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 contribution to the overall free energy of complexation was generated in correlative binding and X-ray studies. The present study demonstrates that defined mutations in highly preorganized inhibitors provide an attractive alternative to site-directed mutagenesis in exploring molecular-recognition phenomena at enzyme active sites.