A novel series of cyanoguanidine-piperazine P2X(7) antagonists was designed based upon the structure of A-740003. Structure-activity relationship (SAR) studies focused on the piperazine moiety and the right hand side substitution. Compounds were assayed for activity at human and rat P2X7 receptors and compound 29 was found to possess potent activity (IC50 = 30-60 nM) at both species. (c) 2008 Elsevier Ltd. All rights reserved.

A novel series of cyanoguanidine-piperazine P2X(7) antagonists were identified and structure-activity relationship (SAR) studies described. Compounds were assayed for activity at human and rat P2X(7) receptors in addition to their ability to inhibit IL-1 beta release from stimulated human whole blood cultures. Compound 27 possesses potent activity (0.12 microM) in this latter assay and demonstrates moderate clearance in-vivo.

We describe the identification, SAR, and in vivo pharmacology of a new series of Src-family selective Lck inhibitors. These thienopyridines were designed based on a desire to access the unique residues in the extended hinge region of Lck.

Structure-activity relationships for new members of a class of nonpeptidic. low-molecular-weight inhibitors of thrombin, a key serine protease in the blood coagulation cascade, are described. These compounds, which originate from X-ray-structure-based design, feature a conformationally rigid, bi- or tricyclic core from which side chains diverge into the four major binding pockets (distal D, proximal P, recognition or specificity S1, and oxyanion hole O) at the thrombin active site (Fig. 1). Phenylamidinium is the side chain of choice for the S1 pocket, while the most active inhibitors orient an i-Pr group into the P-pocket (Table 1). The key step in the synthesis of the inhibitors is the construction of the central bi- or tricyclic scaffold by 1,3-dipolar cycloaddition of an in situ prepared azomethine ylide and an N-substituted maleimide (Schemes 1 3, and 8-10). One series of compounds was designed to explore the binding features of the large hydrophobic D pocket. This pocket provides space for lipophilic residues as bulky as benzhydryl groups. A new strategy was developed, allowing introduction of these sterically demanding substituents very late in the synthesis (Schemes 5 and 6). Benzhydryl derivative (+/-)-2 was found to be the most selective member (K-i (trypsin)/K-i (thrombin) - 1200) of this class of nonpeptidic thrombin inhibitors. while the 'dipipcronyl' analog (+/-)-3 (K-i - 9 nm, 7.60-fold selectivity) displays the highest potency of all compounds prepared so far ( Table 1). A second series of inhibitors features side chains designed to orient into the oxyanion hole and to undergo H-bonding with the backbone NH groups lining the catalytic site of the enzyme. Unfortunately, neither activity nor selectivity could be substantially improved by introduction of these substituents (Table 2). Presumably. the high degree of pre-organization and the rigidity of the tightly bound scaffolds prevents the new substituents from assuming a position that would allow favorable interactions in the oxyanion hole. However, the oxyanion hole and the SI' pocket next to it were found to be capable of accommodating quite large groups, which leaves much room for further exploration.

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 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 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 K-i values for thrombin inhibition of 18 mu M and 0.67 mu M, respectively Conformationally more restricted second-generation analogs (Scheme 2)were more active ((+/-)-22i: K-i = 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 ( K-i = 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 (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 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 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-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.