Inhibition of Inflammation-Associated Thrombosis with ROS-Responsive Heparin-DOCA/PVAX Nanoparticles.

Inflammation-associated thrombosis is a non-negligible source of mortalities and morbidities worldwide. To manipulate inflammation-associated coagulation, nanoparticles that contain anti-inflammatory polymer (copolyoxalate containing vanillyl alcohol, PVAX) and anti-thrombotic heparin derivative deoxycholic acid (Hep-DOCA) are prepared. The strategy takes advantage of the reducted side effects of heparin through heparin conjugation, achievement of long-term anti-inflammation by inflammation-trigged release of anti-inflammatory agents, and formation of PVAX/heparin-DOCA nanoparticles by co-self-assembly. It is demonstrated that the Hep-DOCA conjugate and PVAX are synthesized successfully; PVAX and Hep-DOCA nanodrugs (HDP) are obtained by co-assembly; the HDP nanoparticles effectively reduce the inflammation and coagulation without inducing lethal bleeding both in vivo and in vitro. The method provided here is versatile and effective, which paves new way to develop nanodrugs to treat inflammation-associated thrombosis safely.

Fluorinated Analog NMR s of Organosulfur Compounds from Garlic (Allium sativum): Synthesis, Chemistry and Anti-Angiogenesis and Antithrombotic Studies.

We describe the synthesis, reactivity, and antithrombotic and anti-angiogenesis activity of difluoroallicin (S-(2-fluoroallyl) 2-fluoroprop-2-ene-1-sulfinothioate) and S-2-fluoro-2-propenyl-l-cysteine, both easily prepared from commercially available 3-chloro-2-fluoroprop-1-ene, as well as the synthesis of 1,2-bis(2-fluoroallyl)disulfane, 5-fluoro-3-(1-fluorovinyl)-3,4-dihydro-1,2-dithiin, trifluoroajoene ((E,Z)-1-(2-fluoro-3-((2-fluoroallyl)sulfinyl)prop-1-en-1-yl)-2-(2-fluoroallyl)disulfane), and a bis(2-fluoroallyl)polysulfane mixture. All tested organosulfur compounds demonstrated effective inhibition of either FGF or VEG-mediated angiogenesis (anti-angiogenesis activity) in the chick chorioallantoic membrane (CAM) or the mouse Matrigel® models. No embryo mortality was observed. Difluoroallicin demonstrated greater inhibition (p < 0.01) versus organosulfur compounds tested. Difluoroallicin demonstrated dose-dependent inhibition of angiogenesis in the mouse Matrigel® model, with maximal inhibition at 0.01 mg/implant. Allicin and difluoroallicin showed an effective antiplatelet effect in suppressing platelet aggregation compared to other organosulfur compounds tested. In platelet/fibrin clotting (anti-coagulant activity), difluoroallicin showed concentration-dependent inhibition of clot strength compared to allicin and the other organosulfur compounds tested.

Introducing multiple bio-functional groups on the poly(ether sulfone) membrane substrate to fabricate an effective antithrombotic bio-interface.

It has been widely recognized that functional groups on biomaterial surfaces play important roles in blood compatibility. To construct an effective antithrombotic bio-interface onto the poly(ether sulfone) (PES) membrane surface, bio-functional groups of sodium carboxylic (-COONa), sodium sulfonic (-SO3Na) and amino (-NH2) groups were introduced onto the PES membrane surface in three steps: the synthesis of PES with carboxylic (-COOH) groups (CPES) and water-soluble PES with sodium sulfonic (-SO3Na) groups and amino (-NH2) groups (SNPES); the introduction of carboxylic groups onto the PES membrane by blending CPES with PES; and the grafting of SNPES onto CPES/PES membranes via the coupling of amino groups and carboxyl groups. The physical/chemical properties and bioactivities were dependent on the proportions of the additives. After introducing bio-functional groups, the excellent hemocompatibility of the modified membranes was confirmed by the inhibited platelet adhesion and activation, prolonged clotting times, suppressed blood-related complement and leukocyte-related complement receptor activations. Furthermore, cell tests indicated that the modified membranes showed better cytocompatibility in endothelial cell proliferation than the pristine PES membrane due to the synergistic promotion of the functional groups. To sum up, these results suggested that modified membranes present great potential in fields using blood-contacting materials, such as hemodialysis and surface endothelialization.

Synthesis and Characterization of Tissue Plasminogen Activator-Functionalized Superparamagnetic Iron Oxide Nanoparticles for Targeted Fibrin Clot Dissolution.

Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted great attention in many biomedical fields and are used in preclinical/experimental drug delivery, hyperthermia and medical imaging. In this study, biocompatible magnetite drug carriers, stabilized by a dextran shell, were developed to carry tissue plasminogen activator (tPA) for targeted thrombolysis under an external magnetic field. Different concentrations of active tPA were immobilized on carboxylated nanoparticles through carbodiimide-mediated amide bond formation. Evidence for successful functionalization of SPIONs with carboxyl groups was shown by Fourier transform infrared spectroscopy. Surface properties after tPA immobilization were altered as demonstrated by dynamic light scattering and ζ potential measurements. The enzyme activity of SPION-bound tPA was determined by digestion of fibrin-containing agarose gels and corresponded to about 74% of free tPA activity. Particles were stored for three weeks before a slight decrease in activity was observed. tPA-loaded SPIONs were navigated into thrombus-mimicking gels by external magnets, proving effective drug targeting without losing the protein. Furthermore, all synthesized types of nanoparticles were well tolerated in cell culture experiments with human umbilical vein endothelial cells, indicating their potential utility for future therapeutic applications in thromboembolic diseases.

Synthesis, antiplatelet and antithrombotic activities of resveratrol derivatives with NO-donor properties.

Resveratrol (RVT) is a stilbene with a protective effect on the cardiovascular system; however, drawbacks including low bioavailability and fast metabolism limit its efficacy. In this work we described new resveratrol derivatives with nitric oxide (NO) release properties, ability to inhibit platelet aggregation and in vivo antithrombotic effect. Compounds (4a-f) were able to release NO in vitro, at levels ranging from 24.1% to 27.4%. All compounds (2a-f and 4a-f) have exhibited platelet aggregation inhibition using as agonists ADP, collagen and arachidonic acid. The most active compound (4f) showed reduced bleeding time compared to acetylsalicylic acid (ASA) and protected up to 80% against in vivo thromboembolic events. These findings suggest that hybrid resveratrol-furoxan (4f) is a novel lead compound able to prevent platelet aggregation and thromboembolic events.

Identification of a New Morpholine Scaffold as a P2Y12 Receptor Antagonist.

The P2Y12 receptor is critical for platelet activation and is an attractive drug target for the prevention of atherothrombotic events. Despite the proven antithrombotic efficacy of P2Y12 inhibitors, these thienopyridine scaffolds are prodrugs that lack important features of the ideal antithrombotic agent. For this reason, ticagrelor-a new chemical class of P2Y12 receptor antagonist-was developed, but it can cause shortness of breath and various types of bleeding. Moreover, ticagrelor is a cytochrome P450 3A4 substrate/inhibitor and, therefore, caution should be exercised when it is used concomitantly with strong CYP3A4 inducers/inhibitors. There is a need for novel P2Y12 receptor antagonist scaffolds that are reversible and have high efficacy without associated side effects. Here, we describe a novel antagonist containing a morpholine moiety that was identified by screening libraries of commercially available compounds. The molecule, Compound E, acted on P2Y12, but not P2Y1 and P2Y13, and exhibited pharmacological characteristics that were distinct from those of ticagrelor, acting instead on P2Y12 via an allosteric mechanism. These results provide a basis for the development/optimization of a new class of P2Y12 antagonists.

Efficient Double Suzuki Cross-Coupling Reactions of 2,5-Dibromo-3-hexylthiophene: Anti-Tumor, Haemolytic, Anti-Thrombolytic and Biofilm Inhibition Studies.

The present study describes several novel 2,5-biaryl-3-hexylthiophene derivatives (3a-i) synthesized via a Pd(0)-catalyzed Suzuki cross-coupling reaction in moderate to good yields. The novel compounds were also analyzed for their anti-thrombolytic, haemolytic, and biofilm inhibition activities. In addition, the anti-tumor activity was also evaluated in vitro for newly-synthesized compounds, where 3-hexyl-2,5-bis(4-(methylthio)phenyl)thiophene exhibited the best anti-tumor activity against 4T1 cells with IC50 value of 16 µM. Moreover, 2,5-bis(4-methylphenyl)-3-hexylthiophene showed the highest activity against MCF-7 cells with an IC50 value of 26.2 µM. On the other hand, the compound 2,5-bis(4-chloropheny)-3-hexylthiophene exhibited excellent biofilm inhibition activity. Furthermore, the compound 2,5-bis(3-chloro-4-fluorophenyl)-3-hexylthiophene also exhibited better anti-thrombolytic and hemolytic activity results as compared to the other newly-synthesized compounds.

Synthesis and biological evaluation of methylated scutellarein analogs based on metabolic mechanism of scutellarin in vivo.

Scutellarin (1) could be hydrolyzed into scutellarein (2) in vivo and then converted into methylated, sulfated and glucuronidated forms. In order to investigate the biological activities of these methylated metabolites, eight methylated analogs of scutellarein (2) were synthesized via semi-synthetic methods. The antithrombotic activities of these compounds were evaluated through the analyzation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB). Their antioxidant activities were assessed by measuring their scavenging capacities toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and the ability to protect PC12 cells against H2O2-induced cytotoxicity. Furthermore, the physicochemical properties of these compounds including aqueous solubility and lipophilicity were also investigated. The results showed that 6-O-methylscutellarein (5) demonstrated potent antithrombotic activity, stronger antioxidant activity and balanced solubility and permeability compared with scutellarin (1), which warrants further development of 5 as a promising lead for the treatment of ischemic cerebrovascular disease.

Anticoagulant and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor Xase.

Fucosylated chondroitin sulfate (FCS), a structurally unusual glycosaminoglycan, has distinct anticoagulant properties, and is an especially strong inhibitor of the intrinsic factor Xase (anti-Xase). To obtain a highly selective inhibitor of human Xase, we purified six native FCSs with various sulfation patterns, prepared a series of FCS derivatives, and then elucidated the relationship between the structures and the anticoagulant activities of FCSs. FCSs 1-3 containing higher Fuc2S4S exhibit stronger AT-dependent anti-IIa activities, whereas 4-6 containing more Fuc3S4S produce potent HCII-dependent anti-IIa activities. Saccharides containing a minimum of 6-8 trisaccharide units, free carboxyl groups, and full fucosylation of GlcA may be required for potent anti-Xase activity, and approximately six trisaccharide units and partial fucosylation of GlcA may contribute to potent HCII-dependent activity. Decreasing of the molecular weights markedly reduces their AT-dependent anti-IIa activities, and even eliminates human platelet and factor XII activation. Furthermore, in vitro and in vivo studies suggested that fractions of 6-12 kDa may be very promising compounds as putative selective intrinsic Xase inhibitors with antithrombotic action, but without the consequences of major bleeding and factor XII activation.

Nonthrombogenic, biodegradable elastomeric polyurethanes with variable sulfobetaine content.

For applications where degradable polymers are likely to have extended blood contact, it is often important for these materials to exhibit high levels of thromboresistance. This can be achieved with surface modification approaches, but such modifications may be transient with degradation. Alternatively, polymer design can be altered such that the bulk polymer is thromboresistant and this is maintained with degradation. Toward this end a series of biodegradable, elastic polyurethanes (PESBUUs) containing different zwitterionic sulfobetaine (SB) content were synthesized from a polycaprolactone-diol (PCL-diol):SB-diol mixture (100:0, 75:25, 50:50, 25:75 and 0:100) reacted with diisocyanatobutane and chain extended with putrescine. The chemical structure, tensile mechanical properties, thermal properties, hydrophilicity, biodegradability, fibrinogen adsorption and thrombogenicity of the resulting polymers was characterized. With increased SB content some weakening in tensile properties occurred in wet conditions and enzymatic degradation also decreased. However, at higher zwitterionic molar ratios (50% and 75%) wet tensile strength exceeded 15 MPa and breaking strain was >500%. Markedly reduced thrombotic deposition was observed both before and after substantial degradation for both of these PESBUUs and they could be processed by electrospinning into a vascular conduit format with appropriate compliance properties. The mechanical and degradation properties as well as the acute in vitro thrombogenicity assessment suggest that these tunable polyurethanes could provide options appropriate for use in blood contacting applications where a degradable, elastomeric component with enduring thromboresistance is desired.

Design, synthesis and biological evaluation of hydrogen sulfide releasing derivatives of 3-n-butylphthalide as potential antiplatelet and antithrombotic agents.

In the present study, a series of hydrogen sulfide (H2S) releasing derivatives (8a­g and 9a­f) of 3-n-butylphthalide (NBP) were designed, synthesized and biologically evaluated. The most promising compound 8e significantly inhibited the adenosine diphosphate (ADP) and arachidonic acid (AA)-induced platelet aggregation in vitro, superior to NBP, ticlopidine hydrochloride and aspirin. Furthermore, 8e could slowly produce moderate levels of H2S in vitro, which could be beneficial for improving cardiovascular and cerebral circulation. Most importantly, 8e protected against the collagen and adrenaline induced thrombosis in mice, and exhibited greater antithrombotic activity than NBP and aspirin in rats. Overall, 8e could warrant further investigation for the treatment of thrombosis-related ischemic stroke.

[1, 2, 4]-oxadiazoles: synthesis and biological applications.

In the present article synthesis and medicinal applications of [1, 2, 4] oxadiazoles are reviewed. The oxadiazoles have a wide range of applications such as Antitussive, Anti-inflammatory, Anaesthetic, Vasodilator, anthelmintic, antiallergic, antiplatelet effects in vitro, antithrombotic properties in vivo, etc. Many researchers have synthesized novel heterocyclic compounds containing oxadiazoles with the concept of bioisosterism.

Discovery of Mer specific tyrosine kinase inhibitors for the treatment and prevention of thrombosis.

The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined. Subsequent SAR studies identified compound 23 (UNC2881) as a lead compound for in vivo evaluation. When applied to live cells, 23 inhibits steady-state Mer kinase phosphorylation with an IC50 value of 22 nM. Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric receptor containing the intracellular domain of Mer fused to the extracellular domain of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation, suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.

Discovery of diarylurea P2Y(1) antagonists with improved aqueous solubility.

Preclinical data suggests that P2Y1 antagonists, such as diarylurea compound 1, may provide antithrombotic efficacy similar to P2Y12 antagonists and may have the potential of providing reduced bleeding liabilities. This manuscript describes a series of diarylureas bearing solublizing amine side chains as potent P2Y1 antagonists. Among them, compounds 2l and 3h had improved aqueous solubility and maintained antiplatelet activity compared with compound 1. Compound 2l was moderately efficacious in both rat and rabbit thrombosis models and had a moderate prolongation of bleeding time in rats similar to that of compound 1.

Discovery of 2-(phenoxypyridine)-3-phenylureas as small molecule P2Y1 antagonists.

Two distinct G protein-coupled purinergic receptors, P2Y1 and P2Y12, mediate ADP-driven platelet activation. The clinical effectiveness of P2Y12 blockade is well established. Recent preclinical data suggest that P2Y1 and P2Y12 inhibition provide equivalent antithrombotic efficacy, while targeting P2Y1 has the potential for reduced bleeding liability. In this account, the discovery of a 2-(phenoxypyridine)-3-phenylurea chemotype that inhibited ADP-mediated platelet aggregation in human blood samples is described. Optimization of this series led to the identification of compound 16, 1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-4-(trifluoromethoxy)phenylurea, which demonstrated a 68 ± 7% thrombus weight reduction in an established rat arterial thrombosis model (10 mg/kg plus 10 mg/kg/h) while only prolonging cuticle and mesenteric bleeding times by 3.3- and 3.1-fold, respectively, in provoked rat bleeding time models. These results suggest that a P2Y1 antagonist could potentially provide a safe and efficacious antithrombotic profile.

Discovery of a potential anti-ischemic stroke agent: 3-pentylbenzo[c]thiophen-1(3H)-one.

The development of novel antithrombotic agents with strong free radical scavenging activity is of great significance for the treatment of ischemic stroke. In the present study, 3-alkyl/arylalkyl-substituted benzo[c]thiophen-1(3H)-ones (5a-h) were designed and synthesized. The most active compound 5d significantly inhibited the adenosine diphosphate (ADP) induced and arachidonic acid (AA) induced in vitro platelet aggregation, superior to clinically used antiplatelet drug aspirin (ASP) and anti-ischemic stroke drugs 3-n-butylphthalide (NBP) and edaravone (Eda). More importantly, in comparison with both NBP and Eda, 5d exhibited stronger antithrombotic and free radical scavenging activities and better or comparable neuroprotective effects against ischemia/reperfusion (I/R) in rats by ameliorating neurobehavioral function, reducing infarct size and brain-water content, attenuating cerebral damage, and normalizing the levels of oxidative enzymes. Overall, our findings may provide an alternative strategy for the design of novel anti-ischemic stroke agents more potent than drugs like NBP and Eda.

Synthesis and evaluation of anti-thrombotic activity of benzocoumarin amide derivatives.

A series of novel benzocoumarin amide derivatives have been synthesized and evaluated for their anti-thrombotic activity. Amongst these, compounds 5, 7 and 8 exhibited promising anti-thrombotic profile in an established model of mouse thrombosis. Hence, comprehensive profiling on platelet aggregation and coagulation parameters was carried out to assess its potential as a lead candidate. In vitro treatment of these compounds in mice plasma resulted into significant reduction in ADP (p<0.01) and collagen (p<0.001) induced platelet aggregation. Moreover, Compounds 5, 7 and 8 also significantly increased thrombin time (p<0.05). Thus, in the present study, these benzocoumarin amide derivatives exhibited anti-thrombotic profile via both anti-platelet as well as anti-coagulant action.

The novel platelet activation receptor CLEC-2.

The c-type lectin-like receptor 2 (CLEC-2) was first identified from a bio-informatic screen for c-type lectin-like receptors. However, neither its function nor its ligand(s) had been elucidated for several years. In 2006, we reported that the receptor is expressed on the surface of platelets and serves as a receptor for the snake venom rhodocytin, which potently stimulates platelet aggregation. Since then CLEC-2 has been intensively investigated, and its endogenous/exogenous ligands and several physiological/pathological roles have been clarified. In this article and its accompanying poster, we outline the structure, distribution, signal transduction mechanism and functions of CLEC-2.

Design, synthesis and evaluation of nitric oxide releasing derivatives of 3-n-butylphthalide as antiplatelet and antithrombotic agents.

Novel nitric oxide (NO) releasing derivatives (7a-7l) of 3-n-butylphthalide (NBP) were designed and synthesized. Compound 7e inhibited the adenosine diphosphate (ADP), thrombin (TH) and arachidonic acid (AA)-induced in vitro platelet aggregation, superior to NBP and aspirin, released moderate levels of NO, and improved aqueous solubility relative to NBP. Furthermore, 7e exhibited greater antithrombotic activity than NBP and aspirin in rats, and protected against collagen and adrenaline-induced thrombosis in mice. Therefore, NO-releasing NBP derivatives possessed potent antiplatelet aggregation and antithrombotic activity. Our findings may aid in the design of new therapeutic agents for the treatment of thrombosis-related ischemic stroke.

The discovery and development of rivaroxaban.

Thromboembolic conditions present a considerable challenge to healthcare services because they are associated with substantial morbidity and mortality. The mainstays of prevention and treatment are anticoagulants and antiplatelet agents. Established anticoagulants have drawbacks that make their use difficult to manage and sustain. This has stimulated the search for new oral anticoagulants that are more convenient and yet still effective. This paper describes the development and future potential of rivaroxaban (Xarelto; Bayer Schering Pharma AG, Berlin, Germany)-the first oral, direct Factor Xa inhibitor to be approved for clinical use in the prevention of venous thromboembolism in adult patients undergoing elective hip or knee replacement surgery.