Design, synthesis and SAR of novel ethylenediamine and phenylenediamine derivatives as factor Xa inhibitors

We previously reported on a series of cyclohexanediamine derivatives as highly potent factor Xa inhibitors. Herein, we describe the modification of the spacer moiety to discover an alternative scaffold. Ethylenediamine derivatives possessing a substituent at the C1 position in S configuration and phenylenediamine derivatives possessing a substituent at the C5 position demonstrated moderate to strong anti-fXa activity. Further SAR studies led to the identification of compound 30h which showed both good in vitro activity (fXa IC₅₀ = 2.2 nM, PTCT2 = 3.9 μM) and in vivo antithrombotic efficacy.     

Orally bioavailable factor Xa inhibitors containing alpha-substituted gem-dimethyl P4 moieties

In an effort to identify a potential back-up to apixaban (Eliquis®), we explored a series of diversified P4 moieties. Several analogs with substituted gem-dimethyl moieties replacing the terminal lactam of apixaban were identified which demonstrated potent FXa binding affinity (FXa K_i), good human plasma anticoagulant activity (PT EC_2x), cell permeability, and oral bioavailability.     

Arylsulfonamidopiperidone derivatives as a novel class of factor Xa inhibitors

The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and selective FXa inhibitors is reported. The arylsulfonamide–valerolactam scaffold was derived based on the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC₅₀ of 7 nM and EC_2×PT of 1.7 μM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide–valerolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4 pockets.     

Structure-activity relationships of anthranilamide-based factor Xa inhibitors containing piperidinone and pyridinone P4 moieties

Introduction of the phenyl piperidinone and phenyl pyridinone P4 moieties in the anthranilamide scaffold led to potent, selective, and orally bioavailable inhibitors of factor Xa. Anthranilamide 28 displayed comparable efficacy to apixaban in the rabbit arteriovenous-shunt (AV) thrombosis model.     

Investigation of the terminal P4 domain in a series of D-phenylglycinamide-based factor Xa inhibitors

Several P4 domain derivatives of the general d-phenylglycinamide-based scaffold (2) were synthesized and evaluated for their ability to bind to the serine protease factor Xa. Some of the more potent compounds were evaluated for their anticoagulant effects in vitro. A select subset containing various P1 indole constructs was further evaluated for their pharmacokinetic properties after oral administration to rats.     

Interactions between heparin and factor Xa inhibition of prothrombin activation

The effects of heparin on prothrombin activation have been examined. Heparin was found to inhibit the rate of prothrombin activation by Factor Xa, calcium and phospholipid. In the absence of phospholipid, heparin had no effect on the rate of prothrombin activation. In contrast, heparin was found to increase the rate of activation of prethrombin-1 and prethrombin-2. Initial velocity studies indicated that heparin blocks lipid stimulation of prothrombin activation. In accord with this, binding studies demonstrated that heparin could displace Factor Xa, and in separate experiments, prothrombin, from phospholipid vesicles.     

Factor Xa (FXa) inhibitor from the nymphs of the camel tick Hyalomma dromedarii

An inhibitor of factor Xa (FXa) was isolated from the nymphs of the camel tick Hyalomma dromedarii by a combination of chromatography on DEAE-cellulose and Sephacryl S-300 columns. The isolated nymphal FXa inhibitor turned out to be a homogenous preparation of a single polypeptide chain (15 kDa) as judged by both the native and denatured SDS-PAGE. Its pI value ranged from 7.7 to 7.9. The inhibitor is a potent anticoagulant since it prolonged both the activated partial thromboplastin time (APTT) and the prothrombin time (PT) of the camel plasma in a concentration-dependent manner. Its activity was threefold lower toward thrombin than FXa, but it did not inhibit any of the proteases; trypsin, α-chymotrypsin, papain, pepsin and subtilisin. The inhibitor binds at two sites on FXa uncompetitively with an inhibition constant (K_i) value of 134 nM.     

Orally efficacious thrombin inhibitors with cyanofluorophenylacetamide as the P2 motif

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K_i = 1.2 nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.     

Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection.     

Semisynthesis of ent-norstrobane diterpenoids as potential inhibitor for factor Xa

A semisynthesis of two ent-strobane diterpenoids strobols C (7) and D (14) was accomplished via a Wagnar-Meerwein rearrangement. Compounds 7, 14, and the intermediate products were evaluated for their inhibition on factor Xa (FXa). Among all the compounds screened for FXa inhibitory activity, three compounds 6, 7, and 9 showed significant inhibitory activities with IC₅₀ values of 1067?±?164, 81?±?11, 1023?±?89?nM, respectively. The inhibitory activity on FXa described in this study highlight the importance of structural modification based on natural products in the development of FXa inhibitors.     

Novel strategy to boost oral anticoagulant activity of blood coagulation enzyme inhibitors based on biotransformation into hydrophilic conjugates

The blood coagulation cascade represents an attractive target for antithrombotic drug development, and recent studies have attempted to identify oral anticoagulants with inhibitory activity for enzymes in this cascade, with particular attention focused on thrombin and factor Xa (fXa) as typical targets. We previously described the discovery of the orally active fXa inhibitor darexaban (1) and reported a unique profile that compound 1 rapidly transformed into glucuronide YM-222714 (2) after oral administration. Here, we propose a novel strategy towards the discovery of an orally active anticoagulant that is based on the bioconversion of a non-amidine inhibitor into the corresponding conjugate to boost ex vivo anticoagulant activity via an increase in hydrophilicity. Computational molecular modeling was utilized to select a template scaffold and design a substitution point to install a potential functional group for conjugation. This strategy led to the identification of the phenol-derived fXa inhibitor ASP8102 (14), which demonstrated highly potent anticoagulant activity after biotransformation into the corresponding glucuronide (16) via oral dosing.     

The discovery of potent and long-acting oral factor Xa inhibitors with tetrahydroisoquinoline and benzazepine P4 motifs

The discovery and evaluation of potent and long-acting oral sulfonamidopyrrolidin-2-one factor Xa inhibitors with tetrahydroisoquinoline and benzazepine P4 motifs are described. Unexpected selectivity issues versus tissue plasminogen activator in the former series were addressed in the later, delivering a robust candidate for progression towards clinical studies.     

Orally active factor Xa inhibitors: 4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine derivatives

In our investigation of factor Xa inhibitors, a series of 1-(6-chloronaphthalen-2-yl)sulfonyl-4-(4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carbonyl)piperazines 3a-i were synthesized. In vitro inhibitory activities of the compounds against factor Xa and coagulation are summarized. Among the compounds, 3c and 3d, possessing a carbamoyl or N-methylcarbamoyl moiety, showed potent inhibitory activities when administered orally to rats.     

Enhanced fibrinolysis by proteolysed coagulation factor Xa

We previously showed that coagulation factor Xa (FXa) enhances activation of the fibrinolysis zymogen plasminogen to plasmin by tissue plasminogen activator (tPA). Implying that proteolytic modulation occurs in situ, intact FXa (FXaα) must be sequentially cleaved by plasmin or autoproteolysis, producing FXaβ and Xa33/13, which acquire necessary plasminogen binding sites. The implicit function of Xa33/13 in plasmin generation has not been demonstrated, nor has FXaα/β or Xa33/13 been studied in clot lysis experiments. We now report that purified Xa33/13 increases tPA-dependent plasmin generation by at least 10-fold. Western blots confirmed that in situ conversion of FXaα/β to Xa33/13 correlated to enhanced plasmin generation. Chemical modification of the FXaα active site resulted in the proteolytic generation of a product distinct from Xa33/13 and inhibited the enhancement of plasminogen activation. Identical modification of Xa33/13 had no effect on tPA cofactor function. Due to its overwhelming concentration in the clot, fibrin is the accepted tPA cofactor. Nevertheless, at the functional level of tPA that circulates in plasma, FXaα/β or Xa33/13 greatly reduced purified fibrin lysis times by as much as 7-fold. This effect was attenuated at high levels of tPA, suggesting a role when intrinsic plasmin generation is relatively low. FXaα/β or Xa33/13 did not alter the apparent size of fibrin degradation products, but accelerated the initial cleavage of fibrin to fragment X, which is known to optimize the tPA cofactor activity of fibrin. Thus, coagulation FXaα undergoes proteolytic modulation to enhance fibrinolysis, possibly by priming the tPA cofactor function of fibrin.     

Structure and property based design of factor Xa inhibitors: pyrrolidin-2-ones with aminoindane and phenylpyrrolidine P4 motifs

The rational design, syntheses and evaluation of potent sulfonamidopyrrolidin-2-one-based factor Xa inhibitors incorporating aminoindane and phenylpyrrolidine P4 motifs are described. These series delivered highly potent anticoagulant compounds with excellent oral pharmacokinetic profiles; however, significant time dependant P450 inhibition was an issue for the aminoindane series, but this was not observed with the phenylpyrrolidine motif, which produced candidate quality molecules with potential for once-daily oral dosing in humans.     

Mapping the active site of factor Xa by selective inhibitors: An NMR and MD study

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH₂ and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH₂, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site. Proteins 30:264–279, 1998. © 1998 Wiley-Liss, Inc.     

Selective and dual action orally active inhibitors of thrombin and factor Xa

The synthetic entry to new classes of dual fXa/thrombin and selective thrombin inhibitors with significant oral bioavailability is described. This was achieved through minor modifications to the sulfonamide group in our potent and selective fXa inhibitor (E)-2-(5-chlorothien-2-yl)-N-{(3S)-1-[(1S)-1-methyl-2-(morpholin-4-yl)-2-oxoethyl]-2-oxopyrrolidin-3-yl}ethenesulfonamide and these observed activity changes have been rationalised using structural studies.     

Design,synthesis and biological evaluation of anthranilamide derivatives as potential factor Xa (fXa) inhibitors

Factor Xa (fXa) is a crucial player in various thromboembolic disorders. Inhibition of fXa can provide safe and effective antithrombotic effects. In this study, a series of anthranilamide compounds were designed by utilizing structure-based design strategies. Optimization at P1 and P4 groups led to the discovery of compound 16g: a highly potent, selective fXa inhibitor with pronounced in vitro anticoagulant activity. Moreover, 16g also displayed excellent in vivo antithrombotic activity in the rat venous thrombosis (VT) and arteriovenous shunt (AV-SHUNT) models. The bleeding risk evaluation showed that 16g had a safer profile than that of betrixaban at 1?mg/kg and 5?mg/kg dose. Additionally, 16g also exhibited satisfactory PK profiles. Eventually, 16g was selected to investigate its effect on hypoxia-reoxygenation- induced H9C2 cell viability. MTT results showed that H9C2 cell viability can be remarkably alleviated by 16g.     

Discovery of novel aminobenzisoxazole derivatives as orally available factor IXa inhibitors

Using structure based drug design, novel aminobenzisoxazoles as coagulation factor IXa inhibitors were designed and synthesized. Highly selective inhibition of FIXa over FXa was demonstrated. Anticoagulation profile of selected compounds was evaluated by aPTT and PT tests. In vitro ADMET and pharmacokinetic (PK) profiles were also evaluated.     

Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa by preventing interaction(s) of enzyme with polysaccharide

Platelet factor 4 (PF4) is a heparin-binding protein which exhibits anti-heparin activities through the inhibition of antithrombin (AT)-dependent reactions with the serine proteases thrombin and factor Xa. PF4 also neutralizes heparan sulfate (HS), a glycosaminoglycan (GAG) present on the surface of endothelial cells, thereby possibly modulating an anticoagulant response. Previous models of PF4 mechanism did not distinguish whether PF4 causes steric hindrance of AT binding to fXa or of AT binding to the surface of the GAG chain. To shed light on the mechanism of PF4, studies of HS/heparin-catalyzed fXa inactivation by AT were undertaken. The results were consistent with PF4 directly interfering with AT binding to fXa rather than AT binding to the GAG chain, since PF4 did not prevent the heparin-dependent increase in AT intrinsic fluorescence. In fact, PF4 mechanism was competitive with respect to AT and non-competitive with respect to fXa, suggesting inhibition of important regulatory/catalytic interactions of fXa with the polysaccharide. Altogether, the results suggested a model by which PF4 bound to proximal (but distinct) sites to AT, resulting in steric interference of fXa binding to both polysaccharide and AT. It is proposed that PF4 inhibited the sequence of events recapitulated in the template mechanism describing heparin-dependent inhibition of fXa.