**3.1. FXa structural target points**

An ideal anticoagulant would prevent thrombosis without inducing systemic hypocoagulation, and would thereby prevent undesired bleeding complications. Thus, a factor Xa inhibitor could potentially have the properties of a desirable anticoagulant. In the search for new drugs, anticoagulant serine protease activated factor Xa is a particularly promising target and

FXa plays an important role in first and secondary hemostasis. It produces the core catalyzing reaction that results in thrombin enzyme formation by means of the blood coagulation cascade which results in clot formation and wound closure [33, 47]. Moreover, FXa was found to play a central role in the coagulation process leading to hemostasis in the original extrinsic/intrinsic model [33] as well as in the newly proposed cell-based model. Factor X can be activated through either the intrinsic or extrinsic pathway. Initiation of both pathways activates the inactive precursor FX to FXa. Considering that one molecule of FXa catalyzes the formation of 1000 thrombin molecules, this amplification step can be substantial. Moreover, both pathways

lead to the propagation and amplification of coagulation through the activation of FX.

The perfect antithrombotic agent would not induce systemic hypocoagulation and thus provides equilibrium between clot formation and secondary problems such as bleeding. The investigation into finding new anticoagulant agents reveals that serine protease FXa is an important validated pharmaceutical achievement whose use has grown remarkably since the beginning of the twenty-first century [42–44]. Thus, an FXa inhibitor combined with an antiplatelet moiety could possibly provide the features of an effective drug, thus preventing the platelet aggregation during the hemostasis process, avoiding the thrombus formation and

As explained above, FXa performs a crucial function in the coagulation process. Thus, FXa provides a specific target for novel anticoagulant agents. The synthesis of direct FXa inhibitors that are able to effectively inhibit prothrombinase-associated and clot-bound FXa, and therefore provide greater potential anticoagulant activity, is therefore a significantly important advance. There is enough evidence to imply that inhibition earlier during primary hemostasis in the coagulation cascade at the FXa level could provide higher antithrombotic potential by using inhibition of platelet adhesion drugs. Furthermore, preclinical studies indicate that FXa inhibitors possibly possess a broader therapeutic index. Therefore, there is a significant number of pharmaceutical companies, which are working to discover new anticoagulant drugs, and have finally decided to focus on small molecules such as direct FXa inhibitors [54–59].

FXa is a serine protease which catalyzes the production of 1000 thrombin molecules involving the interaction on the platelets surface, Ca2+ ions, and FVa called the prothrombinase complex. The prothrombinase complex acts on the natural substrate producing the catalytic coagula-

Structurally FXa, like trypsin, belongs among the family of serine proteases within the catalytic domain, which is formed by two antiparallel β-barrel folds that act in tandem to produce the catalytic triad and the substrate binding site. Schechter and Berger (**Figure 6**) have provided a nomenclature adopted by scientists, which describes the prototypical binding site of a serine protease. Consequently, each protein subsite, labeled Si, binds its related amino acid

has attracted a strong interest in the last 5 years.

18 Anticoagulant Drugs

inhibiting the catalyzing FXa reaction [33, 48–53].

tion process.

substrate, labeled Pi [60].

As it was exposed before, FXa plays a critical role in coagulation. Together with FVa and calcium ions on a phospholipid surface, FXa forms the prothrombinase complex, which is responsible for the conversion of prothrombin to thrombin, the final effector of coagulation (**Figure 1**).

Oral anticoagulant drug discovery efforts initially focused on the development of smallmolecule anticoagulants that target thrombin directly—the oral DTIs. But, there is some evidence to suggest that inhibition earlier in the coagulation cascade at the level of FXa may have greater antithrombotic potential. In addition preclinical studies suggest that FXa inhibitors may possess a wider therapeutic index than DTIs. Thus, it is understandable that a great number of pharmaceutical companies dedicated to the discovery of this oral anticoagulant drug have finally and determinedly concentrated on small-molecule, direct FXa inhibitors [33, 56, 57, 74–78].

It is worth considering briefly some of the significant molecular characteristics of the target protein. FXa belongs to the family of serine proteases such as trypsin; the catalytic domain consists of two antiparallel β-barrel folds that together form the catalytic triad and the substrate binding site. Accordingly to the Schetcher and Berger nomenclature each protein subsite (Si) binds the amino acid (Pi) residue [79]. Specifically, FXa is composed by four principal subsites S1, S2, S3, and S4.

S1 is an anionic pocket—hydrophobic and deep cleft—formed by Tyr228, Ser195, and Asp189; and S4 subsite has three domains to link with the ligand: one hydrophobic pocket defined by Tyr99, Trp215, and Phe174, one cationic hole formed by Glu97 and Lys96, and a water pocket where the natural substrate is trapped under the following amino acids: Thr98, Ile175, and

**4. Direct FXa inhibitors development**

Xa inhibitors in animal models of thrombosis.

and development of new synthetic oral FXa inhibitors.

pathologies.

therapy [79, 88–91].

In spite of the fact that as early as the beginnings of the 1980s, the factor Xa had already been recognized as an auspicious target for the development of new anticoagulants, its viability inhibition was not tested until the late 1980s. It was in 1987 that the first factor Xa inhibitor—naturally occurring compound antistasin—was extracted from the salivary glands of the Mexican leech *Haementeria officinalis*. Antistasin is a 119 amino-acid polypeptide; kinetic studies revealed that it is a slow, tight-binding, potent factor Xa inhibitor [83–85]. Similar properties show another factor Xa inhibitor—the tick anticoagulant peptide (tAP) [86]. This anticoagulant peptide is a single-chain, amino-acid peptide which was isolated in 1990 from extracts of the soft tick *Ornithodoros moubata*. The antithrombotic effects of these compounds were compared with those of direct thrombin inhibitors, and of indirect thrombin and factor

FXa Direct Synthetic Inhibitors

21

http://dx.doi.org/10.5772/intechopen.76518

The discovery of FXa's role in the clotting cascade produced an increasing interest in this enzyme due to its pharmacological target for the treatment of a diverse number of hemostatic

The first FXa inhibitory studies were carried by using natural anticoagulants obtained from ticks (antistatin), leeches (Yagin), and bats (Draculin). These natural anticoagulant proteins had indirect activity on FXa. Antistatin produce a slow-release FXa-complex which reduces the cascade amplification. Besides, Draculin directly inhibits FXa without activity on thrombin [87].

Several investigation groups started designing and synthesizing novel small molecules for the treatment of thrombotic-related pathologies such as deep vein thrombus (DVT), acute coronary syndrome (ACS), stroke, as well as for the prevention of clot production during surgeries. Moreover, pharmaceuticals companies have been financing strongly in the research

For example, fondaparinux (**Figure 5c**), is selective for FXa but acts indirectly via binding to antithrombin and has demonstrated similar clinical benefit over LMWHs in venous thrombotic indications. The safety and efficacy of one provided the first clinical proof of the principle that targeting FXa would be an important advancement in the area of anticoagulation

A second generation of synthetic derivatives, idrabiotaparinux, is in late-stage clinical trials for treatment of VTE and for stroke prevention in patients with AF [92]. Early efforts to identify inhibitors of FXa stemmed from the prior discoveries of thrombin inhibitors such as compounds showing in **Figure 6** are examples of early FXa inhibitors. Because of the success of indirect dual factor Xa and thrombin inhibitors, such as LMWHs, indirect inhibitors of factor Xa with greater selectivity, such as fondaparinux, were developed in parallel with oral direct factor Xa inhibitors, such as rivaroxaban [93] (**Figure 7c**) and apixaban [94] (**Figure 7d**).

These last two small molecules have been demonstrated to have the best pharmacokinetics characteristics, and they have a fully complete preclinical characterization as an oral direct FXa inhibitor. Furthermore, rivaroxaban was approved in Canada, Europe, and other countries for the prevention of VTE in adults undergoing hip and knee surgery, and it has a predictable anticoagulant response avoiding the need for monitoring. Moreover, apixaban was

**Figure 6.** Nomenclature based on the Schechter and Berger convention. Figure adapted from Berg et al [67].

**Figure 7.** Early and more recent direct FXa inhibitors.

Tyr175. Besides, the S2 subsite is not well defined, has a slightly profound pocket, and is fused with the S4 subsite (**Figure 8**). Finally, the S3 pocket is exposed to the solvent, and it is situated at the borderline of the S1 subsite region [78, 80].

All reported data indicates that small-molecule serine protease inhibitors bind one or more of the subsites. **Figure 5** shows the most important serine protease subsites responsible for the design molecules recognition and binding characteristics.

In view of the small size of these synthetic inhibitors, they allow inhibiting both bound prothrombinase and free FXa. In addition, these drugs are able to penetrate the blood clot and inhibit FXa [61, 81, 82].

**Figure 8.** Most important amino acid residues in S1 and S4 pockets.
