Factor Xa Inhibitory Activity Research Articles

Design and synthesis of tricyclic benzo[1, 3]oxazinyloxazolidinones as blood coagulation factor Xa inhibitors

The coagulation enzyme factor Xa (FXa) is a valuable target for developing anticoagulants to treat thrombotic diseases. In this work, we designed and synthesized a series of conformationally restricted compounds containing the benzo[1,3]oxazinyloxazolidinone scaffold. The target compounds were synthesized by 11 steps reaction. The preliminary SAR showed that on the phenyl ring of benzo[1,3]oxazinyloxazolidinone, lactam replacements at the 6-position were favorable for FXa inhibition. Compound 16b showed good FXa inhibitory activity with an IC50 of 0.43 μM. The results indicate that benzo[1,3]oxazinyloxazolidinone is a promising scaffold for developing FXa inhibitors as antithrombotic agents.

Inhibition of factor Xa activity, platelet aggregation, and experimentally induced thrombosis by Sparstolonin B

Background: Sparstolonin B (SsnB) is an isocumarin compound extracted from medicinal plants such as Sparganium stoloniferum and Scirpus yagara with well documented anti-inflammatory activity. Here we examined if SsnB also possesses antithrombotic activity and the underlying mechanisms. Methods: Anti-thrombotic effects of SsnB were determined by measuring in vitro/ex vivo/in vivo clotting times, platelet aggregation assay, production and activity of factor Xa, nitric oxide, and expressions of relative proteins. Results: Treatment with SsnB prolonged the clotting time of human platelet-poor serum at concentrations comparable to the clinical anticoagulant rivaroxaban (as a positive control) and inhibited human platelet aggregation induced by adenosine diphosphate (ADP) or the thromboxane A2 analog U46619. SsnB also inhibited U46619-induced and ADP-induced phosphorylation of phospholipase C (PLC)γ2/protein kinase C (PKC) and intracellular calcium mobilization, both of which are required for platelet aggregation. In addition, SsnB inhibited expression of the cell adhesion factors P-selectin and PAC-1. SsnB increased production of the vasodilator nitric oxide and suppressed secretion of the vasoconstrictor endothelin-1 from ADP- or U46619-treated human umbilical vein endothelial cells. Further, SsnB reduced coagulation factor Xa (FXa) catalytic activity and production by endothelial cells as well as FXa-induced platelet aggregation. Conclusion: Finally, SsnB injection reduced thrombus formation time, number, size, and related mortality in mouse models of thromboembolism. SsnB is a promising antithrombotic agent targeting both FXa and platelet aggregation pathways, which can overcome the side effects of existing antithrombotic agents.

An integration strategy combined progressive multivariate statistics with anticoagulant activity evaluation for screening anticoagulant quality markers in Chinese patent medicine

Ethnopharmacological relevanceThe cardiovascular and cerebrovascular diseases affect human health globally. Naoxintong capsules (NXTs), a famous Chinese Patent Medicine, has been especially applied to treat cerebral infarction and coronary heart disease in clinical practice. The anticoagulant activity of this prescription plays an important role in this course of treatment. Aim of the studyThrombin and factor Xa (FXa) are two key targets considering the anticoagulant activity. The purpose of this investigation is to screen the quanlity markers as key thrombin and FXa inhibitors for the anticoagulant activity oriented quality control of Chinese patent medicine. Materials and methodsSimple multi-polar solvent extraction processes using various proportions of solvents were conducted and their thrombin/FXa inhibitory activities were evaluated in vitro. Bivariate correlation analysis (BCA), grey correlation analysis (GCA), and orthogonal partial least squares discriminate analysis (OPLS-DA) were adopted for screening the potential active markers related to the anticoagulant activity. The chemical structures of these active compounds were identified by UHPLC–Q-TOF-MS/MS and their thrombin/FXa inhibitory activity was determined. The molecular docking technology was applied to explore the interaction between the compounds and targets. The contribution of these anticoagulant active ingredients in NXT was also investigated. Last but not the least, the contents of these markers in NXT were determined by liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (HPLC-ESI-MS/MS) method. ResultsThe results showed that the NXT extract exhibited great activity against thrombin and FXa, especially extracted by 75% methanol (v/v). Six marker compounds with potential anticoagulant activity were screened out. Therein, four of the active compounds owing thrombin inhibitory activity (paeoniflorin, lithospermic acid, salvianolic acid B, Z-ligustilide) and five of the active compounds owing FXa inhibitory activity (3,5-dicaffeoylquinic acid, rosmarinic acid, lithospermic acid, salvianolic acid B and Z-ligustilide). In addition, these active compounds accounted for a large proportion of thrombin/FXa inhibitory activity of NXTs. The binding energy also showed the strong interaction formed by close connection of the compounds to the residues of targets. ConclusionsThe proposed integrated stategy could be an efficient strategy to screen potential thrombin/FXa inhibitors for the bioactivity related quanlity control of Chinese patent medicine.

Pituitary Apoplexy in the Setting of Oral Anticoagulation Therapy With Apixaban

Background: Several medical conditions require chronic anticoagulation, but there are potential long-term complications, including intracranial hemorrhages. Pituitary apoplexy (PA), caused by infarction or bleeding into the pituitary gland, is a rare condition that commonly occurs in the setting of a pituitary adenoma. However, several reports have shown an increased risk of PA associated with anticoagulation therapy. Recent case reports have shown that even the newer oral anticoagulants may increase the risk of PA. Typically patients present with an acute headache, visual disturbances and pituitary hormonal deficiencies. Management is controversial and includes either a conservative medical treatment versus a more aggressive surgical approach. We present a case of a PA induced by Apixaban, an orally active factor Xa inhibitor.Clinical Case: This is the case of a 45 years-old male patient with prior medical history of hyperthyroidism treated with radioiodine ablation, systolic heart failure and atrial fibrillation, receiving Apixaban for stroke risk reduction. The patient presented initially to the ER after developing an acute, pulsatile frontal headache, associated with nausea and vomiting. He was treated for a suspected acute gastroenteritis and was discharged home. Two days later the patient returned to the ER, with persistent headaches unresponsive to oral analgesics. This time, the patient also complained of decreased energy, difficulty concentrating, and memory problems. He denied visual changes, galactorrhea, decreased libido or polyuria. Vital signs were unremarkable and there was no orthostatic hypotension. Physical examination showed a male in mild distress due to pain. There were no visual field defects on confrontation, no neurological deficits, and an intact mental status. Brain imaging showed hemorrhage within the pituitary gland with associated edema, compatible with PA. There was no obvious evidence of a pre-existing pituitary adenoma. Laboratory workup did not reveal any hormonal deficiencies. The patient was managed conservatively with close neurological follow up and empiric high dose dexamethasone. Headaches improved significantly after treatment and eventually resolved. After clinical improvement, the patient was discharged home on physiologic replacement of glucocorticoids with outpatient follow up and plans for re-evaluation of hormonal axis.Conclusion: Oral anticoagulants can increase the risk of PA, even in the absence of a pre-existing pituitary adenoma, as other case reports have shown. Management is controversial, and although there are agents for reversal of Apixaban effects (recombinant factor Xa), their use in PA has not been described. This case was managed conservatively with excellent results. Although we cannot exclude a pre-existing pituitary adenoma in this patient, this case shows that Apixaban increases the risk of PA.

The influence of feeding and gastroprotectant medications on the Factor Xa inhibitory activity of orally administered rivaroxaban in normal dogs

Rivaroxaban is a new anticoagulant option for dogs, yet its reported oral bioavailability is as low as 60%. The objective of this study was to examine the influence of feeding and gastroprotectant medications on the bioactivity (anti-Xa activity) of rivaroxaban in healthy dogs. Prospective experimental study. University research laboratory. Five healthy neutered male purpose-bred Beagles. Dogs were administered a median dose of 1.8mg/kg rivaroxaban (range, 1.6-1.8mg/kg) orally once daily for 2 consecutive days with either (1) no food, (2) food, (3) sucralfate 30 minutes before rivaroxaban, or (4) omeprazole at the same time as rivaroxaban. Blood was collected from preplaced jugular catheters immediately before and at 6 time points after rivaroxaban administration (2, 4, 8, 24, 36, and 48 hours). A rivaroxaban calibrated anti-Xa activity assay (RIVA) was used to monitor anticoagulant effect. Rivaroxaban administration resulted in significant increases in RIVA (P=0.02), with peak activities occurring 2 to 4hours after dosingduring each study arm. No feeding was associated with significantly higher RIVA at the 36-hour time point compared to all other treatment arms (P<0.0001), and feeding resulted in high RIVA at the 48-hour time point compared with sucralfate administration (P=0.003). No significant changes in RIVA were otherwise identified with respect to feeding or gastroprotectant administration (P=0.2). Although administration without food demonstrated an apparent increase in RIVA 36hours after drug administration, clinically relevant differences among treatment groups were not identified in combined analyses of time points. Based on these results, dogs treated with rivaroxaban do not require special modification of feeding practices or gastroprotectant drug administration.

Effects of Hypericum perforatum (St John's wort) on the pharmacokinetics and pharmacodynamics of rivaroxaban in humans.

To investigate the influence of a cytochrome P450 CYP3A4 and efflux transporter P-glycoprotein (P-gp) inducing Hypericum perforatum extract on the pharmacokinetics and pharmacodynamics of rivaroxaban. Open-label, nonrandomized, sequential treatment interaction study. Following CYP3A4 and P-gp phenotyping using low-dose midazolam and fexofenadine, 12 healthy volunteers received a single oral dose of 20 mg rivaroxaban and rivaroxaban plasma concentrations and inhibition of the activated coagulation factor X (factor Xa) activity were measured prior to and up to 48 h postdosing. The procedures were repeated after 2 weeks' treatment with the H. perforatum extract. The geometric mean ratios for the area under the concentration-time curve and Cmax of rivaroxaban after/before induction with the H. perforatum extract were 0.76 (90% confidence interval [CI] 0.70, 0.82) and 0.86 (90% CI 0.76, 0.97), respectively. Inhibition of factor Xa activity was reduced with a geometric mean area under the effect-time curve ratio after/before induction of 0.80 (90% CI 0.71, 0.89). No clinically significant differences were found regarding Tmax (median 1.5 vs 1 h, P = .26) and terminal elimination half-life (mean 10.6 vs 10.8 h, P = .93) of rivaroxaban. The H. perforatum extract significantly induced CYP3A4 and P-gp activity, as evidenced by phenotyping. The CYP3A4/P-gp inducing H. perforatum extract caused a decrease of rivaroxaban exposure with a proportional decrease of the pharmacodynamic effect. Although the data do not justify a contraindication for the combination or a systematic adjustment of rivaroxaban dosage, avoidance of the combination or laboratory monitoring should be considered in patients taking hyperforin-containing H. perforatum extracts with rivaroxaban.

Characterization of thrombin/factor Xa inhibitors in Rhizoma Chuanxiong through UPLC-MS-based multivariate statistical analysis

BackgroundThe dry root and rhizome of Ligusticum chuanxiong Hort., or Chuanxiong, has been used as a blood-activating and stasis-removing traditional Chinese medicine for 1000 years. Our previous studies have shown the inhibitory activity on platelet and thrombin (THR) of Chuanxiong. THR and factor Xa (FXa) play significant roles in the coagulation cascade and their inhibitors are of valuable in the treatment of thromboembolic diseases. The aim of the present study is to screen THR and FXa inhibitors from Chuanxiong.MethodsFour extracts [ethyl acetate (EA), butanol (BA) and remained extract (RE) from 75% ethanol extract, and water extract (WE)] of Chuanxiong were prepared, and their THR/FXa inhibitory activities were assessed in vitro. Following silica-gel column chromatography (SC), the active EA extract and BA extract was further partitioned, respectively. Their active fractions (EA-SC1 to EA-SC5; BA-SC1 to BA-SC5) were obtained and analyzed by LC–MS. After modeling by the principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA), the specific marker compounds were predicted and identified. Their enzyme inhibitory was assessed in vitro and interactions with THR/FXa were investigated by molecular docking analysis.ResultsChuanxiong EA extract showed strong activity against THR and BA extract was more effective in inhibiting FXa activity, and their fractions exhibited obvious difference in enzyme inhibitory activity. Furthermore, marker compounds a–h were predicted by PCA and OPLS-DA, and their chemical structures were identified. Among them, senkyunolide A, Z-ligustilide, ferulic acid and senkyunolide I (IC50 was determined as 0.77 mM) with potential THR inhibitory activity, as well as isochlorogenic acid A with FXa inhibitory activity were screened out. It was found that the four components could interact with the active site of THR, and the binding energy was lower than − 5 kcal/mol. Isochlorogenic acid A were bound to the active site of FXa, and the binding energy was − 9.39 kcal/mol. The IC50 was determined as 0.56 mM.ConclusionsTHR/FXa inhibitory components in different extracts of Chuanxiong were successfully characterized by the method of enzyme inhibition activity assays with ultra performance liquid chromatography-quadrupole time of flight mass spectrometry-based multivariate statistical analysis.

Phenanthrenes isolated from diocorea batatas Decne peel with anti-platelet aggregation activity via direct factor Xa inhibitory activity

Abstract Phenanthrene is a class of phenolic metabolites present in Dioscorea batatas which has bioactivities such as antifungal, anti-inflammatory and antioxidative activities. Two phenanthrenes isolated from D. batatas Decne peel, exhibited inhibition against factor Xa and platelet aggregation. They were evaluated for their anti-FXa and anti-platelet aggregation properties by monitoring clotting time, platelet aggregation, FXa activity, and thrombus formation. They exhibited inhibition of the catalysis of FXa and inhibition of adenosine diphosphate- and U46619- induced platelet aggregation. They also inhibited phosphorylation of myristoylated alanine-rich C kinase substrate induced by ADP- and U46619 and the expression of P-selectin, while activating GP IIb/IIIa 1 in platelets. Improved production of nitric oxide and inhibition of endothelin-1 oversecretion were observed. In vivo studies demonstrated the enhanced antithrombotic effects and elicited anticoagulant effects of the compounds. Their anti-thrombotic efficacy were equivalent to rivaroxaban.

Differential Neutralization of Apixaban, Betrixaban, Edoxaban, and Rivaroxaban By Andexanet Alfa As Measured By Whole Blood Thromboelastographic Analysis

Introduction/Background: Recombinant coagulation factor Xa (FXa), inactivated Zh-zo, also known as andexanet alfa (AA), is a modified version of human FXa that has been developed as an antidote to neutralize the bleeding effects of oral FXa inhibitors such as, Apixaban and Rivaroxaban. The relative biological effect of these drugs have been investigated using various clot based and amidolytic methods for FXa inhibition. This Factor Xa inhibitory activity acts as a surrogate marker for the circulating level of these agents. We have recently reported that the FXa activity of these Anti-Xa agents does not fully reflect their biologic spectrum (JCath 25,1-11,2019). Whole blood assays such as thromboelastographic analysis represent a global assay which takes into account both the plasmatic and cellular components of blood and provides a more physiologic endpoint to study the anticoagulant effects of these drugs. The purpose of this study was to investigate the anticoagulant effects of currently available oral Anti-Xa agents such as Apixaban, Betrixaban, Edoxaban, and Rivaroxaban and their relative neutralization by AA in terms of such thromboelastographic parameters as R, K, Angle and MA. Materials and Methods: Analysis was carried out in whole blood using thromboelastography (TEG) using the TEG 5000 Hemostasis System (Haemonetics Corp, Massachusetts). Blood was drawn from healthy donors in individual groups (n=5-10) into 3.2% citrated tubes. In the TEG cup for testing, 0.2 M CaCl2, saline (with a filler and control), each of the individual FXa Inhibitors at a final concentration(FC) of 1 ug/mL, AA at FC of both 100 ug/mL and 50 ug/mL were tested for the relative neutralization of the anticoagulant effects. TEG parameters such as R-time, K-time, angle and MA were measured. All results were compiled individually for the saline control, 100 ug/mL AA and 50 ug/mL AA supplemented systems. Statistical analysis was carried out via an F test for equality of variances followed by the appropriate t test for equal or unequal variances. Results: When comparing the anticoagulants directly to one another, it was observed that Edoxaban shows the strongest anticoagulant effects in both R and K time followed by Betrixaban, then Rivaroxaban which were very similar in their anticoagulative effects, with Apixaban showing the weakest anticoagulant effect as shown in Table 1A. In the reversal studies as shown in Table 1B, as measured by various TEG parameters, R-Time, AA (FC=100 ug/mL) showed full neutralization effects in Apixaban (p=.027), Betrixaban(p=&lt;.01), Edoxaban(p&lt;.01), and Rivaroxaban(p&lt;.01). In K-time, Betrixaban and Edoxaban were fully neutralized (respectfully p=.049 and p=.035) with partial neutralizations of Apixaban and Rivaroxaban. No significant neutralization was noted in the Angle and MA. AA at 50 ug/mL showed full neutralizations, in R-time, in Betrixaban and Rivaroxaban (Betrixaban[p&lt;.01] and Rivaroxaban[p=.0287]), AA at this concentration showed partial neutralizations of Apixaban and Edoxaban. In K-time, AA showed full neutralization of Betrixaban and Edoxaban (Betrixaban[p=&lt;.01] and Edoxaban[p&lt;.01]). Apixaban and Rivaroxaban saw no neutralization effect by AA at FC=50 ug/mL in K-time. AA did not exhibit any significant neutralization effects in the Angle or MA parameters. Summary and Conclusion: All of the 4 Agents produced measurable anticoagulative activities at 1 ug/mL as measured by the TEG parameters. Edoxaban exhibited the strongest anticoagulative effect followed by Betrixaban and Rivaroxaban whereas Apixaban showed much weaker anticoagulant effects. AA FC=100 ug/mL showed much stronger, consistent, and complete neutralization effects of all of the 4 FXa Inhibitors when compared to AA at FC=50 ug/mL. These results strongly suggest that regardless of the variable anticoagulative effect exhibited by the FXa Inhibitors, AA at FC=100ug/mL fully neutralized the anticoagulant effects of this agent as measured by the TEG parameters. AA is shown to be the most effective in neutralizing Betrixaban in R-Time and K-Time at both concentrations of AA. AA was seen to neutralize Apixaban the least. It can be concluded that the effect of AA as a neutralizing agent is both drug and donor dependent and therefore dosage adjustment may be needed for the optimal clinical outcome with this antidote. Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

Edoxaban Exerts Antioxidant Effects Through FXa Inhibition and Direct Radical-Scavenging Activity.

The interplay between oxidative stress, inflammation, and tissue fibrosis leads to the progression of chronic kidney disease (CKD). Edoxaban, an activated blood coagulation factor Xa (FXa) inhibitor, ameliorates kidney disease by suppressing inflammation and tissue fibrosis in animal models. Interestingly, rivaroxaban, another FXa inhibitor, suppresses oxidative stress induced by FXa. Thus, FXa inhibitors could be multitargeted drugs for the three aforementioned risk factors for the progression of CKD. However, the exact mechanism responsible for eliciting the antioxidant effect of FXa inhibitors remains unclear. In this study, the antioxidant effect of edoxaban was evaluated. First, the intracellular antioxidant properties of edoxaban were evaluated using human proximal tubular cells (HK-2 cells). Next, direct radical scavenging activity was measured using the electron spin resonance and fluorescence analysis methods. Results show that edoxaban exhibited antioxidant effects on oxidative stress induced by FXa, indoxyl sulfate, and angiotensin II in HK-2 cells, as well as the FXa inhibitory activity, was involved in part of the antioxidant mechanism. Moreover, edoxaban exerted its antioxidative effect through its structure-specific direct radical scavenging activity. Edoxaban exerts antioxidant effects by inhibiting FXa and through direct radical-scavenging activity, and thus, may serve as multitargeted drugs for the three primary risk factors associated with progression of CKD.

Pharmacokinetic and Pharmacodynamic Drug Monitoring of Direct-Acting Oral Anticoagulants: Where Do We Stand?

For decades, oral anticoagulation has been based on vitamin K antagonist such as warfarin, which requires pharmacodynamic (PD) drug monitoring to guide the therapy. The drug effect is measured by the clotting test prothrombin time and expressed as international normalized ratio. New direct oral anticoagulants are increasingly used in fixed-dose regimens but are licensed without any therapy monitoring. However, extensive clinical experiences have demonstrated that interindividual variations in the response to the therapy with direct oral anticoagulants do exist. In situations such as bleeding or thrombosis, therapeutic drug monitoring could be useful. Unfortunately, global coagulation assays such as the prothrombin time or the activated partial thrombin time are not suitable for this purpose. To measure drug concentrations, more specific coagulation test can be used if they are externally calibrated with the respective drugs. For the direct thrombin inhibitor dabigatran etexilate, a calibrated diluted thrombin time or ecarin clotting time can be used, whereas for anti-factor Xa drugs such as rivaroxaban, apixaban, edoxaban, and betrixaban, calibrated anti-factor Xa assays are appropriate. However, the gold standard to measure drug concentrations is LC-MS/MS. The variation in bleeding and thrombotic events noted with both drug classes under fixed-dose conditions suggests additional interindividual PD differences. Therefore, PD monitoring to individualize the therapy may be an option. For dabigatran, this is the inhibition of thrombin formation and for anti-factor Xa drugs, the inhibition of factor Xa activity, which can be followed using the functional assays mentioned above but without calibration. Alternatively, thrombin generation assays have been proposed for both drug classes. So far, not many clinical data have been published about the potentially beneficial effects of PD monitoring for dose individualization. The assay platforms for PD monitoring are present in many clinical laboratories, but efforts are needed to validate and standardize available assays to perform appropriate clinical trials.

Differential Effects of Dabigatran, Rivaroxaban, Apixaban, Edoxaban and Betrixaban on Fibrinokinetics and their Modulation by FEIBA

ObjectiveNew direct oral anticoagulants have a safety profile that differentiates them from traditionally used oral anticoagulants. DOACs with the similar mechanisms of action and structural similarities are being marketed by numerous companies under varied brands. Such widespread use warrants differentiation of these DOACs on clot formation and their neutralization profile by antidotes. Fibrinokinetics is a method in which thrombin induced clot formation can be measured. The purpose of this study is to compare the effect of Dabigatran, Rivaroxaban, Edoxaban, Betrixaban and Apixaban on thrombin induced fibrinokinetics in normal human plasma. Furthermore, the feasibility of factor eight inhibitor bypass agent (FEIBA) as an antidote to modulate these DOACs is investigated.Materials and MethodsCitrated pool blood plasma was obtained from Loyola blood bank. Purified thrombin was used as a trigger to generate clot at a concentration of 5U/ml. Powdered forms of Dabigatran, Rivaroxaban, and Apixaban were obtained commercially and were used at a working dilution of 1ug/mL. Fibrinokinetics was measured in microtiter plates using 175 microliter of plasma supplemented with the drugs to be tested at a concentration of 25uL of 5U of thrombin followed by 25uL of 0.025M of CaCl2. For the control well plates 25uL of saline was added. To study the FEIBA neutralization, 25uL of FEIBA at a concentration of 0.1U/mL was added in the other set of experimental well plates. The rate of clot formation was monitored for 30 minutes at A45 nm. Both kinetic and endpoint methods were used to calculate the rate of clot formation.ResultsIn the saline control system the time dependent increase in the clot formation was observed for a period of 30 minutes reaching the maximum of 1. Dabigatran, Edoxaban, Rivaroxaban produced a concentration dependent inhibition of clot formation whereas Apixaban and Betrixaban did not produce any significant inhibition of clot formation at concentration up to 1ug/mL. FEIBA supplementation at a final concentration 0.1U/mL produced an increase in clot density in the case of Betrixaban and Apixaban whereas FEIBA reversed the effect of Rivaroxaban, Edoxaban and Dabigatran at a lower concentration up to 0.125U/mL.ConclusionsThree groups can be stipulated from the results of this experiment. The first group includes Dabigatran and has the strongest effect on clot formation and experienced significant inhibition by FEIBA. The second group includes Rivaroxaban and Edoxaban with a strong factor Xa inhibitory activity and moderate neutralization by FEIBA. The third group includes Betrixaban and Apixaban with a weak factor Xa activity and insignificant modulation by FEIBA. The successful modulation by FEIBA on certain DOACs indicate that antidotes for these drugs in the future can stem from procoagulants that are already in use such as FEIBA.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

2‐Aminobenzamide‐Based Factor Xa Inhibitors with Novel Mono‐ and Bi‐Aryls as S4 Binding Elements

AbstractFactor Xa, a key serine protease in the coagulation cascade, has attracted a great deal of attention as a target for developing new antithrombotic agents. A series of novel alkyl, benzyl, biphenyl and substituted piperazine derivatives as S4 binding elements have been synthesized as potential human factor Xa (FXa) inhibitors with 2‐amino‐5‐chloropyridine as the S1 binding moiety. All the synthesized compounds were tested in vitro for FXa inhibitory activity by chromogenic substrate hydrolysis assay. The active compounds were selected to assess their ex vivo prothrombin time prolonging activity. Keeping in mind the promising in vitro and ex vivo profile of N‐(5‐chloropyridin‐2‐yl)‐2‐(4‐(2‐cyanophenyl)benzylamino)benzamide (16 f), it was selected for further in vivo evaluation of its antithrombotic potential. Structure‐activity relationship and molecular interaction analysis by the docking and dynamics studies within the series are discussed.

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.

Synthesis and evaluation of anthranilamide-based derivatives as FXa inhibitors.

Factor Xa (FXa) plays a significant role in the blood coagulation cascade and is a promising target for anticoagulation drugs. Three oral FXa inhibitors have been approved by FDA for treating thrombotic diseases. In this study, 43 novel compounds were synthesized anthranilamide-based FXa inhibitors aiming to ameliorate the toxicity of traditional FXa inhibitors in clinic. The data indicated that the compounds 6a, 6a-b, 6a-e, 6k, 6k-a and 6k-b showed remarkable FXa inhibitory activity and excellent selectivity over thrombin in vitro. Selected compounds also exhibited anticoagulant activities in vitro consequently and were potent novel anti-coagulators in further.

Identification of AcAP5 as a novel factor Xa inhibitor with both direct and allosteric inhibition

Ancylostoma caninum anticoagulant peptide 5 (AcAP5) is a potent inhibitor for coagulation factor Xa (FXa). Previous studies show that AcAP5 binds to FXa at the active site, and/or the exosite. The active site-binding contributes to direct blocking of FXa catalytic activity, but the effect of exosite-binding and the underlying mechanism remain unknown. To investigate whether and how the exosite-binding affects FXa function, we prepared several AcAP5 mutants with modifications to the active site-binding or exosite-binding region. Their FXa-inhibiting and anticoagulant activities were examined both in vitro and in rabbit plasma, and the interactions with FXa were analyzed using in silico molecular modeling, docking, and molecular dynamics simulation. Mutants abolishing either active site- or exosite-binding resulted in a dramatic decrease in their anti-FXa and anticoagulant activities. Elongation of AcAP5 exosite-binding region also impaired the FXa-inhibiting activity. Computational analysis demonstrated that the conformation of FXa becomes more rigid due to exosite-binding with AcAP5, which consequently affects its catalytic activity. Our results suggest that both active site- and exosite-binding contribute to the FXa inhibitory activity of AcAP5.

Managing transitions from oral factor Xa inhibitors to unfractionated heparin infusions.

Published evidence regarding the effects of oral factor Xa inhibitors on anticoagulation monitoring tests is reviewed with a focus on monitoring concerns that can arise during transitions to i.v. heparin therapy. Assays that measure inhibition of factor Xa activity (i.e., anti-Xa assays) are widely used in U.S. institutions to monitor i.v. heparin therapy and, in some cases, for monitoring other types of anticoagulation therapy. Clinicians have raised concerns that the use of anti-Xa assays to monitor heparin levels in hospitalized patients who must be transitioned from oral factor Xa inhibitor therapy to i.v. unfractionated heparin (UFH) infusions could yield unquantifiable or inaccurate results, leading to unnecessary UFH dose reductions and potential treatment failures; the manufacturer labeling of oral factor Xa inhibitors (apixaban, edoxaban, and rivaroxaban) does not provide specific guidance on this issue. Results of a literature review indicated that residual effects of oral factor Xa inhibitor use can result in substantial interference with the currently available chromogenic anti-Xa assays but negligible to moderate effects on global coagulation assays, which measure activated partial thromboplastin time (aPTT) or prothrombin time. Therefore, during the transition from an oral factor Xa inhibitor to i.v. UFH therapy, it may be prudent to consider an aPTT assay for anticoagulation monitoring. The use of oral factor Xa inhibitors appears to affect the accuracy of anti-Xa assay results, with results of global coagulation assays affected to a lesser degree.

Novel Anthranilamide-Based FXa Inhibitors: Drug Design, Synthesis and Biological Evaluation.

Factor Xa (FXa) plays a significant role in the blood coagulation cascade and it has become a promising target for anticoagulation drugs. Three oral direct FXa inhibitors have been approved by the FDA for treating thrombotic diseases. By structure-activity relationship (SAR) analysis upon these FXa inhibitors, a series of novel anthranilamide-based FXa inhibitors were designed and synthesized. According to our study, compounds 1a, 1g and 1s displayed evident FXa inhibitory activity and excellent selectivity over thrombin in in vitro inhibition activities studies. Compounds 1g and 1s also exhibited pronounced anticoagulant activities in in vitro anticoagulant activity studies.

Oxidized antithrombin is a dual inhibitor of coagulation and angiogenesis: Importance of low heparin affinity

Endogenous proteins that promote vascular endothelial cell based inhibition of angiogenesis are an attractive option for antitumor therapy. Inactive cleaved and latent conformations of antithrombin (AT) are antiangiogenic, but not its native form which is an inhibitor of proteases involved in blood coagulation. Unlike native, the cleaved and latent conformations are reactive center loop inserted conformations which binds heparin with very low affinity. We use a sulfoxy modified AT to assess the role of reactive center loop insertion and heparin affinity in antiangiogenic function. Chorioallantoic membrane assay (CAM) shows that antiangiogenic activity of latent and oxidized AT are better than thalidomide, a potent antiangiogenic drug. Wound healing experiments suggest that latent and oxidized conformations can influence endothelial cell migration. Latent and cleaved conformations of AT shows an increase in α-helical content in the presence of unfractionated heparin, but not the oxidized AT. Unlike the loop inserted polymer, cleaved and latent conformations, oxidized AT has factor Xa inhibitory activity indicating that loop insertion is not necessary for antiangiogenic role. The results of our study establish that active conformation of AT can become antiangiogenic while maintaining its anticoagulant activity possibly through chelation of low affinity heparin in the vicinity of endothelial cell.

Design, synthesis and structure–activity relationship of oxazolidinone derivatives containing novel S4 ligand as FXa inhibitors

A novel series of potent and efficacious factor Xa inhibitors which possesses pyrrole/indole/thiazole moieties as S4 binding element was identified. Compound 7b showed strong human factor Xa inhibitory activity (IC50 = 2.01 nM) and anticoagulant activities in both human (PTCT2 = 0.15 μM, APPTCT2 = 0.30 μM) and rabbit plasma (PTCT2 = 0.46 μM, APPTCT2 = 0.75 μM). The SARs analyses indicated that the size and water solubility of different alkylamino group at the position of S4 ligand were responsible for the anticoagulant activity.