**3. Clotting cascade: factor Xa function**

Factor X or Stuart-Prower Factor named after the male patient named Stuart in 1957 and a female patient named Prower with FX deficiency [45, 46]. Moreover, Factor X has long been known to have a key role in hemostasis and plays a central part in the blood-clotting cascade by catalyzing the production of thrombin, which leads to clot formation and wound closure [33, 47].

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 has attracted a strong interest in the last 5 years.

As the discovery of small-molecule protease inhibitors has progressed, this convention has been amplified to denote drug substructures that similarly bind to substrate amino acids

FXa Direct Synthetic Inhibitors

19

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

In the 1980s, early attempts to identify FXa inhibitors were prompted by prior thrombin inhibitor discoveries such as the compounds illustrated in **Figure 7** (**a** and **b**), which are examples of early [61] and most recent direct FXa inhibitors rivaroxaban (**Figure 7c**) and apixaban

Development of rivaroxaban was a major breakthrough in anticoagulation drug discovery and was the first approved orally active direct FXa inhibitor. However, recently studies have shown that rivaroxaban and apixaban discontinuation could result in thromboembolic events, and the use of rivaroxaban associated with warfarin increases the risk of major bleeding in non-valvular atrial fibrillation patients [62–64]. Through the study of the chemical structures of these inhibitors illustrated below, it also became evident that various substitutes could be

In spite of extensive knowledge about the clotting mechanism, its complexity poses a considerable challenge to the research and development of powerful anticoagulants that are both

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

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

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

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

(**Figure 6**) [54].

safe and effective.

(**Figure 1**).

[33, 56, 57, 74–78].

subsites S1, S2, S3, and S4.

(**Figure 7d**) chemical structures.

**3.1. FXa structural target points**

accepted in both the S1 and S4 regions [45, 65–73].

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

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 coagulation process.

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 substrate, labeled Pi [60].

As the discovery of small-molecule protease inhibitors has progressed, this convention has been amplified to denote drug substructures that similarly bind to substrate amino acids (**Figure 6**) [54].

In the 1980s, early attempts to identify FXa inhibitors were prompted by prior thrombin inhibitor discoveries such as the compounds illustrated in **Figure 7** (**a** and **b**), which are examples of early [61] and most recent direct FXa inhibitors rivaroxaban (**Figure 7c**) and apixaban (**Figure 7d**) chemical structures.

Development of rivaroxaban was a major breakthrough in anticoagulation drug discovery and was the first approved orally active direct FXa inhibitor. However, recently studies have shown that rivaroxaban and apixaban discontinuation could result in thromboembolic events, and the use of rivaroxaban associated with warfarin increases the risk of major bleeding in non-valvular atrial fibrillation patients [62–64]. Through the study of the chemical structures of these inhibitors illustrated below, it also became evident that various substitutes could be accepted in both the S1 and S4 regions [45, 65–73].

In spite of extensive knowledge about the clotting mechanism, its complexity poses a considerable challenge to the research and development of powerful anticoagulants that are both safe and effective.
