Development of New Peptidomimetic NADPH Oxidase Inhibitors with Antithrombotic Properties.

The ability of synthetic peptides inhibitors of NOX1 to effectively block the production of ROS by the enzyme was studied with different methodologies. Specifically, taking advantage of our understanding of the active epitope of the regulatory NOX1 subunit NOXA1 as a potent inhibitor of NOX1-derived O2⋅- formation, a panel of peptidomimetic derivatives of this peptide were designed and synthesized with the aim of improving their activity and increasing their stability in plasma. The results highlighted that improved efficacy and potency was found for both the peptide-peptoid hybrid GS2, whereas stapled peptide AC5 and its precursor showed higher stability despite lower biological potency. This study showed that minimal structural modifications of NOXA1 peptides are required to improve both their potency and stability to finally achieve best candidates as new potential anti-thrombotic drugs.

Discovery of Milvexian, a High-Affinity, Orally Bioavailable Inhibitor of Factor XIa in Clinical Studies for Antithrombotic Therapy.

Factor XIa (FXIa) is an enzyme in the coagulation cascade thought to amplify thrombin generation but has a limited role in hemostasis. From preclinical models and human genetics, an inhibitor of FXIa has the potential to be an antithrombotic agent with superior efficacy and safety. Reversible and irreversible inhibitors of FXIa have demonstrated excellent antithrombotic efficacy without increased bleeding time in animal models (Weitz, J. I., Chan, N. C. Arterioscler. Thromb. Vasc. Biol. 2019, 39 (1), 7-12). Herein, we report the discovery of a novel series of macrocyclic FXIa inhibitors containing a pyrazole P2' moiety. Optimization of the series for (pharmacokinetic) PK properties, free fraction, and solubility resulted in the identification of milvexian (BMS-986177/JNJ-70033093, 17, FXIa Ki = 0.11 nM) as a clinical candidate for the prevention and treatment of thromboembolic disorders, suitable for oral administration.

Facile green approach towards the synthesis of some phenyl piperazine based dithiocarbamates as potent hemolytic and thrombolytic agents.

The facile and efficient protocol for the synthesis of N-phenyl piperazine based di-thio-carbamates has been reported under neat conditions. A library of novel piperazine based di-thio-carbamates (3a-h) in excellent yields has been prepared. Solvent free, catalyst free and easy work up conditions make this protocol an attractive synthetic protocol to achieve novel biologically active di-thio-carbamates. The synthesized molecules have been characterized by FT-IR, 1HNMR and 13CNMR spectroscopic techniques. The pharmacological aspects of these derivatives have been evaluated via hemolysis and thrombolysis. All the target molecules (3a-h) exhibit mild to medium potential as hemolytic and thrombolytic agents. Among the synthesized derivatives, compound 3c showed least cytotoxicity and better thrombolytic potential.

Synthesis and Bioactivities of Marine Pyran-Isoindolone Derivatives as Potential Antithrombotic Agents.

2,5-Bis-[8-(4,8-dimethyl-nona-3,7-dienyl)-5,7-dihydroxy-8-methyl-3-keto-1,2,7,8-teraahydro-6H-pyran[a]isoindol-2-yl]-pentanoic acid (FGFC1) is a marine pyran-isoindolone derivative isolated from a rare marine microorganism Stachybotrys longispora FG216, which showed moderate antithrombotic(fibrinolytic) activity. To further enhance its antithrombotic effect, a series of new FGFC1 derivatives (F1-F7) were synthesized via chemical modification at C-2 and C-2' phenol groups moieties and C-1″ carboxyl group. Their fibrinolytic activities in vitro were evaluated. Among the derivatives, F1-F4 and F6 showed significant fibrinolytic activities with EC50 of 59.7, 87.1, 66.6, 82.8, and 42.3 µM, respectively, via enhancement of urokinase activity. Notably, derivative F6 presented the most remarkable fibrinolytic activity (2.72-fold than that of FGFC1). Furthermore, the cytotoxicity of derivative F6 was tested as well as expression of Fas/Apo-1 and IL-1 on HeLa cells. The results showed that, compared to FGFC1, derivative F6 possessed moderate cytotoxicity and apoptotic effect on HeLa cells (statistical significance p > 0.1), making F6 a potential antithrombotic agent towards clinical application.

Phosphinanes and Azaphosphinanes as Potent and Selective Inhibitors of Activated Thrombin-Activatable Fibrinolysis Inhibitor (TAFIa).

Selective and potent inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) have the potential to increase endogenous and therapeutic fibrinolysis and to behave like profibrinolytic agents without the risk of major hemorrhage, since they do not interfere either with platelet activation or with coagulation during blood hemostasis. Therefore, TAFIa inhibitors could be used in at-risk patients for the treatment, prevention, and secondary prevention of stroke, venous thrombosis, and pulmonary embolisms. In this paper, we describe the design, the structure-activity relationship (SAR), and the synthesis of novel, potent, and selective phosphinanes and azaphosphinanes as TAFIa inhibitors. Several highly active azaphosphinanes display attractive properties suitable for further in vivo efficacy studies in thrombosis models.

Discovery of novel (6S/12aS)-heptachpyridone capable of inhibiting thrombosis in vivo.

The in vitro conversion of (1S,3S)-1-dimethoxylethyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid, (1S,3S)-DCCA, in rat plasma is monitored by HPLC-FT-ICR-MS. We show that the in vitro conversion of (1S,3S)-DCCA in rat plasma for 1 h leads to forming (6S/12aS)-bisdimethoxyethylheptachpyridone, reflecting intermolecular condensation of (1S,3S)-DCCA, and the in vitro conversion of (6S/12aS)-bisdimethoxyethylheptachpyridone in rat plasma for 1 h leads to forming (6S/12aS)-heptachpyridone, reflecting hydrolysis of (6S/12aS)-bisdimethoxyethylheptachpyridone. At a dose of 1.0 µmol/kg (6S/12aS)-heptachpyridone orally inhibits venous thrombosis and arterial thrombosis in vivo. Bleeding time, clotting time and international normalized ratio show that at this dose (6S/12aS)-heptachpyridone has no bleeding risk, does not lengthen clotting time and does not change the exogenous coagulation pathway. We also show that the reactions promoted by rat plasma are easy to practice by chemical synthesis. Thus our findings build a bridge across the in vivo conversion and the application.

(Semi)-Synthetic Fucosylated Chondroitin Sulfate Oligo- and Polysaccharides.

Fucosylated chondroitin sulfate (fCS) is a glycosaminoglycan (GAG) polysaccharide with a unique structure, displaying a backbone composed of alternating N-acetyl-d-galactosamine (GalNAc) and d-glucuronic acid (GlcA) units on which l-fucose (Fuc) branches are installed. fCS shows several potential biomedical applications, with the anticoagulant activity standing as the most promising and widely investigated one. Natural fCS polysaccharides extracted from marine organisms (Echinoidea, Holothuroidea) present some advantages over a largely employed antithrombotic drug such as heparin, but some adverse effects as well as a frequently found structural heterogeneity hamper its development as a new drug. To circumvent these drawbacks, several efforts have been made in the last decade to obtain synthetic and semi-synthetic fCS oligosaccharides and low molecular weight polysaccharides. In this Review we have for the first time collected these reports together, dividing them in two topics: (i) total syntheses of fCS oligosaccharides and (ii) semi-synthetic approaches to fCS oligosaccharides and low molecular weight polysaccharides as well as glycoclusters displaying multiple copies of fCS species.

(E)-N'-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide protecting rat heart tissues from isoproterenol toxicity: Evidence from in vitro and in vivo tests.

The current study was aimed to assess the protective effect of a new molecule (E)-N'-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide, denoted 1c, against cardiac remodeling process in isoproterenol (Isop) induced myocardial infarction (MI) in rats. Male Wistar rats were randomly divided into four groups, control, Isop (85 mg/kg body weight was injected subcutaneously into rats at an interval of 24 h for 2 days (6th and 7th day) to induce MI and pretreated animals with acenocoumarol (Ace) (150 µg/kg bw) and 1c (150 µg/kg bw) by oral administration during 7 days and injected with isoproterenol (Isop + Ace) and (Isop + 1c) groups. Results in vitro showed that 1c is endowed with potent inhibition of angiotensin-converting enzyme (ACE) with an IC50 39.12 µg/ml. The in vivo exploration evidenced alteration in the ECG pattern, notable cardiac hypertrophy and increase in plasma level of fibrinogen, troponin-T, CK-MB and LDH, AST and ALT by 171%, 300%, 50%, 64% and 75% respectively with histological myocardium necrosis and cells inflammatory infiltration. However, pre-treatment with 1c improved the ECG pattern reduced significantly the cardiac dysfunction markers and ameliorated the thrombolytic process by decreasing fibrinogen level as compared to untreated infracted rats. Overall, (E)-N'-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide 1c could be used as anticoagulant agent to prevent thrombosis in acute myocardial infarction.

Potent, Orally Bioavailable, and Efficacious Macrocyclic Inhibitors of Factor XIa. Discovery of Pyridine-Based Macrocycles Possessing Phenylazole Carboxamide P1 Groups.

Factor XIa (FXIa) inhibitors are promising novel anticoagulants, which show excellent efficacy in preclinical thrombosis models with minimal effects on hemostasis. The discovery of potent and selective FXIa inhibitors which are also orally bioavailable has been a challenge. Here, we describe optimization of the imidazole-based macrocyclic series and our initial progress toward meeting this challenge. A two-pronged strategy, which focused on replacement of the imidazole scaffold and the design of new P1 groups, led to the discovery of potent, orally bioavailable pyridine-based macrocyclic FXIa inhibitors. Moreover, pyridine-based macrocycle 19, possessing the phenylimidazole carboxamide P1, exhibited excellent selectivity against relevant blood coagulation enzymes and displayed antithrombotic efficacy in a rabbit thrombosis model.

Synthesis, in vitro and in silico studies of HO-1 inducers and lung antifibrotic agents.

Aim: Dimethyl fumarate (DMF) analogs were synthesized to obtain inducers of HO-1 and antifibrotic agents. Methods: HO-1 expression levels were measured on lung fibroblasts (MRC5). NMR and docking studies were performed. Heme oxygenase activity, gene levels and protein expression have been measured for the most active compound 1a. Collagen production by fibroblast after exposure to TGF-ß was measured. Results: Compound 1a showed to be a strong HO-1 inducer. Its activity seems to be mediated by activation of nuclear factor erythroid 2 related factor 2 (Nrf2). TGF-ß-induced collagen production was significantly decreased on MRC5, pretreated with DMF or 1a. DMF and 1a have a high potential for treatment of lung fibrotic injuries.

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.

Antithrombotic Activity of a Novel Diazepino[1,2-α] Benzimidazole Derivative on Arterial Thrombosis Model in Rats without Concomitant Pathology and in Rats with Experimental Myocardial Infarction.

Antithrombotic activity of a novel tricyclic derivative of diazepino[1,2-α]benzimidazole (DAB-15) was examined on the model of arterial thrombosis developed in rats without concomitant pathology and in rats with experimental myocardial infarction. DAB-15 demonstrated high antithrombotic efficacy in modeled thrombosis of carotid artery in rats without the concomitant pathology surpassing that of the reference drugs acetylsalicylic acid and clopidogrel by 5.1 and 4.8 times, respectively. In rats with experimental noncoronary myocardial infarction, DAB-15 increased the thrombus formation time by 86.2% in comparison with experimental control level in non-treated rats with similar myocardial infarction.

Targeted thrombolysis by using c-RGD-modified N,N,N-Trimethyl Chitosan nanoparticles loaded with lumbrokinase.

Lumbrokinase (LK) has strong fibrinolytic and thrombolytic activities, but it has a short half-life, can be easily inactivated, and may cause hemorrhage as a side effect. This study develops a potential thrombolytic therapy by fabricating N,N,N-Trimethyl Chitosan (TMC) nanoparticles modified with the cyclic Arg-Gly-Asp-Phe-Lys peptide (c-RGD) and loaded with LK (i.e. c-RGD-LK-NPs). The binding of c-RGD to platelet membrane GPIIb/IIIa receptors is expected to enable targeted delivery of the c-RGD-conjugated TMC to the thrombus. The synthesized c-RGD-LK-NPs had a mean particle size of 232.0 nm, zeta potential of 19.8 mV, entrapment efficiency of 52.7% ± 2.5%, and loading efficiency of 17.4% ± 0.65%. Transmission electron microscopy showed that they were generally spherical. The c-RGD-LK-NPs gave a cumulative in vitro LK release of 80.6% over 8 h, and the activity of LK was close to 80%, indicating that the nanoparticles protected the activity of LK. In vitro blood clot lysis assays were carried out and in vivo thrombolysis effect was tested in Sprague-Dawley rats carotid artery thrombus model. In all cases, the c-RGD-LK-NPs showed superior performance compared with the free LK and the unmodified TMC nanoparticles loaded with LK. The c-RGD-LK-NPs reagent is expected to be potentially useful in treating thromboembolic diseases.

Role of Bedside Preparation in Reducing "Door-to-Needle" Tissue-Type Plasminogen Activator (Alteplase) Administration Times and Association with Patient Outcomes.

INTRODUCTION: The purpose of this study is to assess the benefit of bedside alteplase preparation as a component of the acute stroke process. METHODS: A retrospective, single center study, designed to evaluate the impact of a bedside alteplase preparation protocol. Stroke patients receiving intravenous (IV) alteplase prepared at bedside were compared to pre-bedside alteplase preparation patients. The primary outcome was to compare door-to-needle (DTN) times between the groups. The secondary outcomes included comparison of pre-bedside alteplase preparation to post-bedside alteplase preparation on the following variables: imaging-to-drug times, order entry to drug administration times, percentage of patients achieving the 60 minute DTN goal, rate of intracranial hemorrhage (ICH), and patient discharge disposition. RESULTS: Patients in the pre-bedside preparation group included those who received IV alteplase between Jan. 1, 2012 and Jan. 31, 2015 and post-bedside preparation patients between Feb. 1, 2015 and March 31, 2016. Thirty-one patients were enrolled in the study, 16 in the pre-bedside preparation group and 15 in the post-beside preparation group. The mean DTN time in the post-bedside alteplase preparation group was significantly reduced, as compared to the pre-bedside preparation group (66.6 minutes vs. 95.9 minutes, p=0.024). Percent of patients meeting the 60 minute DTN time goal was significantly improved when alteplase was prepared at bedside (53.3 percent vs. 18.8 percent) (p=0.044). Rates of ICH were not significantly different between the two populations. CONCLUSIONS: Bedside alteplase preparation significantly reduced DTN times in an academic hospital emergency department.

A biological, fluorescence and computational examination of synthetic coumarin derivatives with antithrombotic potential.

BACKGROUND: Scientists still look for new drugs, which have anticoagulant properties. This is so important because existing anticoagulant drugs give many side effects, for example major bleeding. In this study we examined nine coumarin derivatives - candidates to be future antithrombotic drugs, which were synthetized and crystallized in our previous paper. METHODS: Here we show the fluorescence and fluorescence quenching of coumarin derivatives with di- or trimethoxybenzylamine moieties in C-3 position. All nine compounds were checked by lactate dehydrogenase assay to examine their cytotoxic activity on hepatic cells. We also investigated the other biological properties (bioactivity, drug-likeness and blind docking) using computational tools. Lipophilicity coefficient logP of all obtained compounds was determined using by RP-TLC and compared to theoretical predictions. RESULTS: The obtained coumarins exhibited low lipophilic character. The substances bound with HSA and did not demonstrate cytotoxicity against isolated liver cells. The most interesting compound (3b) possessed two methoxy- group in 2- and 4-position in benzene ring, ability to interact with two HSA binding sites and probably smaller steric hindrance in comparison to other synthesized derivatives. CONCLUSIONS: Our present study shows that after examination of fluorescence, cytotoxic activity, lipophilicity, theoretical bioactivity, drug-likeness and blind docking of our synthesized compounds they have potential as antithrombotic medicines and may be candidates to be drugs after further studies.

The preparation of heparin-like hyperbranched polyimides and their antithrombogenic, antibacterial applications.

1,3,5-Tris(4-aminophenoxy) benzene (TAPOB) and 2,2-bis [4-(3,4-dicarboxyphenoxy) phenyl] propane dianhydride (BPADA) were used to synthesize an amino-terminated hyperbranched polyimide (AM-HBPI). Then, the 2-methacryloyloxyethyl phosphorylcholine-modified hyperbranched polyimide (HBPI-MPC) was obtained through the graft modification of MPC onto AM-HBPI by Michael addition. The infrared spectroscopy and X-ray photoelectron spectroscopy spectra showed MPC molecules were successfully grafted onto the HBPI molecules. The HBPI-MPC films exhibited slightly decreased thermal stabilities with 5% weight loss temperature in the range of of 418-483 °C in nitrogen, compared with the pure HBPI film. With the increase of MPC grafting amount, the static water contact angles decreased from average 84.0° of the pure HBPI film to average 45.0° of the HBPI-MPC film with 20% MPC. Meanwhile, the increased surface roughness of the HBPI-MPC films increased the contact areas with the platelets, enhancing their anticoagulant efficiency. The number of platelet adhesion declined and the shape of platelet changed from flat to round. The recalcification times grew from average 300 s of pure HBPI to average 551 s of the HBPI-MPC film with 20% MPC, indicating improved anticoagulant properties and biocompatibility. Bacterial adhesion test also demonstrated the number of bacterial adhesion was significantly reduced and antibacterial properties were improved. Thus, the HBPI-MPC films have great application prospects as biomedical anticoagulant materials.

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.

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.

Developments in the synthesis of the antiplatelet and antithrombotic drug (S)-clopidogrel.

S-(+)-Methyl 2-(2-chlorophenyl)-2-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate, also known as (S)-clopidogrel, is marketed under the trade names Plavix and Iscover. It is a potent thienopyridine-class of antithrombotic and antiplatelet drug (antiaggregant). Among the two available stereoisomers of clopidogrel, for pharmaceutical activities only the S-enantiomer is applicable, as no antithrombotic activity is observed in the R-enantiomer and causes political upheavals and social turmoil in animal experiments. Worldwide sales of Plavix amounted to $6.4 billion yearly, which ranks second. Attributed to the increased demand of (S)-clopidogrel drug, it provoked the synthetic community to devise facile synthetic approaches. This review aims to summarize the synthetic methods of (S)-clopidogrel drug reported in the literature. The present review discusses the pros and cons of each synthetic methodology, which would be beneficial to the scientific community for further developments in the synthetic methodologies for (S)-clopidogrel. In addition, the compilation approach of literature-reported synthetic strategies of (S)-clopidogrel in one platform is advantageous, supportive, and crucial for the synthetic community to elect the best synthetic methodology of (S)-clopidogrel and to create new synthesis ideas.

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.