Dose-dependent antithrombotic activity of an orally active tissue factor/factor VIIa inhibitor without concomitant enhancement of bleeding propensity.

The discovery of a highly potent and selective tissue factor/factor VIIa inhibitor is described. Upon oral administration of its double prodrug in the guinea pig, a dose-dependent antithrombotic effect is observed in an established model of arterial thrombosis without prolonging bleeding time. The pharmacodynamic properties of this selective inhibitor are compared to the behaviour of a mixed factor VIIa/factor Xa inhibitor.

Selective and orally bioavailable phenylglycine tissue factor/factor VIIa inhibitors.

We describe the structure-based design and synthesis of highly potent, orally bioavailable tissue factor/factor VIIa inhibitors which interfere with the coagulation cascade by selective inhibition of the extrinsic pathway.

Design of selective phenylglycine amide tissue factor/factor VIIa inhibitors.

Proof of concept experiments have shown that tissue factor/factor VIIa inhibitors have antithrombotic activity without enhancing bleeding propensity. Starting from lead compounds generated by a biased combinatorial approach, phenylglycine amide tissue factor/factor VIIa inhibitors with low nanomolar affinity and good selectivity against other serine proteases of the coagulation cascade were designed, using the guidance of X-ray structural analysis and molecular modelling.

Fluorine interactions at the thrombin active site: protein backbone fragments H-C(alpha)-C=O comprise a favorable C-F environment and interactions of C-F with electrophiles.

In a systematic fluorine scan of a rigid inhibitor to map the fluorophilicity/fluorophobicity of the active site in thrombin, one or more F substituents were introduced into the benzyl ring reaching into the D pocket. The 4-fluorobenzyl inhibitor showed a five to tenfold higher affinity than ligands with other fluorination patterns. X-ray crystal-structure analysis of the protein-ligand complex revealed favorable C-F...H-C(alpha)-C=O and C-F...C=O interactions of the 4-F substituent of the inhibitor with the backbone H-C(alpha)-C=O unit of Asn98. The importance of these interactions was further corroborated by the analysis of small-molecule X-ray crystal-structure searches in the Protein Data Base (PDB) and the Cambridge Structural Database (CSD). In the C--F...C=O interactions that are observed for both aromatic and aliphatic C-F units and a variety of carbonyl and carboxyl derivatives, the F atom approaches the C=O C atom preferentially along the pseudotrigonal axis of the carbonyl system. Similar orientational preferences are also seen in the dipolar interactions C--F.C[triple chemical bond]N, C-F.C-F, and C-F...NO(2), in which the F atoms interact at sub-van der Waals distances with the electrophilic centers.

A fluorine scan of the phenylamidinium needle of tricyclic thrombin inhibitors: effects of fluorine substitution on pKa and binding affinity and evidence for intermolecular C-F...CN interactions.

The H-atoms of the phenylamidinium needle of tricyclic thrombin inhibitors, which interacts with Asp189 at the bottom of the selectivity pocket S1 of the enzyme, were systematically exchanged with F-atoms in an attempt to improve the pharmacokinetic properties by lowering the pK(a) value. Both the pK(a) values and the inhibitory constants K(i) against thrombin and trypsin were decreased upon F-substitution. Interestingly, linear free energy relationships (LFERs) revealed that binding affinity against thrombin is much more affected by a decrease in pK(a) than the affinity against trypsin. Surprising effects of F-substitutions in the phenylamidinium needle on the pK(a) value of the tertiary amine centre in the tricyclic scaffold of the inhibitors were observed and subsequently rationalised by X-ray crystallographic analysis and ab initio calculations. Evidence for highly directional intermolecular C-F...CN interactions was obtained by analysis of small-molecule X-ray crystal structures and investigations in the Cambridge Structural Database (CSD).

Can the calculation of ligand binding free energies be improved with continuum solvent electrostatics and an ideal-gas entropy correction?

The prediction of a ligand binding constant requires generating three-dimensional structures of the complex concerned and reliably scoring these structures. Here, the scoring problem is investigated by examining benzamidine-like inhibitors of trypsin, a system for which errors in the structures are small. Precise and consistent binding free energies for the inhibitors are determined experimentally for this test system. To examine possible improvement of scoring methods, we test the suitability of continuum electrostatics to account for solvation effects and use an ideal-gas entropy correction to account for the changes in the degrees of freedom of the ligand. The small observed root-mean-square deviation of 0.55 kcal/mol of the calculated relative to the experimental values indicates that the essentials of the binding process have been captured. Even though all six ligands make the same salt bridge and H-bonds to the protein, the electrostatic contribution varies among the ligands by as much as 2 kcal/mol. Moreover, although the ligands are rigid and similar in size, the entropic terms also significantly affect the relative binding affinities (by up to 2.7 kcal/mol). The present approach to solvation and entropy may allow the ranking of the ligands to be considerably improved at a cost that makes the method applicable to the optimization of lead compounds or to the screening of small collections of ligands.