Synthesis of novel nonpeptidic thrombin inhibitors

A novel class of nonpeptidic. active, and selective thrombin inhibitors has resulted from X-ray-structure based design and subsequent improvement of t he initial lead molecules. These inhibitors possess a bi- or tricyclic cent ral core structure with attached side chains to reach the three binding poc kets (selectivity S1 pocket, distal D pocket, and proximal P pocket) presen t in the active site of the enzyme. The key step in the preparation of thes e compounds is the 1,3-dipolar cycloaddition between an azomethine ylide, p repared in situ by the decarboxylative method from an aromatic aldehyde and an a-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 fo r incorporation into the S1 pocket, which was introduced in the last step o f the synthesis by a Pinner reaction. The compounds were tested in biologic al assays for activity against thrombin and the related serine protease try psin. The first-generation lead compounds (+/-)-11 and (+/-)-19 (Scheme 1) with a bicyclic central scaffold showed K-i values for thrombin inhibition of 18 mu M and 0.67 mu M, respectively Conformationally more restricted sec ond-generation analogs (Scheme 2)were more active ((+/-)-22i: K-i = 90 nM(T able 1)); yet the selectivity for thrombin over trypsin remained weak. In t he third-generation compounds, a small lipophilic side chain for incorporat ion into the hydrophobic P pocket was introduced (Schemes 7 and 8). Since t his pocket is present in thrombin but not in trypsin, an increase in bindin g affinity was accompanied by an increase in selectivity for thrombin over trypsin. The most selective inhibitor ( K-i = 13 nM, 760-fold selectivity f or thrombin over trypsin, Table 2) was (+/-)-1 with an i-Pr group for incor poration into the P pocket. Optical resolution of (+/-)-1 (Scheme 9) provid ed (+)-1 with a Ki value of 7 nM and a 740-fold selectivity, whereas (-)-1 was 800-fold less active (K-i= 5.6 mu M, 21-fold selectivity). The absolute configuration of the stronger-binding enantiomer was assigned based on the Xray crystal structure of the complex formed between thrombin and this inh ibitor. Compound (+)-1 mimics the natural thrombin substrate, fibrinogen, w hich 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 argi nine residue rphenylamidinium 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-st ructure analyses of free inhibitors confirmed the high degree of preorganiz ation of these compounds in the unbound state. Since all inhibitors prefer similar modes of association with thrombin, detailed information on the str ength of individual intermolecular bonding interactions and their increment al 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.