Structure-activity relationships within a series of highly potent 2-carboxyindole-based factor Xa inhibitors incorporating a neutral P1 ligand are described with particular emphasis on the structural requirements for addressing subpockets of the factor Xa enzyme. Interactions with the subpockets were probed by systematic substitution of the 2-carboxyindole scaffold, in combination with privileged P1 and P4 substituents. Combining the most favorable substituents at the indole nucleus led to the discovery of a remarkably potent factor Xa inhibitor displaying a K(i) value of 0.07 nM. X-ray crystallography of inhibitors bound to factor Xa revealed substituent-dependent switching of the inhibitor binding mode and provided a rationale for the SAR obtained. These results underscore the key role played by the P1 ligand not only in determining the binding affinity of the inhibitor by direct interaction but also in modifying the binding mode of the whole scaffold, resulting in a nonlinear SAR.

Abstract-Rivaroxaban is a direct inhibitor of factor Xa, a coagulation factor at a critical juncture in the blood coagulation pathway leading to thrombin generation and clot formation. It is selective for human factor Xa, for which it has Ͼ10 000-fold greater selectivity than for other biologically relevant serine proteases (half-maximal inhibitory concentration [IC 50 ], Ͼ20 mol/L). Rivaroxaban inhibits factor Xa in a concentration-dependent manner (inhibitory constant [K i ], 0.4 nmol/L) and binds rapidly (kinetic association rate constant [k on ], 1.7ϫ10 7 mol/L Ϫ1 s Ϫ1 ) and reversibly (kinetic dissociation rate constant [k off ], 5ϫ10 Ϫ3 s Ϫ1 ). By inhibiting prothrombinase complex-bound (IC 50 , 2.1 nmol/L) and clot-associated factor Xa (IC 50 , 75 nmol/L), rivaroxaban reduces the thrombin burst during the propagation phase. In animal models of venous and arterial thrombosis, rivaroxaban showed dose-dependent antithrombotic activity. In healthy individuals, rivaroxaban was found to have predictable pharmacokinetics and pharmacodynamics across a 5-to 80-mg total daily dose range, inhibiting factor Xa activity and prolonging plasma clotting time. In phase III clinical trials, rivaroxaban regimens reduced rates of venous thromboembolism in patients after total hip or knee arthroplasty compared with enoxaparin regimens, without significant differences in rates of major bleeding, showing that rivaroxaban has a favorable benefit-to-risk profile. (Arterioscler Thromb Vasc Biol. 2010;30:376-381.) Key Words: anticoagulants Ⅲ blood coagulation Ⅲ factor Xa Ⅲ rivaroxaban Ⅲ venous thromboembolism F actor Xa is a coagulation factor that acts at the convergence point of the intrinsic and extrinsic pathways in the blood coagulation system. 1 It catalyzes the cleavage of prothrombin and, therefore, is critical for thrombin generation (Figure 1). 1,2 Indirect factor Xa inhibitors, such as fondaparinux and biotinylated idraparinux, exert their thrombotic effect by binding to antithrombin; therefore, their efficacy depends on the circulating level of antithrombin. They are parenteral agents and cannot be administered orally. 3 Rivaroxaban is the first direct factor Xa inhibitor to be licensed (in the European Union 4 and several other countries) for the prevention of venous thromboembolism (VTE) in adult patients after elective hip or knee arthroplasty. Studies of rivaroxaban in the treatment of VTE, prevention of cardiovascular events in patients with acute coronary syndrome, prevention of stroke in those with atrial fibrillation, and prevention of VTE in hospitalized medically ill patients are ongoing (Supplementary Table I, available at http:// atvb.ahajournals.org). Other direct factor Xa inhibitors are also in advanced development; apixaban and edoxaban (DU-176b) are undergoing phase III study, whereas betrixaban and YM150 have passed clinical phase II testing. This review discusses the properties of rivaroxaban and findings from clinical trials. See accompanying article on page 369 Pharmacological PropertiesCompound C...

The design, synthesis, and structure-activity relationship of 3-oxybenzamides as potent inhibitors of the coagulation protease factor Xa are described on the basis of X-ray structures, privileged structure motifs, and SAR information. A total of six X-ray structures of fXa/inhibitor complexes led us to identify the major protein-ligand interactions. The binding mode is characterized by a lipophilic dichlorophenyl substituent interacting with Tyr228 in the protease S1 pocket, while polar parts are accommodated in S4. This alignment in combination with docking allowed derivation of 3D-QSAR models and tailored scoring functions to rationalize biological affinity and provide guidelines for optimization. The resulting models showed good correlation coefficients and predictions of external test sets. Furthermore, they correspond to binding site topologies in terms of steric, electrostatic, and hydrophobic complementarity. Two approaches to derive tailored scoring functions combining binding site and ligand information led to predictive models with acceptable predictions of the external set. Good correlations to experimental affinities were obtained for both AFMoC (adaptation of fields for molecular comparison) and the novel TScore function. The SAR information from 3D-QSAR and tailored scoring functions agrees with all experimental data and provides guidelines and reasonable activity estimations for novel fXa inhibitors.

Patients with haemophilia (PWH) are usually monitored by the one-stage activated partial thromboplastin time (aPTT) factor VIII (FVIII) assay. Different aPTT activators may affect clotting time (CT) and FVIII:C levels in patients treated with PEGylated FVIII. To evaluate the characteristics of PEGylated FVIII (BAY 94-9027) in various aPTT clotting assays, and to identify suitable aPTT reagents for monitoring BAY 94-9027 during the treatment of PWH, BAY 94-9027 and World Health Organization (WHO) 8th FVIII standards (WHO-8) were spiked into pooled and individual severe haemophilia A plasma at 1.0, 0.25 and 0.05 IU mL(-1) . Five commercial aPTT reagents widely used in clinical laboratories were compared and evaluated for BAY 94-9027 activity in plasma from PWH. BAY 94-9027 and WHO-8 bestowed similar CT and excellent precision when ellagic acid (SynthAFax, Dade Actin, and Cephascreen) aPTT reagents were used. In contrast, BAY 94-9027 showed significantly prolonged CT and poor precision compared with WHO-8 using silica aPTT reagents (APTT-SP and STA PTT 5). Furthermore, free 60-kDa polyethylene glycol (PEG), used for the conjugation of FVIII, showed a dose-dependent prolongation of CT in the APTT-SP assay. There was no effect on the SynthAFax-APTT, prothrombin time, or FXIa-initiated thrombin generation assay, demonstrating that the PEG moiety on FVIII has no general effect on the coagulation cascade. In summary, ellagic aPTT reagents (SynthAFax, Dade Actin, and Cephascreen) are most suitable for evaluating potency of BAY 94-9027 and should be the preferred aPTT reagents used in clinical laboratories for monitoring FVIII activity after infusion of BAY 94-9027 to PWH.

The extent of reversal by aPCC, PCC and rFVIIa was dependent on the parameter measured in rivaroxaban-anticoagulated plasma or blood. ETP measurements may have predictive power for assessing the reversal potential of PCC or aPCC and may be used to indicate an increased prothrombotic risk.