐ray crystal structure of the complex formed between thrombin and this inhibitor. Compound (+)‐1 mimics the natural thrombin substrate, fibrinogen, which binds to the enzyme with the Ph group of a phenylalanine (piperonyl in (+)‐1) in the distal D pocket, with the i‐Pr group of a valine (i‐Pr in (+)‐1) in the proximal P pocket, and with a guanidinium side chain of an arginine residue (phenylamidinium group in (+)‐1) in the selectivity S1 pocket of thrombin. A series of analogs of (±)‐1 with the phenylamidinium group replaced by aromatic and aliphatic rings bearing OH or NH2 groups (Schemes 10 – 14) were not effectively bound by thrombin. A number of X‐ray crystal‐structure analyses of free inhibitors confirmed the high degree of preorganization of these compounds in the unbound state. Since all inhibitors prefer similar modes of association with thrombin, detailed information on the strength of individual intermolecular bonding interactions and their incremental con...

Structure-Based Design of Nonpeptidic Thrombin Inhibitors: Exploring the D-Pocket and the Oxyanion Hole

“…Fig. 1 shows inhibitor ()-1 (K i 7 nm, selectivity over trypsin K i (trypsin)/ K i (thrombin) 740) developed in our laboratory and its binding mode in the thrombin active site according to an X-ray crystal structure of the protein complex [9] [10]. The thrombin active site features four pockets: The distal (D) pocket is a large hydrophobic pocket that preferentially accommodates lipophilic residues such as aromatic rings; it is occupied by a Phe side chain in the case of the natural substrate fibrinogen.…”

“…The second Ph ring (b) of the active enantiomer of (AE)-2 was predicted to adopt a location in the D-pocket similar to that of the piperonyl residue in ()-1. As a difference, however, Ph ring b in (AE)-2 was expected to undergo parallel p-p stacking with the indole ring of Trp215, whereas the piperonyl residue in ()-1 prefers an edgeto-face orientation [9] [10]. Finally, the active enantiomer of compound (AE)-4 combines the recognition features of both piperonyl and diarylmethyl residues.…”

“…Synthesis. Synthesis of the N-benzyl-substituted inhibitor (AE)-3 was accomplished according to the method used for (AE)-1 [10]. N-Benzylmaleimide (5) [18] was reacted in a 1,3-dipolar cycloaddition with the azomethine ylide [19], generated in situ from l-proline (6) and 4-bromobenzaldehyde (7), to yield nearly equal amounts of the desired endo-cycloadduct (AE)-8a (42%) and the undesired exo-derivative (AE)-8b (40%; Scheme 1).…”

“…N-Benzylmaleimide (5) [18] was reacted in a 1,3-dipolar cycloaddition with the azomethine ylide [19], generated in situ from l-proline (6) and 4-bromobenzaldehyde (7), to yield nearly equal amounts of the desired endo-cycloadduct (AE)-8a (42%) and the undesired exo-derivative (AE)-8b (40%; Scheme 1). Regio-and stereoselective reduction (Li[Et 3 BH], THF) of (AE)-8a afforded hydroxy lactam (AE)-9, which was converted to sulfone (AE)-10 [10]. Nucleophilic displacement of the arylsulfonyl group according to the protocol (ZnCl 2 /i-PrMgCl) introduced by Ley and co-workers [20] gave ± under retention of configuration ± isopropyl derivative (AE)-11.…”

“…Hence, it was decided to replace proline (6) by achiral a-aminoisobutyric acid (18) as the amine component in the azomethine ylide formation [10]. This would eliminate one stereogenic center in the cycloadducts, while ultimately leading to bicyclic inhibitors close in potency to their tricyclic congeners.…”