RATIONALE:Mast cells and neutrophils are key contributors to the pathophysiological inflammatory processes that underpin asthma and chronic obstructive pulmonary disease, partly through the release of noxious serine proteases, including cathepsin G (Cat G) and chymase. From this standpoint, a dual inhibitor of neutrophil Cat G and mast cell chymase could protect against these disease-related inflammatory responses.OBJECTIVES:We examined the antiinflammatory pharmacology of RWJ-355871, a dual inhibitor of Cat G and chymase, in animal models of inflammation that evince pathophysiological pathways relevant to asthma and chronic obstructive pulmonary disease to determine the therapeutic potential of this compound.METHODS:In an ovalbumin (OVA)-sensitized rat model, RWJ-355871 was administered to block the mast-cell-mediated increase in paw volume caused by OVA injection. In a sheep asthma model, antigen-induced airway responses were assessed with and without aerosol treatment with RWJ-355871. In a murine tobacco-smoke model of airway inflammation, the effect of RWJ-355871 on smoke-induced neutrophilia was determined.MEASUREMENTS AND MAIN RESULTS:Intravenous treatment of OVA-sensitized rats with RWJ-355871 provided dose-dependent reduction in the increase in rat paw volume. In allergic sheep, aerosol pretreatment with RWJ-355871 showed dose-dependent inhibition of the antigen-induced early response, late response, and post-antigen-induced airway hyperreponsiveness. In tobacco-smoke-exposed mice, nebulized RWJ-355871 significantly reduced the smoke-induced neutrophilia from the levels observed in untreated mice.CONCLUSIONS:The preclinical antiinflammatory effects of RWJ-355871 in these animal models of inflammation indicate that this dual inhibitor may have therapeutic utility for treating airway inflammatory diseases involving mechanisms that depend on Cat G and/or chymase.

Whereas heparin functions as an antithrombotic agent by promoting antithrombin III-based inhibition of thrombin and factor Xa, there is less appreciation for the combination behavior with small-molecule, direct inhibitors of these proteases. We conducted a study in a high-shear arterial environment to explore the potential for a cooperative antithrombotic effect with a thrombin inhibitor (argatroban), a factor Xa inhibitor (YM-60828), and a dual thrombin/factor Xa inhibitor (RWJ-445167). We employed a platelet-dependent vascular injury model in which rats were subjected to an acute electrical injury to the carotid artery. Antithrombotic efficacy was measured for thrombin inhibitor argatroban and factor Xa inhibitor YM-60828 administered alone or in combination. The results indicate that there is a cooperative antithrombotic effect in vivo when both thrombin and factor Xa are inhibited simultaneously. The dual thrombin/factor Xa inhibitor RWJ-445167 was found to have potent antithrombotic activity in this high-shear environment. A comparison of results for RWJ-445167 and argatroban showed additional efficacy with RWJ-445167, suggestive of drug synergy.

We have discovered two related chemical series of nonpeptide urotensin-II (U-II) receptor antagonists based on piperazino-phthalimide (5 and 6) and piperazino-isoindolinone (7) scaffolds. These structure types are distinctive from those of U-II receptor antagonist series reported in the literature. Antagonist 7a exhibited single-digit nanomolar potency in rat and human cell-based functional assays, as well as strong binding to the human U-II receptor. In advanced pharmacological testing, 7a blocked the effects of U-II in vitro in a rat aortic ring assay and in vivo in a rat ear-flush model. A discussion of U-II receptor antagonist pharmacophores is presented, and a specifically defined model is suggested from tricycle 13, which has a high degree of conformational constraint.

Whereas the activation and aggregation of blood platelets are crucial to normal hemostasis, this ensemble is also a key factor in serious cardiovascular disorders, such as myocardial infarction, unstable angina, transient ischemic attack, stroke, peripheral arterial disease, and artherosclerosis. Indeed, abnormal thrombosis is a root cause of adverse cardiovascular events that are responsible for death and disability in humans. As a consequence, plate ACHTUNGTRENNUNGlets are targeted by clinically useful antithrombotic drugs, such as the cyclooxygenase-1 inhibitor aspirin, the GPIIb/IIIa antagonist abciximab, and the adenosine 5’-diphosphate (ADP) receptor antagonist clopidogrel. ADP is an important agonist of platelet activation and aggregation because it induces platelet shape change accompanied by the activation of fibrinogen receptors (GPIIb/IIIa). On platelets, there are three types of cell-surface receptors for ADP, which are members of the P2 purinergic class: P2X1, P2Y1, and P2Y12. [4, 5] P2Y1 and P2Y12 are G protein-coupled receptors (GPCRs), whereas P2X1 is a ligand-gated ion channel. The Gqcoupled P2Y1 receptor initiates ADP-induced platelet activation and the Gi-coupled P2Y12 receptor amplifies activation processes, including aggregation, granule secretion, and procoagulant activity, as caused by various agonists. From this perspective, antagonists of P2Y12 can be therapeutically effective by markedly inhibiting platelet function independent of the activating stimulus. Additionally, because of the restricted distribution of P2Y12 in humans, this receptor is an attractive antiplatelet target for drug discovery. The widespread clinical use of clopidogrel has demonstrated the relevance of inhibiting the platelet-specific P2Y12 receptor to prevent untoward cardiovascular events. However, clopidogrel is a prodrug that requires metabolic conversion in vivo to a highly unstable, reactive species that covalently modifies the P2Y12 receptor. [7] On account of this property, there have been observations in humans of slow onset of pharmacological action and of high interpatient variability. Thus, drug discovery efforts have been mounted to identify potent, direct acting, reversible P2Y12 antagonists, and some promising compounds have emerged, including Cangrelor (AR-C69931MX), AZD-6140, and PRT-128. In seeking suitable drug candidates in this area, we have been exploring carba-nucleoside derivatives that are structurally related to AZD-6140 as reversible P2Y12 antagonists. In this paper, we report on the synthesis and biological evaluation of novel compounds, including tetrazole-containing derivatives with high receptor affinity and excellent potency for inhibiting P2Y12-mediated effects on human platelets.

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

Various 4-phenylpiperidine-benzoxazin-3-ones were synthesized and biologically evaluated as urotensin-II (U-II) receptor antagonists. Compound 12i was identified from in vitro evaluation as a low nanomolar antagonist against both rat and human U-II receptors. This compound showed in vivo efficacy in reversing the ear-flush response induced by U-II in rats.

Introduction: Rivaroxaban is an oral, direct Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders, including arterial indications such as acute coronary syndromes (ACS). The aim of this study was to determine whether rivaroxaban can prevent arterial thrombotic occlusion in electrolytically injured rat carotid arteries. Methods: Anesthetized, male Sprague-Dawley rats were equipped with an intravenous (i.v.) catheter to administer drug or vehicle and an intra-arterial catheter for arterial pressure monitoring and blood sampling. The left carotid artery was exposed and a bipolar electrode placed proximally around it. A pulsed Doppler flow probe was then placed distally around the vessel. Blood was obtained before drug or vehicle administration to determine activated clotting time (ACT), prothrombin time (PT), Russell’s viper venom time (RVVT), activated partial thromboplastin time (aPTT) and thrombin-antithrombin (TAT) complex concentration; baseline carotid blood flow was recorded for 20 minutes. Vehicle (polyethylene glycol:ethanol:water or saline, 0.5 mL/kg/min), rivaroxaban (0.3, 1 and 3 mg/kg; dosing volume 0.5 mL/kg/min), or the low molecular weight heparin enoxaparin (10 mg/kg) were infused i.v. over 30 seconds. After infusion, direct current (3 mA) was applied to the left carotid artery for 5 minutes. Blood flow was monitored for 30 minutes after injury. Blood was sampled again 14.5 and 30 minutes after injury for ACT determination, and after 30 minutes for PT, RVVT, TAT and aPTT determination. Results: Rivaroxaban dose-dependently increased the median time to arterial thrombotic occlusion (TTO), and was significantly more effective than vehicle at doses of 1 and 3 mg/kg (vehicle, 13.2 minutes; rivaroxaban 1 and 3 mg/kg, >30 minutes; P<0.05 by Kaplan-Meier analysis). Compared with enoxaparin (10 mg/kg), rivaroxaban inhibited TTO at doses of 1 and 3 mg/kg more effectively. Rivaroxaban (0.3–3 mg/kg i.v.) dose-dependently prolonged ACT, PT and RVVT, and substantially decreased the formation of TAT complexes, but did not substantially affect aPTT. Enoxaparin prolonged ACT, aPTT and RVVT, and decreased TAT complex formation, but had no effect on PT. Conclusions: Rivaroxaban potently inhibited electrolytically induced arterial thrombotic occlusion in rat carotid arteries, resulting in substantial changes in coagulation parameters. The coagulation parameter changes were consistent with inhibition of FXa activity as indexed by the RVVT. These findings suggest that rivaroxaban may be an effective anticoagulant in arterial thrombotic disorders, such as ACS.

Certain leukocytes release serine proteases that sustain inflammatory processes and cause disease conditions, such as asthma and chronic obstructive pulmonary disease. We identified beta-ketophosphonate 1 (JNJ-10311795; RWJ-355871) as a novel, potent dual inhibitor of neutrophil cathepsin G ( K-i = 38 nM) and mast cell chymase ( K-i = 2.3 nM). The x-ray crystal structures of 1 complexed with human cathepsin G ( 1.85 angstrom) and human chymase ( 1.90 angstrom) reveal the molecular basis of the dual inhibition. Ligand 1 occupies the S-1 and S-2 subsites of cathepsin G and chymase similarly, with the 2-naphthyl in S-1, the 1-naphthyl in S-2, and the phosphonate group in a complex network of hydrogen bonds. Surprisingly, however, the carboxamido- N-(naphthalene-2-carboxyl) piperidine group is found to bind in two distinct conformations. In cathepsin G, this group occupies the hydrophobic S-3/S-4 subsites, whereas in chymase, it does not; rather, it folds onto the 1-naphthyl group of the inhibitor itself. Compound 1 exhibited noteworthy anti-inflammatory activity in rats for glycogen-induced peritonitis and lipopolysaccharide-induced airway inflammation. In addition to a marked reduction in neutrophil influx, 1 reversed increases in inflammatory mediators interleukin-1 alpha, interleukin-1 beta, tissue necrosis factor-alpha, and monocyte chemotactic protein-1 in the glycogen model and reversed increases in airway nitric oxide levels in the lipopolysaccharide model. These findings demonstrate that it is possible to inhibit both cathepsin G and chymase with a single molecule and suggest an exciting opportunity in the treatment of asthma and chronic obstructive pulmonary disease.

Thrombin inhibitors are potentially useful in medicine for their anticoagulant and antithrombotic effects. We synthesized and evaluated diverse heterocycle-activated ketones based on the d-Phe-Pro-Arg, and related thrombin active-site recognition motifs, as candidate inhibitors. The peptide-based alpha-ketoheterocycles were typically prepared by either an imidate or a Weinreb amide route (Schemes 1 and 2), the latter of which proved to be more general. Test compounds were generally assayed for inhibition of human alpha-thrombin and bovine trypsin. From a structure-based design standpoint, the heterocycle allows one to explore and adjust interactions within the S1' subsite of thrombin. The preferred alpha-ketoheterocycle is a pi-rich 2-substituted azole with at least two heteroatoms proximal to the carbon bearing the keto group, and a preferred thrombin inhibitor is 2-ketobenzothiazole 3, with a potent K(i) value of 0.2 nM and ca. 15-fold selectivity over trypsin. 2-Ketobenzothiazole 13 exhibited exceedingly potent thrombin inhibition (K(i) = 0.000 65 nM; slow tight binding). Several alpha-ketoheterocycles had thrombin K(i) values in the range 0.1-400 nM. The "Arg" unit in the alpha-ketoheterocycles can be sensitive to stereomutation under mildy basic conditions. For example, 2-ketothiazoles 4 and 59 readily epimerize at pH 7.4, although they are fairly stable stereochemically at pH 3-4; thus, suitable conditions had to be selected for the enzymatic assays. Lead d-Phe-Pro-Arg 2-benzothiazoles 3, 4, and 68 displayed good selectivity for thrombin over other key coagulation enzymes (e.g., factor Xa, plasmin, protein Ca, uPA, tPA, and streptokinase); however, their selectivity for thrombin over trypsin was modest (<25-fold). Compounds 3, 4, and 68 exhibited potent in vitro antithrombotic activity as measured by inhibition of gel-filtered platelet aggregation induced by alpha-thrombin (IC(50) = 30-40 nM). They also proved to be potent anticoagulant/antithrombotic agents in vivo on intravenous administration, as determined in the canine arteriovenous shunt (ED(50) = 0.45-0.65 mg/kg) and the rabbit deep vein thrombosis (ED(50) = 0.1-0.4 mg/kg) models. Intravenous administration of 3, and several analogues, to guinea pigs caused hypotension and electrocardiogram abnormalities. Such cardiovascular side effects were also observed with some nonguanidine inhibitors and inhibitors having recognition motifs other than d-Phe-Pro-Arg. 2-Benzothiazolecarboxylates 4 and 68 exhibited significantly diminished cardiovascular side effects, and benzothiazolecarboxylic acid 4 had the best profile with respect to therapeutic index. The X-ray crystal structures of the ternary complexes 3-thrombin-hirugen and 4-thrombin-hirugen depict novel interactions in the S(1)' region, with the benzothiazole ring forming a hydrogen bond with His-57 and an aromatic stacking interaction with Trp-60D of thrombin's insertion loop. The benzothiazole ring of 3 displaces the Lys-60F side chain into a U-shaped gauche conformation, whereas the benzothiazole carboxylate of 4 forms a salt bridge with the side chain of Lys-60F such that it adopts an extended anti conformation. Since 3 has a 10-fold greater affinity for thrombin than does 4, any increase in binding energy resulting from this salt bridge is apparently offset by perturbations across the enzyme (viz. Figure 4). The increased affinity and selectivity of 2-ketobenzothiazole inhibitors, such as 3, may be primarily due to the aromatic stacking interaction with Trp-60D. However, energy contour calculations with the computer program GRID also indicate a favorable interaction between the benzothiazole sulfur atom and a hydrophobic patch on the surface of thrombin.

RWJ-53308 is a novel nonpeptide glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist that inhibits fibrinogen binding to GPIIb/IIIa with an IC(50) of 0.4+/-0.3 nM. RWJ-53308 inhibits thrombin-induced platelet aggregation in human gel-filtered platelets (IC(50)=60+/-12 nM) and platelet aggregation in human platelet-rich plasma (PRP) in response to collagen, arachidonic acid, ADP, and SFLLRN-NH(2) (IC(50)=60+/-10, 150+/-30, 70+/-4, and 160+/-80 nM, respectively). The potency of RWJ-53308 in dog and guinea pig PRP is similar to human PRP. RWJ-53308 inhibits ex vivo collagen- and ADP-induced platelet aggregation in conscious dogs for up to 4 h following 0.3 mg/kg iv, and through 4 and 6 h following 1 and 3 mg/kg po. Oral bioavailability is 16+/-7%. RWJ-53308 reduces thrombus weight in a canine arteriovenous (AV) shunt model following intravenous (0.01-0.1 mg/kg) and oral (3 mg/kg) administration. In a guinea pig carotid artery pinch-injury model, RWJ-53308 completely suppresses thrombus-induced cyclic flow reductions (CFR) at 0.7 mg/kg iv. RWJ-53308 also blocks thrombus formation in photoactivation- and ferric chloride-induced models of thrombosis in guinea pigs at 0.3 and 1 mg/kg iv, respectively. In summary, RWJ-53308 is a potent orally active GPIIb/IIIa antagonist that may be useful for both acute and chronic treatment of arterial thrombotic disorders.

Human platelets possess two distinct thrombin-activated receptors, PAR-1 (protease-activated receptor-1) and PAR-4, whereas human vascular smooth muscle cells possess only PAR-1. Although such thrombin receptors have been studied extensively in vitro, their physiological roles are still rather ill-defined. We have now employed a potent, selective PAR-1 antagonist, RWJ-58259, to probe the in vivo significance of PAR-1 in thrombosis and vascular injury. RWJ-58259 was examined in two thrombosis models in guinea pigs: the arteriovenous (A-V) shunt assay (monitoring thrombus weight) and the Rose Bengal intravascular photoactivation assay (monitoring time to occlusion). Administration of RWJ-58259 (10 mg/kg, total i.v. dose) did not inhibit thrombus formation in either thrombosis model, although local, intrashunt delivery in the A-V shunt model did elicit a modest antithrombotic effect (thrombus weight reduction from 35 +/- 2 to 24 +/- 4 mg). These results are consistent with the presence of more than one thrombin-sensitive receptor on guinea pig platelets, in analogy with human platelets. Indeed, we were able to establish that guinea pig platelets express three thrombin receptors, PAR-1, PAR-3, and PAR-4. We also examined RWJ-58259 in a vascular restenosis model involving balloon angioplasty in rats. Perivascular administration of RWJ-58259 (10 mg) significantly reduced neointimal thickness (77 +/- 5 microm to 45 +/- 5 microm, P < 0.05), clearly demonstrating an important role for PAR-1 in vascular injury. From these results, it is evident that a PAR-1 antagonist is not especially effective for treating platelet-dependent thrombosis; however, it could well be beneficial for treating restenosis attendant to arterial injury.

Although intravenously administered antiplatelet fibrinogen receptor (GPIIb/IIIa) antagonists have become established in the acute-care clinical setting for the prevention of thrombosis, orally administered drugs for chronic use are still under development. Herein, we present details from our exploration of structure-activity surrounding the prototype fibrinogen receptor antagonist RWJ-50042 (racemate of 1), which was derived from a unique approach involving the gamma-chain of fibrinogen (Hoekstra et al. J. Med. Chem. 1995, 38, 1582). Our analogue studies culminated in the discovery of RWJ-53308 (2), a potent, orally active GPIIb/IIIa antagonist. To progress from RWJ-50042 to a suitable candidate for clinical development, we conducted a series of optimization cycles that employed solid-phase parallel synthesis for the rapid, efficient preparation of nearly 250 analogues, which were assayed for fibrinogen receptor affinity and inhibition of platelet aggregation induced by four different activators. This strategy produced several promising analogues for advanced study, including 3-(3,4-methylenedioxybenzene)-beta-amino acid analogue 3 (significant improved, in vivo potency) and 3-(3-pyridyl)-beta-amino acid 2 (significant;ly improved potency, oral absorption, and duration of action). In dogs, 2 displayed significant ex vivo antiplatelet activity on oral administration at 1.0 mg/kg, 16% systemic oral bioavailability, minimal metabolic transformation, and an excellent safety profile. Additionally, 2 was found to be efficacious in three in vivo thrombosis models: canine arteriovenous (AV) shunt (0.01-0.1 mg/kg, iv), guinea pig photoactivation-induced injury (0.3-3 mg/kg, iv), and guinea pig ferric chloride-induced injury (0.3-1 mg/kg, iv). On the basis of its noteworthy preclinical data, RWJ-53308 (2) was selected for clinical evaluation.

A study of β-turn peptide mimetics, related to the C-terminal γ-chain of fibrinogen and containing a nipecotic acid scaffold, led to RWJ-50042 (1), an interesting fibrinogen receptor (GPIIb/IIIa) antagonist. To enhance potency, we employed solid-phase parallel synthesis for the preparation of over 200 analogues in a protocol of optimization cycles. This strategy produced several promising nipecotamide analogues, such as 25, which is 35 times more potent than 1 in vitro.