Pharmacological characterization of a novel factor Xa inhibitor, FXV673.

FXV673 is a novel, potent, and selective factor Xa (FXa) inhibitor. FXV673 inhibited human, dog, and rabbit FXa with a K(i) of 0.52, 1.41, and 0.27 nM, respectively. FXV673 also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C (aPC), plasmin, and tissue-plasminogen activator (t-PA). FXV673 prolonged plasma activated partial thromboplastin time (APTT) and prothrombin time (PT) in a dose-dependent fashion. In the APTT assays, the concentrations (microM) required for doubling coagulation time were 0.41 (human), 0.65 (monkey), 1.12 (dog), 0.25 (rabbit), and 0.80 (rat). The concentrations (microM) required in the PT assays were 1.1 (human), 1.32 (monkey), 2.31 (dog), 0.92 (rabbit), and 1.69 (rat). A coupled-enzyme assay was performed to measure thrombin activity following prothrombinase conversion of prothrombin to thrombin. FXV673 showed IC(50)s of 1.38 and 2.55 nM, respectively, when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or fresh platelets were used as the phospholipid source for prothrombinase complex formation. It was demonstrated that FXV673 could inhibit further thrombin generation in the prothrombinase complex using PS/PC liposomes. FXV673 dose-dependently prolonged the time to vessel occlusion and inhibited thrombus formation in well-characterized canine models of thrombosis. Interspecies extrapolation (approximately 2.5-fold higher sensitivity for FXa inhibition in human than in dog) suggested that 100 ng/ml of FXV673 would be an effective plasma concentration for clinical studies. Currently FXV673 is undergoing clinical studies to be developed as an antithrombotic agent.

The design of competitive, small-molecule inhibitors of coagulation factor Xa.

The last five years have seen an explosion of research into inhibitors of Factor Xa as potential antithrombotic agents. Aventis Pharma was a participant in this effort and its two founder companies have substantially contributed to the discovery of new inhibitors over the years. This review traces the systematic development of the former Rhone-Poulenc Rorer factor Xa program from conception to the realization of potent, orally bioavailable inhibitors with exquisite selectivity against other serine proteases. The work on beta-aminoesters described in Part 1 culminates in the development of FXV673 (Ki = 0.5 nM), an effective anticoagulant for acute indications. Part 2.2 details the de novo design of the pyrrolidinone series of inhibitors (RPR120844), within which a group of efficacious i.v. agents were identified (e.g. RPR130737, Ki = 2 nM). The first active and bioavailable benzamidine isostere i.e. the 1-aminoisoquinoline (RPR208815, Ki = 22 nM) was discovered on the pyrrolidinone scaffold (Part 2.3). Ultimately a variety of benzamidine mimics were explored and incorporated into the ketopiperazine series; the 6-substituted aminoquinazolines were found to be the most potent (Part 3). The azaindole, as represented by RPR200443 (Ki = 4 nM), stands out as imparting favorable pharmacokinetic properties to the sulfonamido-ketopiperazines.

In vitro characterization of a novel factor Xa inhibitor, RPR 130737.

RPR 130737 inhibited factor Xa (FXa) with a Ki of 2.4 nM and also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C, plasmin, tissue-plasminogen activator and trypsin. RPR 130737 prolonged plasma activated partial thromboplastin time and prothrombin time in a dose-dependent fashion. In the activated partial thromboplastin time assay, the concentrations required for doubling coagulation time were 0.32 microM (human), 0.61 microM (monkey), 0.44 microM (dog), 0.15 microM (rabbit), and 0.82 microM (rat). The concentrations required to double prothrombin time were 0.86 microM (human), and 1.26 microM (monkey), 1.15 microM (dog), 0.39 microM (rabbit) and 7.31 microM (rat). Kinetic studies revealed that RPR 130737 was a fast binding, reversible and competitive inhibitor for FXa when Spectrozyme FXa, a chromogenic substrate, was used. A coupled-enzyme assay measuring thrombin activity following prothrombinase conversion of prothrombin to thrombin indicated that RPR 130737 was a potent inhibitor for prothrombinase-bound FXa. In this assay, RPR 130737 showed IC50s of 17 nM and 35.9 nM, respectively when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or gel-filtered platelets were used as the phospholipid source. An FX-deficient plasma clotting-time correction assay further demonstrated that RPR 130737 was a specific inhibitor of FXa. RPR 130737 showed no effect on platelet aggregation in vitro. These results indicate that RPR 130737 has the potential to be developed as an antithrombotic agent based on its potent and selective inhibitory effect against FXa.

Crystal structures of human factor Xa complexed with potent inhibitors.

Involved in the coagulation cascade, factor Xa (FXa) is a serine protease which has received great interest as a potential target for the development of new antithrombotics. Although there is a great wealth of structural data on thrombin complexes, few structures of ligand/FXa complexes have been reported, presumably because of the difficulty in growing crystals. Reproducible crystallization conditions for human des-Gla1-45 coagulation FXa have been found. This has led to an improvement in the diffraction quality of the crystals (about 2.1 A) when compared to the previously reported forms (2.3-2.8 A) thus providing a suitable platform for a structure-based drug design approach. A series of crystal structures of noncovalent inhibitors complexed with FXa have been determined, three of which are presented herein. These include compounds containing the benzamidine moiety and surrogates of the basic group. The benzamidine-containing compound binds in a canonical fashion typical of synthetic serine protease inhibitors. On the contrary, molecules that contain surrogates of the benzamidine group do not make direct hydrogen-bonding interactions with the carboxylate of Asp189 at the bottom of the S1 pocket. The structural data provide a likely explanation for the specificity of these inhibitors and a great aid in the design of bioavailable potent FXa inhibitors.

Solid-phase parallel synthesis of azarene pyrrolidinones as factor Xa inhibitors.

A focused library (4 x 14) prepared from 4-aminopyridine and 4-, 5-, and 6-azoindole templates was synthesized using 14 polymer supported 4-amido-2,3,5,6-tetrafluorophenyl (TFP) sulfonate esters inputs. Several compounds were identified as factor Xa inhibitors (IC50< or =0.1 microM) helping to establish the SAR among these four series of azarene pyrrolidinones.

Amido-(propyl and allyl)-hydroxybenzamidines: development of achiral inhibitors of factor Xa.

The design, synthesis and SAR of amido-(propyl and allyl)-hydroxybenzamidine coagulation factor Xa inhibitors is described. These achiral inhibitors are selective for fXa vis a vis structurally related serine proteases and are readily prepared in 6-7 linear steps. The most potent member 9j (fXa Ki = 0.75 nM) is selective (>1000-fold) and an effective anticoagulant in mammalian plasma.

Synthesis, SAR and in vivo activity of novel thienopyridine sulfonamide pyrrolidinones as factor Xa inhibitors.

Thienopyridine sulfonamide pyrrolidinones were found to be potent and selective inhibitors of the coagulation cascade enzyme factor Xa. SAR studies led to several compounds that were selected for further in vivo investigation. These novel aryl binding pocket moieties represent a structural modification to a series of fXa inhibitors. Several compounds proved to be efficacious i.v. antithrombotic agents.

Aminoisoquinolines: design and synthesis of an orally active benzamidine isostere for the inhibition of factor XA.

The design, synthesis and SAR of sulfonamidopyrrolidinone fXa inhibitors incorporating a new benzamidine isostere, namely aminoisoquinolines, is described. These inhibitors have higher Caco-2 cell permeability than comparable benzamidines and attain higher levels of exposure upon oral dosing. The most potent member 14b (fXa Ki=6 nM) is selective against other serine proteases of interest (>600 fold).

Design and structure-activity relationships of potent and selective inhibitors of blood coagulation factor Xa.

The discovery of a series of non-peptide factor Xa (FXa) inhibitors incorporating 3-(S)-amino-2-pyrrolidinone as a central template is described. After identifying compound 4, improvements in in vitro potency involved modifications of the liphophilic group and optimizing the angle of presentation of the amidine group to the S1 pocket of FXa. These studies ultimately led to compound RPR120844, a potent inhibitor of FXa (K(i) = 7 nM) which shows selectivity for FXa over trypsin, thrombin, and several fibrinolytic serine proteinases. RPR120844 is an effective anticoagulant in both the rat model of FeCl(2)-induced carotid artery thrombosis and the rabbit model of jugular vein thrombus formation.

Sulfonamidopyrrolidinone factor Xa inhibitors: potency and selectivity enhancements via P-1 and P-4 optimization.

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa) inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxybenzamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a potent fXa inhibitor (K(i) = 2 nM) with selectivity against structurally related serine proteinases (>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in vivo (e.g. rat FeCl(2)-induced carotid artery thrombosis model).

Characterization of the bisphosphonate recognition site on hydroxyapatite using radioligand binding techniques with [14C]citric acid.

The present studies characterize the binding of [14C]citric acid to synthetic hydroxyapatite (HA) crystals. [14C]Citric acid specifically bound to HA and was dependent upon the concentration of HA in the assay. The binding of [14C] citric acid to HA reached equilibrium within 20 min and remained stable for at least 90 min. Dissociation of bound [14C]citric acid was biphasic in nature since both rapid and more slowly reversible binding components were detected. Saturation experiments also indicated that [14C]citric acid labeled two recognition sites with different affinity (KdH = 42 nM and KdL = 24,000 nM) and density (BmaxH = 161 fmol/micrograms HA and BmaxL = 8.8 pmol/micrograms HA). Ligand competition experiments revealed that compounds that are known to readily bind bone (e.g., sodium pyrophosphate, methylene diphosphonic acid, etidronate) potently inhibited the binding of [14C]citric acid to HA, whereas compounds known to have poorer affinity for bone (e.g., oxalic acid and GABA) did not. Computer analysis of these inhibition curves revealed specific ligand interactions at two different affinity recognition sites. The present results indicate that [14C]citric acid binds discrete sites on synthetic HA in a fashion consistent with a specific labeling of the bisphosphonate recognition site. Analysis of the binding of [14C]citric acid to HA provides a useful method to further explore the structure activity relationships of novel compounds that have binding affinity for bone.

Irreversible inhibition of transglutaminases by sulfonium methylketones: optimization of specificity and potency with omega-aminoacyl spacers.

Sulfonium methylketones, of structure Cbz-Phe-NH(CH2)nCOCH2S+ (CH3)2, n > 2, are specific and potent inactivators of transglutaminases. The length of the -(CH2)n-spacer moiety, n = 1-5, is a critical determinant for both the specificity and potency of the inactivator. The dipeptidyl analog Cbz-Phe-Gly-(CH2)nS+ (CH3)2, n = 1, is a more powerful inactivator of the thiol proteinase cathepsin B, k/K > 3 x 10(5) M-1 min-1, than of transglutaminases, ki(app)/Ki(app) < 1.5 x 10(4) M-1 min-1. In contrast, the gamma-aminobutyryl analog, n = 3, is a very potent transglutaminase inactivator with ki(app)/Ki(app) = 3.1 x 10(6) M-1 min-1, but does not inactivate cathepsin B. In cell studies, the gamma-aminobutyryl and epsilon-aminohexyl analogs inhibited the transglutaminase-mediated process of ionophore-induced cross-linked envelope formation by human malignant keratinocytes and the order of potency was related to that found for enzyme inhibition. The sulfonium methylketones, in equilibrium with the resonance stabilized ylides, are chemically inert towards glutathione under ambient conditions demonstrating the potential utility of this novel class of transglutaminase inhibitors for the study of enzyme inhibition in cellular environments.

Stereocontrolled routes to cis-hydroxyamino sugars, Part VII: Synthesis of daunosamine and ristosamine.

The nitrogen of an allylic amine can serve as the fulcrum for stereocontrolled delivery of oxygen to an adjacent trigonal site, and cis-hydroxyamino sugars can thus be prepared. Methods for achieving the complementary procedure, namely, control of the delivery of nitrogen to an adjacent site by an allylic oxygen, are described. For example, treatment of methyl 2,3,6-trideoxy-alpha-L-erythro-hex-2-enopyranoside with trichloroacetonitrile gave an imidate ester which reacted with iodonium dicollidine perchlorate to give 2-trichloromethyl-(methyl 2,3,4,6-tetradeoxy-2-iodo-alpha-L-altropyranosido)-[3,4-d]-2-oxazo line. Exhaustive reductive dehalogenation of this product followed by hydrolysis led to methyl N-acetyl-alpha-L-ristosaminide. An analogous series of reactions was used to prepare the corresponding daunosaminide.