discovery and development of direct xa inhibitors discovery and development of direct xa inhibitors four drugs from the class of direct xa inhibitors are marketed worldwide. rivaroxaban (xarelto) was the first approved fxa inhibitor to become commercially available in europe and canada in 2008. the second one was apixaban (eliquis), approved in europe in 2011 and in the united states in 2012. the third one edoxaban (lixiana, savaysa) was approved in japan in 2011 and in europe and the us in 2015. betrixaban (bevyxxa) was approved in the us in 2017. history heparin heparin structure heparin was discovered by jay mclean and william henry howell in 1916, it was first isolated from a canine liver, which in greek translates to hepar. heparin targets multiple factors in the blood coagulation cascade, one of them being fxa. at first, it had many side effects but for the next twenty years, investigators worked on heparin to make it better and safer. it entered clinical trials in 1935 and the first drug was launched in 1936. chains of natural heparin can vary from 5.000 to 40.000 daltons. in the 1980s low molecular weight heparin (lmwh) were developed and they only contain chains with an average molecular weight of less than 8.000 da. warfarin warfarin structure in the 1920s there was an outbreak of a mysterious haemorrhagic cattle disease in canada and the northern united states. the disease was named sweet clover disease because the cattle had grazed on sweet clover hay. it wasn't until ten years after the outbreak, that a local investigator, karl p. link and his student wilhelm schoeffel started an intense investigation to find the substance causing the internal bleeding. it took them 6 years to discover dicoumarol, the causing agent. they patented the right for the substance and in 1945 link started selling a coumarin derivative as a rodenticide. he and his colleagues worked on several variations and ended up with a substance they named warfarin in 1948. it wasn't until 1954 that it was approved for medicinal use in humans making warfarin the first oral anticoagulant drug. need for newer and better oral drugs warfarin treatment requires blood monitoring and dose adjustments regularly due to its narrow therapeutic window. if supervision isn't adequate warfarin poses a threat in causing, all too frequent, haemorrhagic events and multiple interactions with food and other drugs. currently, the main problem with low molecular weight heparin (lmwh) is the administration route, as it has to be given subcutaneously. because of these disadvantages there has been an urgent need for better anticoagulant drugs. for a modern society, convenient and fast drug administration is the key to a good drug compliance. in 2008 the first direct xa inhibitor was approved for clinical use. direct xa inhibitors are just as efficacious as lmwh and warfarin but they are given orally and don't need as strict monitoring. other xa inhibitors advantages are rapid onset/offset, few drug interactions and predictable pharmacokinetics. the rapid onset/offset effect greatly reduces the need for “bridging” with parenteral anticoagulants after surgeries. today there are four factor xa inhibitors marketed: rivaroxaban, apixaban, edoxaban and betrixaban. antistasin and tick anticoagulant peptide (tap) factor xa was identified as a promising target for the development of new anticoagulants in the early 1980s. in 1987 the first factor xa inhibitor, the naturally occurring compound antistasin, was isolated from the salivary glands of the mexican leech haementeria officinalis. antistasin is a polypeptide and a potent xa inhibitor. in 1990 another naturally occurring xa inhibitor was isolated, tick anticoagulant peptide (tap) from extracts of the tick ornithodoros moubata. tap and antistasin were used to estimate factor xa as a drug target. mechanism of action coagulation in vivo blood coagulation is a complex process by which the blood forms clots. it is an essential part of hemostasis and works by stopping blood loss from damaged blood vessels. at the site of injury, where there is an exposure of blood under the endothelium, the platelets gather and immediately form a plug. that process is called primary hemostasis. simultaneously, a secondary hemostasis occurs. it is defined as the formation of insoluble fibrin by activated coagulation factors, specifically thrombin. these factors activate each other in a blood coagulation cascade that occurs through two separate pathways that interact, the intrinsic and extrinsic pathway. after activating various proenzymes, thrombin is formed in the last steps of the cascade, it then converts fibrinogen to fibrin which leads to clot formation. factor xa is an activated serine protease that occupies a key role in the blood coagulation pathway by converting prothrombin to thrombin. inhibition of factor xa leads to antithrombotic effects by decreasing the amount of thrombin. directly targeting factor xa is suggested to be an effective approach to anticoagulation. development antistasin, the first discovered direct xa-inhibitor in 1987 antistasin was tested as the first direct xa inhibitor. antistasin is a protein made up of 119 amino acid residues, of which 20 are cysteines involved in 10 disulfide bonds. it acts as a slow, tight-binding inhibitor of factor xa with a ki value of 0.3–0.6 nm but it also inhibits trypsin. recombinant antistasin can be produced by genetically modified yeast, saccharomyces cerevisiae. another natural occurring direct xa-inhibitor, the tick anticoagulant peptide (tap), was discovered in 1990. it is a single-chain, 60 amino acid peptide and like antistasin it is a slow, tight-binding inhibitor with a similar ki value (~0.6 nm). these two proteins were mostly used to validate factor xa as a drug target. animal studies suggested direct xa-inhibition to be a more efficient approach to anticoagulation compared to direct thrombin inhibitors, especially offering a wider therapeutic window and reducing the risk of rebound thrombosis, (increase in thromboembolic events occurring shortly after the withdrawal of an antithrombotic medication) compared to direct and indirect thrombin inhibitors. during the 1990s several low-molecular-weight substances were developed, such as dx-9065a and ym-60828. ym-60828 (left) and dx-9065a (right) were two of the first small-molecule direct xa-inhibitors developed in the 1990s. yet both had highly basic amidine groups that were thought to be necessary for the binding of factor xa but caused a low oral bioavailability. dx-9065a was the first synthetic compound that inhibited fxa without inhibiting thrombin. that was attained by inserting a carboxyl group which seemed to be the most important moiety for a selective binding to fxa. those early developed small molecules yet had amidine-groups or even higher-basic functions, which were thought to be necessary as mimics for an arginine residue in prothrombin, the natural substrate of factor xa. nevertheless, these basic functions are also related to a very poor oral bioavailability (e.g. 2–3% for dx-9065a). in 1998 bayer healthcare, a pharmaceutical company started searching for low-molecular-weight direct factor xa inhibitors with higher oral bioavailability. high-throughput screening and further optimisation at first lead to several substances from the class of isoindolinones demonstrating that much less basic substances can also act as potent xa inhibitors to an ic50 value of up to 2 nm. although isoindolinones have a better oral bioavailability than the original compounds it wasn't sufficient enough. however, the project later lead to the class of n-aryloxazolidinones that provides substances with both high potency of inhibiting factor xa and high bioavailability. one compound of this class, rivaroxaban (ic50 = 0.7 nm, bioavailability: 60%), was granted marketing authorization for the prevention of venous thromboembolism in europe and canada in september 2008. chemistry factor xa: structure and binding sites 3d structure of factor xa factors iia, xa, viia, ixa and xia are all proteolytic enzymes that have a specific role in the coagulation cascade. factor xa (fxa) is the most promising one due to its position at the intersection of the intrinsic and extrinsic pathway as well as generating around 1000 thrombin molecules for each xa molecule which results in a potent anticoagulant effect. fxa is generated from fx by cleavage of a 52 amino acid activation peptide, as the "a" in factor xa means activated. fxa consists of 254 amino acid catalytic domain and is also linked to a 142 amino acid light chain. the chain contains both gla domain and two epidermal growth factor domains (egf like domains). the active site of fxa is structured to catalyze the cleavage of physiological substrates and cleaves phepheasnproarg-thrphe and tyrileaspglyarg-ileval in prothrombin. fxa has four so-called pockets which are targets for substrates to bind to factor xa. these pockets are lined up by different amino acids and xa inhibitors target these pocket when binding to factor xa. the two most relevant pockets regarding affinity and selectivity for the xa inhibitors are s1 and s4. s1: the s1 pocket is a hydrophobic pocket and contains an aspartic acid residue (asp-189) which can serve as a recognition site for a basic group. fxa has a residual space in the s1 pocket and is lined by residues tyr-228, asp-189 and ser-195. s2: the s2 pocket is a small and shallow pocket. it merges with the s4 pocket and has room for small amino acids. tyr-99 seems to block access to this pocket, so this pocket is not as important as s1 and s4. s3: the s3 pocket is located on the rim of the s1 pocket and is flat and exposed to the solvent. this pocket is not as important as s1 and s4. s4: the s4 pocket is hydrophobic in nature and the floor of the pocket is formed by trp-215 residue. the residues phe-174 and tyr-99 of fxa join trp-215 to form an aromatic box that is able to bind aliphatic, aromatic and positively charged fragments. because of the binding to positively charged entities, it can be described as a cation hole. chemical structure and properties of direct xa inhibitors chemical properties of rivaroxaban, apixaban and edoxaban. rivaroxabanapixabanedoxaban mw (g/mol)436460548 molecular formulac19h18cln3o5sc25h25n5o4c24h30cln7o4s shapelll ki0.4 nm0,08 nm0.561 nm ic500.7 nmn/an/a oral bioavailability (%)66–100 (dose-dependent)5062 the structure of apixaban, edoxaban and rivaroxaban. currently existing direct xa inhibitors binding of xa inhibitors to factor xa the xa inhibitors all bind in a so-called l-shape fashion within the active site of factor xa. the key constituents of the factor xa are the s1 and s4 binding sites. it was first noted that the natural compounds, antistasin and tap, which possess highly polar and therefore charged components bind to the target with some specificity. that's why newer drugs were designed with positively charged groups but those resulted in poor bioavailability. nowadays marketed xa inhibitors, therefore contain an aromatic ring with various moieties attached for different interactions with the s1 and s4 binding sites. this also ensures good bioavailability as well as maintaining firm binding strength. the xa inhibitors currently on market today, therefore rely on hydrophobic and hydrogen bonding instead of highly polar interactions. antistasin binding to factor xa antistasin contains an n- and a c-terminal domain which are similar in their amino acid sequences with ~40% identity and ~56% homology. each of them contains a short β-sheet structure and 5 disulfide bonds. only the n-terminal domain is necessary to inhibit xa while the c-terminal domain does not contribute to the inhibitory properties due to differences in the 3 dimensional structure, even though the c-terminal domain has a strongly analogue pattern to the actual active site. the interaction of antistasin with fxa involves both the active site and the inactive surface of fxa. the reactive site of antistasin formed by arg-34 and val-35 in the n-terminal domain suits the binding site of fxa, most likely the s1 pocket. at the same time, glu-15 located outside the reactive site of antistasin fits to positively charged residues on the surface of fxa. the multiple binding is thermodynamically advantageous and leads to sub-nanomolar inhibition (ki = 0.3–0.6 nm). dx-9065a binding to factor xa dx-9065a, the first small molecule direct xa-inhibitor, is an amidinoaryl derivate with a molecular weight of 571.07g/mol. its positively charged amidinonaphtalene group forms a salt bridge to the asp-189 residue in the s1 pocket of fxa. the pyrrolidine ring fits between tyr-99, phe-174 and trp-215 in the s4 pocket of fxa. unlike older drugs, e.g. heparin, dx-9065a is selective for fxa compared to thrombin even though fxa and thrombin are similar in their structure. this is caused by a difference in the amino acid residue in the homologue position 192. while fxa has a glutamine residue in that