Inhibitory effects of ASP6537, a selective cyclooxygenase-1 inhibitor, on thrombosis and neointima formation in rats.

INTRODUCTION: Percutaneous coronary interventions (PCIs), such as balloon angioplasty and stent placement, are effective in the treatment of coronary artery disease. PCI has drawbacks, however, including acute thrombosis after the procedure and restenosis of the vascular lumen due to abnormal neointimal hyperplasia. ASP6537 is a selective COX-1 inhibitor that has been investigated as a possible candidate for clinical development as an antiplatelet agent. In the present study, we evaluated the in vivo antithrombotic effect of ASP6537 and its effect on neointima formation after balloon angioplasty. MATERIAL AND METHODS: The antithrombotic effect of ASP6537 was examined using an arteriovenous shunt thrombosis model in rats while the effect of ASP6537 on neointima formation was evaluated in a rat carotid arterial balloon angioplasty model. RESULTS: In the thrombosis study, ASP6537 dose-dependently decreased the protein content of the thrombus, while no prolongation of template bleeding time was observed. In the neointima study, ASP6537 reduced neointima formation. CONCLUSIONS: ASP6537 may be a promising agent for the prevention of acute thrombosis and restenosis after PCI in place of aspirin.

Biological properties of a specific Galpha q/11 inhibitor, YM-254890, on platelet functions and thrombus formation under high-shear stress.

1 The effects of YM-254890, a specific Galpha(q/11) inhibitor, on platelet functions, thrombus formation under high-shear rate condition and femoral artery thrombosis in cynomolgus monkeys were investigated. 2 YM-254890 concentration dependently inhibited ADP-induced intracellular Ca(2+) elevation, with an IC(50) value of 0.92+/-0.28 microM. 3 P-selectin expression induced by ADP or thrombin receptor agonist peptide (TRAP) was strongly inhibited by YM-254890, with IC(50) values of 0.51+/-0.02 and 0.16+/-0.08 microM, respectively. 4 YM-254890 had no effect on the binding of fibrinogen to purified GPIIb/IIIa, but strongly inhibited binding to TRAP-stimulated washed platelets. 5 YM-254890 completely inhibited platelet shape change induced by ADP, but not that induced by collagen, TRAP, arachidonic acid, U46619 or A23187. 6 YM-254890 attenuated ADP-, collagen-, TRAP-, arachidonic acid- and U46619-induced platelet aggregation with IC(50) values of <1 microM, whereas it had no effect on phorbol 12-myristate 13-acetate-, ristocetin-, thapsigargin- or A23187-induced platelet aggregation. 7 High-shear stress-induced platelet aggregation and platelet-rich thrombus formation on a collagen surface under high-shear flow conditions were concentration dependently inhibited by YM-254890. 8 The antithrombotic effect of YM-254890 was evaluated in a model of cyclic flow reductions in the femoral artery of cynomolgus monkeys. The intravenous bolus injection of YM-254890 dose dependently inhibited recurrent thrombosis without affecting systemic blood pressure or prolonging template bleeding time. 9 YM-254890 is a useful tool for investigating Galpha(q/11)-coupled receptor signaling and the physiological roles of Galpha(q/11).

Pharmacological properties of YM-254890, a specific G(alpha)q/11 inhibitor, on thrombosis and neointima formation in mice.

The pharmacological properties of YM-254890, a specific G(alpha)q/11 inhibitor, on acute thrombosis and chronic neointima formation after vascular injury have been investigated. FeCl3 was used to induce vascular injury in the carotid artery of mice. For the thrombosis studies, the test drug was either intravenously or orally administered before vascular injury. For the neointima studies, the test drug was orally administered 1 h before and twice daily for 1 week after vascular injury. Histological analysis was then performed 3 weeks later. YM-254890 significantly inhibited ex vivo platelet aggregation 5 min after intravenous bolus injection at 0.03 mg/kg or more, and 1 h after oral administration at 1 mg/kg. YM-254890 significantly inhibited thrombus formation after intravenous bolus injection at 0.03 mg/kg as well as after oral administration at 1 mg/kg, but tail transection bleeding time was significantly prolonged at 0.1 mg/kg for intravenous injection and 3 mg/kg for oral administration. Furthermore, oral administration of YM-254890 dose-dependently inhibited neointima formation 3 weeks after vascular injury with significant effects at 1 mg/kg twice daily for 1 week. Clopidogrel also significantly inhibited neointima formation at its antithrombotic dose, but its inhibitory potency was less than that of YM-254890. However, YM-254890 significantly reduced systemic blood pressure at doses 3 times higher than those that produced significant inhibitory effects on thrombosis and neointima formation. Though the systemic use of YM-254890 may be limited, owing to its narrow therapeutic window, this unique compound is a useful research tool for investigating the physiological roles of G(alpha)q/11 .

Combined effects of a factor Xa inhibitor YM466 and a GPIIb/IIIa antagonist YM128 on thrombosis and neointima formation in mice.

Thrombosis and neointima formation limit the efficacy of coronary angioplasty. Factor Xa inhibitors and GPIIb/IIIa antagonists have shown to be effective on acute thrombosis and late neointima formation, however, their combined effects remain to be elucidated. Vascular injury was induced by FeCl(3) in the carotid artery in mice. For thrombosis studies, the test drug was orally administered 1 hour before vascular injury. For neointima studies, the test drug was orally administered 1 hour before and twice daily for 1 week after vascular injury, and then histological analysis was performed 3 weeks after vascular injury. YM466 inhibited thrombotic occlusion at 30 mg/kg with prolongation of prothrombin time (PT), and tail transection bleeding time (BT) was affected at 100 mg/kg. YM466 also inhibited neointima formation at 10 mg/kg. YM128 inhibited thrombotic occlusion and neointima formation at 10 and 30 mg/kg, respectively, with inhibition of platelet aggregation and prolongation of BT. In contrast, the combination of 10 mg/kg YM466 and 3 mg/kg YM128 inhibited thrombotic occlusion and neointima formation without affecting PT, platelet aggregation and BT. Concomitant inhibition of factor Xa and GPIIb/IIIa may provide a safer and more effective therapeutic regimen for treatment of coronary angioplasty.

Antithrombotic and thrombolytic efficacy of YM-254890, a G q/11 inhibitor, in a rat model of arterial thrombosis.

We examined the antithrombotic and thrombolytic effects of the G(q/11) inhibitor YM-254890 in an electrically-induced carotid artery thrombosis model in rats. YM-254890 dose-dependently inhibited ex vivo ADP-induced platelet aggregation after i.v. bolus injection. In the thrombosis study, YM-254890 dosedependently prolonged time to occlusion at doses of 3 and 10 g/kg i.v. and decreased occlusion rate at 10 g/kg i.v. In the thrombolysis study, YM-254890 at 30 micro g/kg i.v. shortened the time to reperfusion and prevented reocclusion after thrombolysis with a modified tissue-type plasminogen activator. YM-254890, at 10 micro g/kg and more, significantly improved carotid patency status after thrombolysis. However, at 30 micro g/kg and more, YM-254890 decreased systemic blood pressure. These results suggest that YM-254890 may be effective for treating G(q)-mediated diseases, and that YM-254890 is a useful tool for investigating the biological roles of G(q/11).

Pharmacological properties of YM-57029, a novel platelet glycoprotein IIb/IIIa antagonist.

The pharmacological properties of YM-57029 [4-[4-(4-carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino]acetic acid monohydrochloride trihydrate), a novel glycoprotein IIb/IIIa antagonist were examined in this study. YM-57029 inhibited fibrinogen binding to purified glycoprotein IIb/IIIa, 1000-fold more potently than the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS). YM-57029 concentration-dependently inhibited ADP-, collagen- and high shear stress-induced platelet aggregation, strongly inhibited ATP release from platelets activated by ADP, and enhanced deaggregation of ADP-induced platelet aggregates. In a pro-aggregatory activity study, RGDS or SC-54701A ((S)-3-[3-[(4-amidinophenyl)carbamoyl]propionamido]-4-pentynoic acid monohydrochloride) caused prominent small aggregate formation. At a higher concentration, RGDS induced medium and large size aggregates, and SC-54701A induced medium aggregates. In contrast, YM-57029 produced only a few small and no larger size aggregates. Ex vivo ADP-induced platelet aggregation and platelet retention to collagen-coated plastic beads were dose-dependently inhibited by YM-57029 after intravenous bolus injection in guinea pigs. YM-57029 produced dose-dependent antithrombotic effects in carotid artery thrombosis and arterio-venous shunt thrombosis models in guinea pigs at 10 and 30 microg/kg, respectively. At these doses, YM-57029 prolonged template bleeding time. These results suggest that YM-57029 is a potent glycoprotein IIb/IIIa antagonist which has less pro-aggregatory effect. It may be a promising antiplatelet agent for thromboembolic diseases, and a good prototype for developing an orally active compound.

YM-254890, a novel platelet aggregation inhibitor produced by Chromobacterium sp. QS3666.

A novel platelet aggregation inhibitor, YM-254890, was isolated from the culture broth of strain QS3666. This strain was isolated from a soil sample collected at Okutama, Tokyo, Japan, and was identified as Chromobacterium sp. by morphological and physiological criteria. YM-254890 was purified from the culture supernatant by solvent extraction, ODS and silica gel flash chromatography, followed by preparative HPLC. YM-254890 inhibited ADP-induced platelet aggregation in human platelet-rich plasma with an IC50 value below 0.6 microM by blocking the P2Y1 receptor-signal transduction pathway.

Biochemical and pharmacological profile of darexaban, an oral direct factor Xa inhibitor.

Darexaban (YM150) is an oral factor Xa inhibitor developed for the prophylaxis of venous and arterial thromboembolic disease. This study was conducted to investigate the biochemical and pharmacological profiles of darexaban and its active metabolite darexaban glucuronide (YM-222714), which predominantly determines the antithrombotic effect after oral administration of darexaban. In vitro activity was evaluated by enzyme and coagulation assays, and a prothrombin activation assay using reconstituted prothrombinase or whole blood clot. In vivo effects were examined in venous thrombosis, arterio-venous (A-V) shunt thrombosis, and bleeding models in rats. Both darexaban and darexaban glucuronide competitively and selectively inhibited human factor Xa with Ki values of 0.031 and 0.020 µM, respectively. They showed anticoagulant activity in human plasma, with doubling concentrations of darexaban and darexaban glucuronide for prothrombin time of 1.2 and 0.95 µM, respectively. Anticoagulant activity was independent of antithrombin. Darexaban and darexaban glucuronide inhibited the prothrombin activation induced by prothrombinase complex or whole blood clot with similar potency to free factor Xa. In contrast, prothrombinase- and clot-induced prothrombin activation were resistant to inhibition by enoxaparin. In venous and A-V shunt thrombosis models in rats, darexaban strongly suppressed thrombus formation without affecting bleeding time, with ID50 values of 0.97 and 16.7 mg/kg, respectively. Warfarin also suppressed thrombus formation in these models, but caused a marked prolongation of bleeding time at antithrombotic dose. In conclusion, darexaban is a selective and direct factor Xa inhibitor and a promising oral anticoagulant for the prophylaxis and treatment of thromboembolic diseases.

Discovery of N-[2-hydroxy-6-(4-methoxybenzamido)phenyl]-4- (4-methyl-1,4-diazepan-1-yl)benzamide (darexaban, YM150) as a potent and orally available factor Xa inhibitor.

Inhibitors of factor Xa (FXa), a crucial serine protease in the coagulation cascade, have attracted a great deal of attention as a target for developing antithrombotic agents. We previously reported findings from our optimization study of a high-throughput screening (HTS) derived lead compound 1a that resulted in the discovery of potent amidine-containing FXa inhibitors represented by compound 2. We also conducted an alternative optimization study of 1a without incorporating a strong basic amidine group, which generally has an adverse effect on the pharmacokinetic profile after oral administration. Replacement of 4-methoxybenzene with a 1,4-benzodiazepine structure and introduction of a hydroxy group at the central benzene led to the discovery of the potent and orally effective factor Xa inhibitor 14i (darexaban, YM150). Subsequent extensive study revealed a unique aspect to the pharmacokinetic profile of this compound, wherein the hydroxy moiety of 14i is rapidly transformed into its glucuronide conjugate 16 (YM-222714) as an active metabolite after oral administration and it plays a major role in expression of potent anticoagulant activity in plasma. The distinctive, potent activity of inhibitor 14i after oral dosing was explained by this unique pharmacokinetic profile and its favorable membrane permeability. Compound 14i is currently undergoing clinical development for prevention and treatment of thromboembolic diseases.

Prodrug-based design, synthesis, and biological evaluation of N-benzenesulfonylpiperidine derivatives as novel, orally active factor Xa inhibitors.

We describe here our investigation of a new series of orally active fXa inhibitors based on a prodrug strategy. Solid-phase parallel synthesis identified a unique series of fXa inhibitors with a substituted benzenesulfonyl group as a novel S4 binding element. This series resulted in compound 39, which exhibited potent inhibitory activity against fXa (IC50 = 13 nM) and excellent selectivity over thrombin (>7000-fold). The masking of its highly hydrophilic groups led to the creation of related prodrug 28, which demonstrated an anticoagulant effect after oral dosing.

Design, synthesis and biological activity of selective and orally available TF/FVIIa complex inhibitors containing non-amidine P1 ligands.

We found the novel selective and orally available non-amidine TF/FVIIa complex inhibitor 21e, 4-({[(1S)-(aminocarbonyl)-3-methylbutyl]amino}carbonyl)-2'-({[4- (aminomethyl)phenyl]amino}carbonyl)-4'-(methylamino)biphenyl-2- carboxylic acid. The derivatives were synthesized by conversions of the isobutyl moiety and the introduction of alkylamino groups to 4'-position of the central phenyl ring of compounds 2a and 2b reported previously. Some compounds show increased in vitro anti-TF/FVIIa and PT prolongation activities. Among them, compound 21e reached and sustained micromolar plasma concentration levels of up to 2h after oral administration in mice. Moreover, compound 21e did not prolong the bleeding time even at the highest dose level in cynomolgus monkeys, while PT was prolonged 3.7-fold increases at this dose.

Potent and selective TF/FVIIa inhibitors containing a neutral P1 ligand.

Inhibition of tissue factor/factor VIIa complex (TF/FVIIa) is an attractive strategy for antithrombotic therapies. We began with an investigation of a non-amidine TF/FVIIa inhibitor based on a modification of amidine compound 1. Optimization of the substituents on the P1 phenyl portion of the compound 1 led to a neutral or less basic alternative for the 4-amidinophenyl moiety. By further optimization of the substituents on the central phenyl ring, a highly potent and selective TF/FVIIa inhibitor 17d was discovered.

Synthesis and biological activity of novel 1,2-disubstituted benzene derivatives as factor Xa inhibitors.

Factor Xa (fXa) is a serine protease that plays a pivotal role in the coagulation cascade. High-throughput screening of the Yamanouchi compound library yielded lead compound 1 with the ability to inhibit fXa at micromolar concentrations. To improve its fXa inhibitory activity and its oral anticoagulant activity, the linker between benzamidine and the central benzene ring was modified and a carboxyl group was introduced at the central benzene ring. The resulting compounds 40b (YM-203552), 41a (YM-202054), and 41c (YM-203558) exhibited potent fXa inhibitory activity and oral anticoagulant activity. In particular, YM-203558 exhibited the most potent oral anticoagulant activity, prolonging PT more than 3-fold at 0.5 and 2.0 h. Additionally, these compounds showed a high degree of selectivity for other serine proteases.

A novel Galphaq/11-selective inhibitor.

YM-254890, which was isolated from the culture broth of Chromobacterium sp., inhibits ADP-induced platelet aggregation and has antithrombotic and thrombolytic effects. YM-254890 blocks Galpha(q/11)-coupled ADP receptor P2Y1-mediated Ca(2+) mobilization. Here we report that YM-254890 is a selective Galpha(q/11) inhibitor. YM-254890 blocked Ca(2+) mobilization mediated by several Galpha(q/11)-coupled receptors but not by Galpha(i)- or Galpha(15)-coupled receptor, indicating that phospholipase Cbeta activation and subsequent signaling molecules are not the target of YM-254890. YM-254890 completely prevented the serum response factor (SRF)-mediated gene transcription induced by Galpha(q)R183C, which is constitutively active in a receptor-dependent manner because of its reduced k(cat) of GTP hydrolysis. Conversely, YM-254890 had only a modest effect on the SRF-mediated gene transcription by Galpha(q)Q209L, which is GTPase-deficient (activated) Galpha(q). These suggested that the acting point of YM-254890 is receptor-Galpha(q) interaction or the subsequent guanine nucleotide exchange step. The fact that YM-254890 (i) inhibited the SRF-mediated gene transcription by Galpha(qi5), which interacts with Galpha(i)-coupled receptor and possesses the effector function of Galpha(q), and (ii) had no effect on the K(d) value of high affinity [(3)H]2MeSADP binding to P2Y1, which reflects the agonist-receptor-Galpha ternary complex, suggested that receptor-Galpha(q/11) interaction is not the target of YM-254890. On the other hand, specific [(35)S]GTPgammaS binding to Galpha(q/11) stimulated by the M1 muscarinic acetylcholine receptor and P2Y1 were inhibited by YM-254890. These data indicate that YM-254890 blocks the exchange of GDP for GTP in Galpha(q/11) activation. This novel Galpha(q/11)-selective inhibitor is a promising and powerful tool for studying Galpha(q/11) protein activation, Galpha(q/11) -coupled receptor signaling, and Galpha(q/11)-mediated biological events.

Orally active factor Xa inhibitor: synthesis and biological activity of masked amidines as prodrugs of novel 1,4-diazepane derivatives.

Factor Xa (fXa) is a serine protease, which plays a pivotal role in the coagulation cascade. To improve the oral anticoagulant activity of fXa inhibitors containing a 1,4-diazepane moiety as the P4 part, a prodrug strategy was examined. Among the compounds evaluated in this study, amidoxime prodrugs bearing an ester moiety, such as compounds 21 and 30, showed effective oral anticoagulant activity in mice.

YM-254890 analogues, novel cyclic depsipeptides with Galpha(q/11) inhibitory activity from Chromobacterium sp. QS3666.

The structure elucidation and biological activity of novel YM-254890 (1) analogues and semi-synthetic derivatives are described. Three natural analogues, YM-254891 (2), YM-254892 (3), and YM-280193 (4), were isolated from the fermentation broth of Chromobacterium sp. QS3666, and two hydrogenated derivatives, YM-385780 (5) and YM-385781 (6), were synthesized from YM-254890. Their structures were determined by one- and two-dimensional NMR studies and mass spectrometry. Among these compounds, two natural analogues 2-3 which possessed acyl groups at beta-HyLeu-1 and one derivative 6 whose conformation was similar to that of 1 showed comparable Galpha(q/11) inhibitory activity to that of 1. This indicates that the acyl beta-HyLeu residue plays an important role in activity and also that the alpha,beta-unsaturated carbonyl group of the N-MeDha residue is not critical to activity. The other hydrogenated derivative 5 had significantly less activity, which could be attributed to conformational differences.

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.

The discovery of YM-60828: a potent, selective and orally-bioavailable factor Xa inhibitor.

Since Factor Xa (FXa) is well known to play a central role in thrombosis and hemostasis, inhibition of FXa is an attractive target for antithrombotic strategies. As a part of our investigation of a non-peptide, orally available FXa inhibitor, we found that a series of N-[(7-amidino-2-naphthyl)methyl]aniline derivatives possessed potent and selective inhibitory activities. Structure--activity relationship (SAR) of the substituent (R(1)) on the central aniline moiety suggested that increasing lipophilicity caused a detrimental effect on anticoagulant activity (prothrombin time assay) in plasma. Several compounds bearing a hydrophilic substituent in R(1) showed not only potent FXa inhibitory activities but also high anticoagulant activities. The best compound in this series was sulfamoylacetic acid derivative (YM-60828) which was a potent, selective and orally bioavailable FXa inhibitor and was chosen for clinical development.

Design, synthesis and biological activity of YM-60828 derivatives: potent and orally-bioavailable factor Xa inhibitors based on naphthoanilide and naphthalensulfonanilide templates.

Factor Xa (FXa) is a serine protease which plays a pivotal role in the coagulation cascade. The inhibition of FXa has received great interest as a potential target for the development of new antithrombotic drug. Herein we describe a series of novel 7-amidino-2-naphthoanilide and 7-amidino-2-naphthalensulfonanilide derivatives which are potent FXa inhibitors. These scaffolds are rigid and are allowed to adopt an L-shape conformation which was estimated as the active conformation based on a docking study of YM-60828 with FXa. Optimization of the side chain at the central aniline nitrogen of 7-amidino-2-naphthoanilide has led to several potent and orally active FXa inhibitors. 5h (YM-169964), the best compound of these series, showed potent FXa inhibitory activity (IC(50)=3.9nM) and effectively prolonged prothrombin time by 9.6-fold ex vivo at an oral dose of 3mg/kg in squirrel monkeys.

Design, synthesis and biological activity of YM-60828 derivatives. Part 2: potent and orally-bioavailable factor Xa inhibitors based on benzothiadiazine-4-one template.

Compound YM-60828 was previously characterized in our laboratory as a potent, selective and orally-bioavailable Factor Xa (FXa) inhibitor. The L-shape conformation of this compound in the active site of FXa was recognized as an important factor in displaying its FXa inhibitory activity. This led to the exploration of conformationally restricted cyclic scaffolds bearing a similar active conformation. The current study investigated a novel series of benzothiadiazine-4-one based compounds as FXa inhibitors. Structure-activity relationship (SAR) investigations revealed some potent FXa inhibitors that were selected for further in vitro and ex vivo anticoagulant studies. Among them, compound 6j (YM-169920) was proved to be most effective anticoagulant in this series. The synthesis and SAR in addition to docking studies of this class of inhibitors are described.