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.[1] The second one was apixaban (Eliquis), approved in Europe in 2011[2] and in the United States in 2012.[3] The third one edoxaban (Lixiana, Savaysa) was approved in Japan in 2011 and in Europe and the US in 2015.[4] Betrixaban (Bevyxxa) was approved in the US in 2017.
History
Heparin
Warfarin
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,
Need for newer and better oral drugs
Warfarin treatment requires blood monitoring and dose adjustments regularly due to its narrow
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
Mechanism of action
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.[10] 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.[11] These factors activate each other in a blood coagulation cascade that occurs through two separate pathways that interact, the intrinsic and extrinsic pathway.[12] 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.[10] 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.[8]
Development
These two proteins were mostly used to validate factor Xa as a
During the 1990s several low-molecular-weight substances were developed, such as DX-9065a[15] and YM-60828.[16]
DX-9065a was the first synthetic compound that inhibited FXa without inhibiting thrombin. That was attained by inserting a
In 1998
Chemistry
Factor Xa: Structure and binding sites
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).[18]
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.[18]
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.[18]
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.[19]
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.[18]
Chemical structure and properties of direct Xa inhibitors
Rivaroxaban | Apixaban | Edoxaban | |
---|---|---|---|
MW (g/mol) | 436 | 460 | 548 |
Molecular formula | C19H18ClN3O5S | C25H25N5O4 | C24H30ClN7O4S |
Shape | L | L | L |
Ki | 0.4 nM | 0,08 nM | 0.561 nM |
IC50 | 0.7 nM | N/A | N/A |
Oral bioavailability (%) | 66–100 (dose-dependent) | 50 | 62 |
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.[20]
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%
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[8]).[13]
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.[21] 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.[22]
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 position, thrombin has a glutamic acid that causes electrostatic repulsion with the carboxyl group of DX-9065a. In addition, a salt bridge between Glu-97 of thrombin and the amidine group fixed in the pyrrolidine ring of DX-9065a reduces the flexibility of the DX-9065a molecule, which now cannot rotate enough to avoid the electrostatic clash. That's why the IC50 value for thrombin is >1000µM while the IC50 value for FXa is 0,16µM.[22]
Rivaroxaban binding to factor Xa
Rivaroxaban binding to FXa is mediated through two hydrogen bonds to the amino acid Gly-219. These two hydrogen bonds serve an important role directing the drug into the S1 and S4 subsites of FXa. The first hydrogen bond is a strong interaction which comes from the carbonyl oxygen of the oxazolidinone core of rivaroxaban. The second hydrogen bond is a weaker interaction and comes from the amino group of the clorothiophene carboxamide moiety.
These two hydrogen bonds result in the drug forming an L-shape and fits in the S1 and S4 pockets. The amino acids residues Phe-174, Tyr-99, and Trp-215 form a narrow hydrophobic channel that is the S4 binding pocket. The morpholinone part of rivaroxaban is “sandwiched” between amino acids Tyr-99 and Phe-174 and the aryl ring of rivaroxaban is oriented perpendicularly across Trp-215. The morpholinone carbonyl group does not have a direct interaction to the FXa backbone, instead, it contributes to a planarization of the morpholinone ring and therefore supports rivaroxaban to be sandwiched between the two amino acids.
The interaction between the chlorine substituent of the thiophene moiety and the aromatic ring of Tyr-228, which is located at the bottom of the S1, it is very important due to the fact that it obviates the need for strongly basic groups for high affinity for FXa. This enables rivaroxaban, which is non-basic, to achieve good oral bioavailability and potency.[8]
Apixaban binding to factor Xa
Apixaban shows a similar binding mode as rivaroxaban and forms a tight inhibitor-enzyme complex when connected to FXa. The p-methoxy group of apixaban connects to S1 pocket of FXa but does not appear to have any interaction with any residues in this region of FXa. The pyrazole N-2 nitrogen atom of apixaban interacts with Gln-192 and the carbonyl oxygen interacts with Gly-216. The phenyl lactam group of apixaban is positioned between Tyr-99 and Phe-174 and due to its orientation, it is able to interact with Trp-215 of the S4 pocket. The carbonyl oxygen group of the lactam moiety interacts with a water molecule and does not seem to interact with any residues in the S4 pocket.[23]
Structure-activity-relationship (SAR)
An important part of designing a compound, that is an ideal inhibitor to a certain target, is to understand the amino acid sequence of the target site for the compound to bind to. Modelling both prothrombin and FXa makes it possible to deduct the difference and identify the amino acids at each binding site. At the bottom of the S1 pocket on FXa the binding amino acid is Asp-189 which amidine moieties can bind to. After X-raying the binding site of FXa, it was revealed that the S1 pocket had a planar shape, meaning that a flat amidinoaryl group should bind to it without steric hindrance.[8]
Modern direct Xa inhibitors are L-shaped molecules whose ends fit perfectly in the S1 and S4 pockets. The long side of the L-shape has to conform to a highly-specific tunnel within the targets active site. To accomplish that, this part of the molecules is designed to have little formal interactions with FXa in that region. As there is no specific bonding, the fit of these agents between the pockets of FXa increases the total specificity of the drugs to the FXa molecule. The interaction between the S1 pocket of FXa and the inhibitor can be both ionic or non-ionic, which is important because it allows the design of the moiety to be adjusted to increase oral bioavailability. Previously designed compounds were charged molecules that are not absorbed well in the gastrointestinal tract and therefore did not reach high serum concentrations. The newer drugs have a better bioavailability as they are not charged and have a non-ionic interaction to the S1 pocket.[20]
Rivaroxaban
During the SAR development of rivaroxaban, researchers realized that adding a 5-chlorothiophene-2-carboxamide group to the oxazolidonine core could increase the potency by 200 fold, which had previously been too weak for medical use. In addition to this discovery, a clear preference for the (S)-configuration was confirmed. This compound had a promising pharmacokinetical profile and did not contain a highly basic amidine group, but that had previously been considered important for the interaction with the S1 pocket. These findings lead to extensive SAR (
Apixaban
During the SAR development of apixaban there were three groups that needed to be tested to attain maximum potency and bioavailability. The first group to be tested was the non-active site as it needs to be stabilized before SAR testing on the p-methoxyphenyl group (S1 binding moiety). There are several of groups that increase the potency of the compound, mostly amides, amines and tetrazoles but also methylsulfonyl and trifluoromethyl groups. Of these groups, carboxamide has the greatest binding and had similar clotting activity as the compounds.[25]
In dog testing, this compound with a carboxamide group called 13F, showed a great pharmacokinetical profile, a low clearance and adequate half-life and volume of distribution. Due to the success of finding a stabilizing group, SAR research for S1 binding moiety (p-methoxyphenyl) was discontinued. In the S4 binding group, N-methylacetyl and lactam analogues proved to have a very high binding affinity for FXa, showed great clotting and selectivity versus other proteases. Orientation turned out to be important as N-methyl acetyl, compared to acetamide, had a 300 fold lower binding ability to FXa due to unfavorable planarity close to the S4 region binding site.[25]
Synthesis
Rivaroxaban
Rivaroxaban chemically belongs to the group of
An industrial preparation of rivaroxaban was registered as a
However, according to the patent the synthesis has “various disadvantages in the reaction management which has particularly unfavourable effects for preparation“. The patent also explains another synthesis starting from a chloro
Various other synthesis pathways of rivaroxaban have been described.[28][29]
Apixaban
The first full synthesis of apixaban was published in 2007.[30] The key step of this reaction is a (3+2)cycloaddition of a p-methoxyphenylchlorohydrazon derivate and a p-iodophenyl-morpholin-dihydropyridin derivate. After the following elimination of HCl and morpholine, the iodine is substituted by 2-piperidinone by copper-catalization and the ethyl esther is converted to an amide (aminolysis). This reaction was registered as a patent in 2009.[31]
Clinical use
Direct factor Xa inhibitors are being used clinically and their usage is constantly increasing. They are gradually taking over
Rivaroxaban | Apixaban | Edoxaban | Betrixaban | |
---|---|---|---|---|
Brand name | Xarelto | Eliquis | Savaysa, Lixiana | Bevyxxa |
Developer and producer | Bayer | Pfizer | Daiichi Sankyo | Portola Pharmaceuticals |
Pharmacokinetics
Rivaroxaban | Apixaban | Edoxaban | |
---|---|---|---|
Metabolism | CYP3A4/5 (major), CYP2J2 (minor) | CYP3A4 (major), CYP1A2, 2C8, 2C19, 2J2 (all minor) | CYP34A (major) |
Protein binding (%) | 92–95 | 87 | 55 |
Half life (hrs) | 5–9 | 6–12 | 5–11 |
Elimination | Renal (66%; 36% as unchanged drug) | Renal (27%), fecal | Renal (35%) |
Absorption (Tmax) | 2–4 hours | 3–4 hours | 1–2 hours |
Distribution (L) | 50 | 21–61 | 107 |
Renal clearance (L/hr) | 2.4 | 7.5 | 11 |
Future perspectives
Direct Xa inhibitors in clinical trials
Rivaroxaban, apixaban, edoxaban and betrixaban are already on the market. As of October 2016, several new direct Xa inhibitors have entered clinical trials. These are letaxaban from Takeda and eribaxaban from Pfizer.[34]
Antidotes
Andexxa (
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