**4. Direct FXa inhibitors development**

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 Xa inhibitors in animal models of thrombosis.

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 pathologies.

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 and development of new synthetic oral FXa inhibitors.

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 therapy [79, 88–91].

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

at the borderline of the S1 subsite region [78, 80].

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

inhibit FXa [61, 81, 82].

20 Anticoagulant Drugs

design molecules recognition and binding characteristics.

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

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

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

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 approved by the Food and Drug Administration (FDA) in December 2012 with an indication of reducing the risk of stroke and dangerous blood clots (systemic embolism) in patients with atrial fibrillation (AF) [95–98].

Based on these discoveries, in the mid-1990s, it was assumed that small-molecule, direct factor Xa inhibitors could most likely become a better option than the antithrombotic therapies used in those days [99].
