Factor VIIa inhibitors: gaining selectivity within the trypsin family

Within the trypsin family of coagulation proteases, obtaining highly selective inhibitors of factor VIIa has been challenging. We report a series of factor VIIa (fVIIa) inhibitors based on the 5-amidino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole (1) scaffold with potency for fVIIa and high selectivity against factors IIa, Xa, and trypsin. With this scaffold class, we propose that a unique hydrogen bond interaction between a hydroxyl on the distal ring of the biaryl system and the backbone carbonyl of fVIIa lysine-192 provides a basis for enhanced selectivity and potency for fVIIa.     

Discovery of benzothiazole guanidines as novel inhibitors of thrombin and trypsin IV

In a project to find novel neutral P1 fragments for the synthesis of thrombin inhibitors with improved pharmacokinetic properties, fragments containing a benzothiazole guanidine scaffold were identified as weak thrombin inhibitors. WaterLOGSY (Water-Ligand Observed via Gradient SpectroscopY) NMR was used to detect fragments binding to thrombin and these fragments were followed up by Biacore A100 affinity measurements and enzyme assays. A crystal structure of the most potent compound with thrombin was obtained and revealed an unexpected binding mode as well as the key interactions of the fragment with the protein. Based on these results, the structure-based design and synthesis of a small series of optimized novel substituted benzothiazole guanidines with comparatively low pK(a) values was accomplished. Testing of these compounds against human trypsin I and human trypsin IV revealed unexpected inhibitory activity and selectivity of some of the compounds, making them attractive starting points for selective trypsin inhibitors.     

Discovery of potent, selective 4-fluoroproline-based thrombin inhibitors with improved metabolic stability

Previous reports from our laboratories described potent tripeptide thrombin inhibitors which incorporate heterocycle-substituted chlorophenyl groups in the P1 position. Using these as lead compounds for further optimization, we identified sites of metabolism and designed analogs with 4-fluoroproline in P2 and cyclopropane-containing side chains in P3 as an approach to reducing metabolism and improving their oral pharmacokinetic performance. The large (300-fold) difference in potency between analogs containing (4R)- and (4S)-4-fluoroproline was rationalized by analyzing inhibitor-enzyme interactions in crystal structures of related compounds and by molecular modeling which indicated that the more potent (4R)-4-fluoroproline isomer stabilizes a proline ring conformation that is preferred for binding to the enzyme. An optimal compound from this work, 41, exhibits high potency in a coagulation assay in human plasma (2xAPTT=190 nM), excellent selectivity versus the digestive enzyme trypsin (K(i)=3300 nM), and excellent oral bioavailability in dogs with moderate clearance (F=100%, CL=12 mL/min/kg).     

Benzimidazole-based DGAT1 inhibitors with a [3.1.0] bicyclohexane carboxylic acid moiety

Previously disclosed benzimidazole-based DGAT1 inhibitors containing a cyclohexane carboxylic acid moiety suffer from isomerization at the alpha position of the carboxylic acid group, generating active metabolites which exhibit DGAT1 inhibition comparable to the corresponding parent compounds. In this report, we describe the design, synthesis and profiling of benzimidazole-based DGAT1 inhibitors with a [3.1.0] bicyclohexane carboxylic acid moiety. Our results show that single isomer 3A maintains in vitro and in vivo inhibition against DGAT1. In contrast to previous lead compounds, 3A does not undergo isomerization during in vitro hepatocyte incubation study or in vivo mouse study.     

Novel bicyclic lactam inhibitors of thrombin: potency and selectivity optimization through P1 residues

Peptidomimetic inhibitors of thrombin lacking the important Ser195–carbonyl interaction have been prepared. The binding energy lost after the removal of the activated carbonyl was recaptured through a series of modifications of the P1 residues of the bicyclic lactam inhibitors. Selected substituted compounds displayed useful pharmacological profiles both in vitro and in vivo.     

D-Phe-Pro-Arg type thrombin inhibitors: unexpected selectivity by modification of the P1 moiety

Synthesis of thrombin inhibitors and their binding mode to thrombin is described. Modification of the P1 moiety leads to an increased selectivity versus trypsin. The observed selectivity is discussed in view of their thrombin-inhibitor complex X-ray structures.     

Azaindoles: moderately basic P1 groups for enhancing the selectivity of thrombin inhibitors

Starting from a 2-amino-6-methylpyridine P1 group and following a strategy of enlarging it whilst reducing its polarity, we have developed a series of potent, moderately basic azaindoles which are intrinsically much more selective for thrombin versus trypsin. Certain pyrazinone acetamide azaindole derivatives have pharmacokinetic parameters after oral administration to dogs, or efficacy in vitro, comparable to an optimized pyrazinone acetamide 2-amino-6-methylpyridine derivative.     

Interaction between alpha-2 macroglobulin and trypsin with synthetic trypsin inhibitors]

The complex formed between trypsin (Tn) and alpha 2 Macroglobulin (alpha 2 M) retains the whole hydrolytic activity of the enzyme for synthetic substrates. Moreover synthetic inhibitors of low molecular weight stiel inhibit this activity. A comparative study of three inhibitors (Benzylamine, Butylamine, Benzamidine) has been carried out and shows that their behavior is similar. These inhibitors bind trypsin when it is bound to alpha 2 M and reciprocally alpha 2 M can bind Tn-inhibitor complex. Nevertheless the dissociation constant of the enzyme-inhibitor complex (Ki) is increased by alpha 2 M. In the case of Benzamidine the value of Ki is 2.22.10(-5) M for native enzyme and 13.4.10(-5) M for Tn-alpha 2 M and in the case of Butylamine this value increases from 0.5.10(-3) M to 2.95.10(-3) M. These variations of the Ki values are due to the modification of the accessibility of the inhibitor to the active site. Unpublished results show that the alpha 2 M molecule undergoes a deep structural modification in the course of the complex formation, which must lead to an increase of the value of Ki. This structural modification is probably irreversible so that the alpha 2 M complex has never been dissociated without altering the alpha 2 M molecule. The increase of the values of Ki cannot therefore result in an effective decrease of the association constant of the Tn-alpha 2 M complex.     

2-Aminoquinazolin-4(3H)-one based plasmepsin inhibitors with improved hydrophilicity and selectivity

2-Aminoquinazolin-4(3H)-ones were previously discovered as perspective leads for antimalarial drug development targeting the plasmepsins. Here we report the lead optimization studies with the aim to reduce inhibitor lipophilicity and increase selectivity versus the human aspartic protease Cathepsin D. Exploiting the solvent exposed area of the enzyme provides an option to install polar groups (R¹) the 5-position of 2-aminoquinazolin-4(3H)-one to inhibitors such as carboxylic acid without scarifying enzymatic potency. Moreover, introduction of R¹ substituents increased selectivity factors of compounds in this series up to 100-fold for Plm II, IV vs CatD inhibition. The introduction of flap pocket substituent (R²) at 7-postion of 2-aminoquinazolin-4(3H)-one allows to remove Ph group from THF ring without notably impairing Plm inhibitory potency. Based on these findings, inhibitors were developed, which show Plm II and IV inhibitory potency in low nanomolar range and remarkable selectivity against Cathepsin D along with decreased lipophilicity and increased solubility.     

Design and structure-activity relationship of thrombin inhibitors with an azaphenylalanine scaffold: potency and selectivity enhancements via P2 optimization

Theoretical and structural studies followed by the directed synthesis and in vitro biological tests lead us to novel noncovalent thrombin pseudopeptide inhibitors. We have incorporated an azapeptide scaffold into the central part of the classical tripeptide D-Phe-Pro-Arg inhibitor structure thus eliminating one stereogenic center from the molecule. A series of compounds has been designed to optimize the occupancy of the S2 pocket of thrombin. Increased hydrophobicity at P2 provides an enhanced fit into this active site S2 pocket. In the present paper, we also report on the structure of these inhibitors in solution and conformational analysis of inhibitors in the active site in order to asses the consequences of the replacement of the central alpha-CH by a nitrogen functionality. In vitro biological testing of the designed inhibitors shows that elimination of R, S stereoisomerism and restriction of conformational freedom influences the binding of inhibitors in a favorable fashion.     

Interactions of thrombin with benzamidine-based inhibitors.

Trypsin and trypsin-like enzymes cleave C-terminal bonds of the basic amino acids Arg and Lys. Inhibitors of these enzymes have been found not only among Arg and Lys derivatives but also with structurally related benzamidines. Especially cyclic amides of 4-amidinophenylalanine were found to be inhibitors of thrombin. The most potent selective thrombin inhibitor of these type is N alpha-(beta-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidine. From the X-ray crystal structures of thrombin and trypsin-inhibitor complexes the thrombin complexes formed with inhibitors derived from amidinophenylalanine have been modeled. These models allow valuable predictions to design inhibitors of improved selection and binding properties. Most recently, also the X-ray crystal structures of complexes of inhibitors with bovine thrombin have been solved.     

Interaction of sheep haptoglobin with trypsin inhibitors

It was found that polymeric sheep haptoglobin C interacts with duck egg ovomucoid and with maize trypsin inhibitor. These inhibitors do not block the region in haptoglobin C molecule which is responsible for the formation of its complex with hemoglobin. The binding of the natural protein inhibitors is suggestive of homology of the haptoglobin site involved in the interaction with the substrate-specific site of trypsin. It is assumed that the regions in these protein molecules adjacent to the active and specific sites also possess a high degree of homology.     

Stabilization of donor blood with thrombin inhibitors

The content of free calcium ions in stored blood can be maintained by stabilizing it with thrombin inhibitors. With regard to a long-term stabilization of blood or blood plasma, satisfactory results can only be achieved with the competitive synthetic thrombin inhibitor 4-amidinophenylpyruvic acid by adding slight amounts of heparin and by storing it in a cool room. By using hirudin (greater than 50 ATE/ml of blood) the stabilization of blood enables a long-lasting anticoagulative effect to be achieved, such as it is required for the purpose of reprocessing blood to plasma fractions.