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.the binding of rivaroxaban to pockets s1 and s4 of factor xa. two hydrogen bonds are formed and serve an important role directing rivaroxaban into the s1 and s4 subsites. due to these hydrogen bonds, rivaroxaban forms a l-shape and fits in the pockets. chlorine substituent of the drug interacts with tyr-228 in the s1 pocket which enables rivaroxaban to achieve good oral bioavailability and potency. 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.the binding of apixaban to pockets s1 and s4 of factor xa. the pyrazole n-2 nitrogen atom of apixaban interacts with amino acid gln-192 at the s4 pocket. the carbonyl oxygen interacts with gly-216 of the s1 pocket as well as interacting with a water molecule. 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. the sar of rivaroxaban including the final moieties. during the sar testing, r1 was defined as the most important group for potency. pyrrolidinone was the first r1 functional group to significantly increase the potency but further researches revealed even higher potency with a morpholinone group instead. groups r2 and r3 had hydrogen or fluorine attached and it was quickly assessed that having hydrogen resulted in highest potency 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. 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. 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 (structure-activity relationship) researches. during the sar testing, r1 was defined as the most important group for potency. pyrrolidinone was the first r1 functional group to significantly increase the potency but further researches revealed even higher potency with a morpholinone group instead. groups r2 and r3 had hydrogen or fluorine attached and it was quickly assessed that having hydrogen resulted in highest potency. groups r2 and r3 were then substituted for various groups, which were all less potent than the hydrogen, so hydrogen was the final result. as the chlorothiophene moiety had an inadequate water solubility, substituting it with another group was attempted but was unsuccessful. the chlorothiophene moiety binds to tyr-228 at the bottom of the s1 pocket, making it a key factor regarding binding to fxa. rivaroxaban has both high affinity and good bioavailability. the structure of apixaban, before adjusting the moiety's for maximum potency apixabanthe 13f moiety intermediate before apixaban was fully developed 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. 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. rivaroxaban rivaroxaban chemically belongs to the group of n-aryloxazolidinones. other drugs of that group are linezolid and tedizolid, both of whom are antibiotics. a synthesis of n-aryloxazolidinones starting with an o-silyl protected ethyl(2,3-dihydroxypropyl)-carbamate was published in 2016. in a one-pot reaction the carbamate cyclisizes to a 2-oxazolidone ring under slightly basic conditions while simultaneously the oxazolidone nitrogen is arylized by copper-catalization. for rivaroxaban in particular, 3-morpholinone substitutes the iodine in p-position of the benzene ring by copper-catalization. afterwards, the silyl protecting group is removed and the resulting alcohol is replaced by an amino group which is then acylated in the last step. an industrial preparation of rivaroxaban was registered as a patent by bayer healthcare in 2005. it starts from n-(4-aminophenol)-morpholinone which is alkylated by a propylene oxide derivate that also contains a primary amine involved in a phthalimide protection group. next, a phosgene equivalent is added to form the 2-oxazolidone ring and the phthalimide is removed. the free amine can now be acylated which leads to rivaroxaban. 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 chlorothiophene derivate that would be more suitable for the industrial process but points out that toxic solvents or reagents have to be removed from the final product. therefore, this way is not an alternative. various other synthesis pathways of rivaroxaban have been described.synthesis of rivaroxaban as patented by bayer healthcare in 2005. 1st step: alkylation of the primary aromatic amine 2nd step: formation of the 2-oxazolinidone ring, using a phosgene equivalent 3rd step: removal of the phthalimide protection group 4th step: acylation of the primary amine apixaban the first full synthesis of apixaban was published in 2007. 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. synthesis of apixaban, as published by pinto, et al. in 2007. the notable key step is a (3+2)cycloaddition of the p-methoxyphenylchlorohydrazon derivate and the p-iodophenyl-morpholin-dihydropyridin derivate in the first reaction. clinical use direct factor xa inhibitors are being used clinically and their usage is constantly increasing. they are gradually taking over warfarin usage and low molecular weight heparins (lmwh). indication for xa inhibitors is preventing deep vein thrombosis (dvt) which can lead to pulmonary embolism. it is also used to treat atrial fibrillation to lower the risk of stroke caused by a blood clot. another indication is a prophylactic treatment for blood clotting (thrombosis) due to atherosclerosis. rivaroxaban was the first fxa inhibitor on the market and then followed by apixaban, edoxaban and betrixaban. rivaroxabanapixabanedoxaban betrixaban brand namexareltoeliquissavaysa, lixiana bevyxxa developer and producerbayerpfizerdaiichi sankyo portola pharmaceuticals pharmacokinetics pharmacokinetics of rivaroxaban, apixaban and edoxaban. rivaroxabanapixabanedoxaban metabolismcyp3a4/5 (major), cyp2j2 (minor)cyp3a4 (major), cyp1a2, 2c8, 2c19, 2j2 (all minor)cyp34a (major) protein binding (%)92–958755 half life (hrs)5–96–125–11 eliminationrenal (66%; 36% as unchanged drug)renal (27%), fecalrenal (35%) absorption (tmax)2–4 hours3–4 hours1–2 hours distribution (l)5021–61107 renal clearance (l/hr)2.47.511 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. antidotes andexxa (andexanet alfa) from portola pharmaceuticals is a recombinant protein that is given intravenously. it works as an antidote to all direct and indirect fxa inhibitors. andexxa acts as a decoy receptor for xa inhibitors.