Synthesis of new class dipeptide analogues with improved permeability and antithrombotic activity

3-(S)-1,2,3,4-Tetrahydro-beta-carboline-3-carboxylic acid isolated from A. Chinese G. Don was found to possess moderate anti-aggregation activity, but with poor bioavailability. To improve its pharmacological property, we designed and synthesized a series of novel dipeptide analogues by incorporating tetrahydro-beta-carboline-3-carboxylic acid skeleton as an amino acid surrogate (*Trp). It turned out these dipeptide analogues exhibited good membrane permeability based on in vitro Caco-2 cell monolayers permeability assay. As a result, the overall biological properties of these molecules were significantly improved depending on the nature of the amino acid residues introduced onto the 3-position of the tetrahydro-beta-carboline moiety. It was very interesting to notice that these dipeptide analogues (5b,c,h,i,n,o,p,q) displayed a remarkable dual antiaggregatory activity in both of ADP- and PAF-induced platelet aggregation assay, and their aggregation response was significantly higher than that of aspirin (p<0.01). In addition, these dipeptide analogues were observed for the dose-dependent antithrombotic effect using in vivo rat arterial thrombosis model. The potency of antithrombotic activity of 5h,i,n,p was significantly higher than that of aspirin (n=12, p<0.01) at equal dose (5 micromol/kg).     

Synthesis and biological activity of novel 1,4-diazepane derivatives as factor Xa inhibitor with potent anticoagulant and antithrombotic activity

Factor Xa (fXa) is a serine protease involved in the coagulation cascade, which has received great interest as a potential target for the development of new antithrombotic drugs. Herein we report a novel series of fXa inhibitors in which the 1,4-diazepane moiety was designed to interact with the S4 aryl-binding domain of the fXa active site. Compound 13 (YM-96765) showed potent fXa inhibitory activity (IC(50) = 6.8 nM) and effective antithrombotic activity without prolonging bleeding time.     

In vivo monitoring of thrombosis in mice by optical coherence tomography

The objective of this study is to establish a novel method for continuously monitoring thrombus progression with various outcome measures and to assess the efficacy of antithrombotic drugs in murine thrombosis model in mice. In the study, thrombus was induced in the femoral vein of mice by FeCl₃ and monitored over time by spectral‐domain optical coherence tomography (OCT). Three‐dimensional images of thrombi with or without heparin as an antithrombotic agent were obtained from OCT angiography. In addition, several parameters of thrombi were analyzed and compared between control and anticoagulant groups. By using OCT, we were able to trace thrombus generation in the same mouse in real time. We found that in our model heparin reduced thrombus size by ~60% and thrombus cross‐sectional area by 50%. OCT results also show that both time to thrombus size (>0.02mm³) and time to occlusion (>30%) were significantly reduced after heparin addition. This study demonstrates that OCT reliably monitors thrombus generation and progression from various aspects including thrombus size. This enables us to measure the kinetic of thrombosis more accurately, and effectively evaluate the efficacy and activities of antithrombotic drugs. This model may represent a useful tool in antithrombotic drug discoveries in preclinical studies.      

Design, synthesis, and evaluation of orally active inhibitors of plasminogen activator inhibitor-1 (PAI-1) production

A novel series of butadiene-imide 1 (T-686) derivatives having an inhibitory activity against PAI-1 production was synthesized and evaluated their biological activities and DMPK profiles, in which 15k (T-2639) was selected as the best compound based on its strong antithrombotic activity and good bioavailability.     

Discovery of novel tetrahydroisoquinoline derivatives as potent and selective factor Xa inhibitors

A series of novel 2,7-disubstituted tetrahydroisoquinoline derivatives were designed and synthesized. Among these derivatives, compounds 1 and 2 (JTV-803) exhibited potent inhibitory activity against FXa and good selectivity with respect to other serine proteases (thrombin, plasmin, and trypsin). In addition, compound 2 exhibited potent anti-FXa activity after intravenous and oral administration to cynomolgus monkey, and showed a dose-dependent antithrombotic effect in a rat model of venous thrombosis.     

Novel antithrombotic therapy.

The prospects for more effective and safe antithrombotic therapy appear promising. I have presented only a portion of all novel antithrombotic agents currently under basic and clinical investigations. A discussion of additional new antithrombotic drugs is presented elsewhere.     

K-134, a Phosphodiesterase 3 Inhibitor,Prevents Brain Damage by Inhibiting Thrombus Formation in a Rat Cerebral Infarction Model

Background

K-134 is a more potent antiplatelet drug with a selective inhibitory effect on phosphodiesterase 3 (PDE3) compared with its analogue, cilostazol.

Objectives

This study was performed to compare the ameliorating effects of K-134 and cilostazol on brain damage in an experimental photothrombotic cerebral infarction model.

Methods and Results

We investigated the effects of oral preadministration of PDE3 inhibitors in a rat stroke model established by photothrombotic middle cerebral artery (MCA) occlusion. K-134 significantly prolonged MCA occlusion time at doses >10 mg/kg, and reduced cerebral infarct size at 30 mg/kg in the stroke model (n = 12, 87.5±5.6 vs. 126.8±7.5 mm³, P<0.01), indicating its potent antithrombotic effect. On the other hand, the effects of cilostazol on MCA occlusion time and cerebral infarct size are relatively weak even at the high dosage of 300 mg/kg. Furthermore, K-134 blocked rat platelet aggregation more potently than cilostazol in vitro. Also in an arteriovenous shunt thrombosis model, K-134 showed an antithrombotic effect greater than cilostazol.

Conclusions

These findings suggest that K-134, which has strong antithrombotic activity, is a promising drug for prevention of cerebral infarction associated with platelet hyperaggregability.     

Androgenic profile and genotoxicity evaluation of testosterone propionate and novel synthesized heterocyclic steroids

In this study, we tested the androgenic activity of three structurally promising novel synthesized heterocyclic steroids compared with testosterone propionate in male mice. Additionally, the possible genotoxic effects of the novel synthesized heterocyclic steroids in comparison with testosterone propionate on male mice using chromosomal analysis of somatic and germ cells as well as RAPD-PCR were investigated. Male mice were administered with two doses of testosterone propionate, pyridoandrostene derivative 4b, pyrimidinoandrostene derivative 9a and thienoandrostene derivative 12 (200 and 400mg/kg b.w.) daily for 2 weeks. Results indicated that compounds 4b and 12 have androgenic activity as well as testosterone propionate. There were no significant differences in the frequencies of total chromosomal aberrations in both somatic and germ cells as well as no alteration in the DNA bands patterns between control, testosterone propionate and pyridoandrostene 4b treated animals. However, the pyrimidinoandrostene derivative 9a caused significant increase in the mean value of total chromosomal aberrations of both somatic and germ cells (P< or =0.01) as well as enhanced the polymorphic bands patterns as compared to the control and the other tested compounds. On the other hand, thienoandrostene derivative 12 induced significant decrease in the mean values of chromosomal aberrations in both somatic and germ cells, decreased sperm morphological abnormalities, increased the sperm count and motility than control. Our data indicate that testosterone propionate; pyridoandrostene 4b and thienoandrostene derivative 12 have no genotoxic activity. However, pyrimidinoandrostene derivative 9a has genotoxic activity possibly due to a modulation of the different expression of the catalyzing enzyme systems which will be investigated in the nearly future.     

Dibasic benzo[b]thiophene derivatives as a novel class of active site directed thrombin inhibitors: 2. Sidechain optimization and demonstration of in vivo efficacy

Potent, subnanomolar thrombin inhibitors 4, 5, and 6 are developed through side chain optimization of novel, benzo[b]thiophene-based small organic entities 2 and 3 and through SAR additivity studies of the new structural elements identified. X-ray crystallographic studies of 4b-thrombin complex revealed a hydrophobic and an electrostatic interaction of these new elements with thrombin at the S2 and S3 binding sites. In vitro and in vivo pharmacological studies showed that 4, 5, and 6 are potent anticoagulants in human plasma with demonstrated antithrombotic efficacy in a rat model of thrombosis.     

Pharmacochemistry of the platelet purinergic receptors

Platelets contain at least five purinergic G protein-coupled receptors, e.g., the pro-aggregatory P2Y₁ and P2Y₁₂ receptors, a P2Y₁₄ receptor (GPR105) of unknown function, and anti-aggregatory A_2A and A_2B adenosine receptor (ARs), in addition to the ligand-gated P2X1 ion channel. Probing the structure–activity relationships (SARs) of the P2X and P2Y receptors for extracellular nucleotides has resulted in numerous new agonist and antagonist ligands. Selective agents derived from known ligands and novel chemotypes can be used to help define the subtypes pharmacologically. Some of these agents have entered into clinical trials in spite of the challenges of drug development for these classes of receptors. The functional architecture of P2 receptors was extensively explored using mutagenesis and molecular modeling, which are useful tools in drug discovery. In general, novel drug delivery methods, prodrug approaches, allosteric modulation, and biased agonism would be desirable to overcome side effects that tend to occur even with receptor subtype-selective ligands. Detailed SAR analyses have been constructed for nucleotide and non-nucleotide ligands at the P2Y₁, P2Y₁₂, and P2Y₁₄ receptors. The thienopyridine antithrombotic drugs Clopidogrel and Prasugrel require enzymatic pre-activation in vivo and react irreversibly with the P2Y₁₂ receptor. There is much pharmaceutical development activity aimed at identifying reversible P2Y₁₂ receptor antagonists. The screening of chemically diverse compound libraries has identified novel chemotypes that act as competitive, non-nucleotide antagonists of the P2Y₁ receptor or the P2Y₁₂ receptor, and antithrombotic properties of the structurally optimized analogues were demonstrated. In silico screening at the A_2A AR has identified antagonist molecules having novel chemotypes. Fluorescent and other reporter groups incorporated into ligands can enable new technology for receptor assays and imaging. The A_2A agonist CGS21680 and the P2Y₁ receptor antagonist MRS2500 were derivatized for covalent attachment to polyamidoamine dendrimeric carriers of MW 20,000, and the resulting multivalent conjugates inhibited ADP-promoted platelet aggregation. In conclusion, a wide range of new pharmacological tools is available to control platelet function by interacting with cell surface purine receptors.     

Antiproliferative effects on human tumor cells and rat aortic smooth muscular cells of 2,3-heteroarylmaleimides and heterofused imides

A series of 2,3-heteroarylmaleimides 9 and polyheterocondensed imides 12 were prepared in good yields and short reaction time using a very efficient procedure consisting in the condensation of the corresponding anhydrides and N,N-diethylethylenediamine and microwave heating. The antiproliferative activity of the novel molecules was tested against human tumor cells (NCI-H460 lung carcinoma) and rat aortic smooth muscle cells (SMCs). The IC50 values for the novel molecules ranged from 0.08 to 13.9 microM in SMCs, and from 0.84 to 9 microM in the tumor cell line. The activity profile for compounds 9 and 12 is comparable to that obtained for amonafide in NCI-H460, except for fused imides 12b,i which proved to be about 10-fold more potent. Whereas, in rat SMCs, only the compound 12b was shown to be 10-fold more potent than amonafide. Instead 12c is equipotent to amonafide. These results suggest that the extended pi-system and the kind of heteroatom are essential in the binding with the molecular target.     

Identification of cryptotanshinone from Tongmai to inhibit thrombosis in zebrafish via regulating oxidative stress and coagulation cascade

BackgroundThromboembolic events are leading causes of mortality and morbidity all over the world. Tongmai (TM) is a botanical drug with valid clinical efficacy and safety in the management of thrombosis and ischemic cardiovascular diseases, however, its active compounds and underlying mechanism are largely unclear.PurposeTo investigate the endogenous effects, therapeutic mechanism and active compounds of TM in thrombus formation.Study designCombined with transgenic zebrafish models and high-content imaging system, this study evaluated the endogenous antithrombotic effects of TM and screened for the active compounds.MethodsThe PHZ-induced thrombotic model in erythrocytes or platelets labeled transgenic zebrafish were established, to dynamically evaluate the antithrombotic effects of TM. The oxidative damage levels were analyzed by specific fluorescent probes, and the expression levels of key factors in coagulation cascades and platelet activation were examined by QPCR. TM were dissected into fractions by reverse phase chromatography and subsequently screened for their antithrombotic effects in the transgenic fish models. The compounds of the active TM fraction were then analyzed by UPLC-Q-TOF analysis and further verified for their antithrombotic effects and mechanisms.ResultsIn PHZ-induced zebrafish thrombotic model, TM incubation markedly increased cardiac blood flow, decreased peripheral erythrocytes aggregation, and recovered peripheral platelet circulation. Besides, the levels of oxidative stress and lipid peroxidation were increased in the PHZ-induced thrombotic fish, which were greatly decreased by TM treatment. Moreover, TM significantly reduced the expression of coagulation factor II (thrombin) and the downstream fibrinogen. In order to identify the active compounds of TM, four fractions were separated from the extract by reverse phase chromatography, which were subsequently screened for their antithrombotic effects in the fish model. As a result, fraction 4 showed the strongest effect in inhibiting thrombosis. Finally, through UPLC-Q-TOF analysis and endogenous screening, cryptotanshione was identified as the main active compound with antithrombotic effects.ConclusionOur study demonstrated the endogenous antithrombotic effects of TM, which is possibly mediated by inhibiting oxidative stress and coagulation cascade. Cryptotanshione was identified as a major compound with antithrombotic activity and is a promising candidate for novel antithrombotic therapy.     

Molecular docking‐assisted screening reveals tannic acid as a natural protein disulphide isomerase inhibitor with antiplatelet and antithrombotic activities

Protein disulphide isomerase (PDI) promotes platelet activation and constitutes a novel antithrombotic target. In this study, we reported that a PDI‐binding plant polyphenol, tannic acid (TA), inhibits PDI activity, platelet activation and thrombus formation. Molecular docking using plant polyphenols from dietary sources with cardiovascular benefits revealed TA as the most potent binding molecule with PDI active centre. Surface plasmon resonance demonstrated that TA bound PDI with high affinity. Using Di‐eosin‐glutathione disulphide fluorescence assay and PDI assay kit, we showed that TA inhibited PDI activity. In isolated platelets, TA inhibited platelet aggregation stimulated by either GPVI or ITAM pathway agonists. Flow cytometry showed that TA inhibited thrombin‐ or CRP‐stimulated platelet activation, as reflected by reduced granule secretion and integrin activation. TA also reduced platelet spreading on immobilized fibrinogen and platelet adhesion under flow conditions. In a laser‐induced vascular injury mouse model, intraperitoneal injection of TA significantly decreased the size of cremaster arteriole thrombi. No prolongation of mouse jugular vein and tail‐bleeding time was observed after TA administration. Therefore, we identified TA from natural polyphenols as a novel inhibitor of PDI function. TA inhibits platelet activation and thrombus formation, suggesting it as a potential antithrombotic agent.     

CXC Chemokine Ligand 12 Protects Pancreatic β-Cells from Necrosis through Akt Kinase-Mediated Modulation of Poly(ADP-ribose) Polymerase-1 Activity

The diabetes prevention paradigm envisages the application of strategies that support the maintenance of appropriate β-cell numbers. Herein we show that overexpression of CXC chemokine ligand12 (CXCL12) considerably improves the viability of isolated rat Langerhans islet cells and Rin-5F pancreatic β-cells after hydrogen peroxide treatment. In rat islets and wt cells hydrogen peroxide treatment induced necrotic cell death that was mediated by the rapid and extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1). In contrast, CXCL12-overexpressing cells were protected from necrotic cell death as a result of significantly reduced PARP-1 activity. CXCL12 downstream signalling through Akt kinase was responsible for the reduction of PARP-1 activity which switched cell death from necrosis to apoptosis, providing increased protection to cells from oxidative stress. Our results offer a novel aspect of the CXCL12-mediated improvement of β-cell viability which is based on its antinecrotic action through modulation of PARP-1 activity.     

Synthesis and biological activity of bi/tricyclic azasugars fused thiazolidin-4-one and thiazinan-4-one by microwave-assisted tandem Staudinger/aza-Wittig/cyclization

A convenient synthesis of novel bi/tricyclic azasugars fused thiazolidin-4-one and thiazinan-4-one by the one-pot tandem Staudinger/aza-Wittig/cyclization reaction under microwave radiation was demonstrated. The reactions were carried out with the azidosugar 1 and mercaptan acids via a key intermediate Schiff base and stereoselectively afforded the titled bi/tricyclic azasugars in good yield. All the dominant products were in the 1,2-trans form and the reaction stereoselectivity mainly depended upon the steric hindrance of the neighboring rigid cyclic isopropylidene groups on C-2, 3 which favors the exo-attack of the sulfur atom (in mercaptan acids) to the intermediate imine. The preliminary biological evaluation of the compounds 10-17 showed that compounds 10b, 11a, 12b, 14b, 16b, 17a, and 17b were found to active the natural killer (NK) cells significantly (immunopotentiating activity) and compounds 10a, 10b, 12a, 16b, and 17b exhibited weak inhibitory activity against β-glucosidase. Yet none of these tested compounds have obvious effects on T cell proliferation, or show inhibition against α-amylase and α-glucosidase.     

低分子肝素的抗炎作用及机制

低分子肝素(low molecular weight heparin, LMWH)除作为抗凝血和抗血栓药在临床上广为应用外,近年来其抗炎活性也颇受重视.LMWH抗炎机制涉及炎症细胞、炎症因子和黏附分子等环节.目前对LMWH的抗炎机制研究还处在初级阶段,但是LMWH独特的性质使其有望成为有效且安全的新型抗炎药物.     

The Bloom's syndrome helicase (BLM) interacts physically and functionally with p12, the smallest subunit of human DNA polymerase delta

Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL δ (hPOL δ). The hPOL δ enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL δ strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL δ. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL δ.     

Design and synthesis of a novel synthetic NAPAP-penta-saccharide conjugate displaying a dual antithrombotic action.

The synthesis of a novel antithrombotic consisting of a heparin pentasaccharide conjugated to the active site inhibitor N-(2-naphtalenesulfonyl)-glycyl-(D)-4-aminophenyl-alanyl-piperidin e (NAPAP) (i.e. compound I) is reported. This conjugate shows a unique pharmacological profile both in vitro and in vivo having direct anti-thrombin and ATIII-mediated anti-Xa activity. Furthermore, conjugate I has a prolonged in vivo half-life compared to NAPAP (1.5 h vs 9 min.).     

Zebrafish model of photochemical thrombosis for translational research and thrombolytic screening in vivo

Acute thromboembolic diseases remain the major global cause of death or disability. Although an array of thrombolytic and antithrombotic drugs has been approved to treat or prevent thromboembolic diseases, many more drugs that target specific clotting mechanisms are under development. Here a novel zebrafish model of photochemical thrombosis is reported and its prospective application for the screening and preclinical testing of thrombolytic agents in vivo is demonstrated. Through photochemical excitation, a thrombus was induced to form at a selected section of the dorsal aorta of larval zebrafish, which had been injected with photosensitizers. Such photochemical thrombosis can be consistently controlled to occlude partially or completely the targeted blood vessel. Detailed mechanistic tests indicate that the zebrafish model of photochemical thrombosis exhibits essential features of classical coagulation and a thrombolytic pathway. For demonstration, tissue plasminogen activator (tPA), a clinically feasible thrombolytic agent, was shown to effectively dissolve photochemically induced blood clots. In light of the numerous unique advantages of zebrafish as a model organism, our approach is expected to benefit not only the development of novel thrombolytic and antithrombotic strategies but also the fundamental or translational research targeting hereditary thrombotic or coagulation disorders.

     

Amino acid based enantiomerically pure 3-substituted benzofused heterocycles: A new class of antithrombotic agents

A diverse group of novel medium ring heterocycles derived from naturally abundant proteinogenic amino acids were evaluated for their potency towards antithrombotic activity. The more potent benzofused oxazepine and oxazocine scaffolds were diversified by incorporating different amino acids at the position number 3. Further the effect of ring size has also been taken into account and it was observed that the eight-membered oxazocines ane more potent compared to the corresponding oxazepines.