Potent, orally active GPIIb/IIIa antagonists containing a nipecotic acid subunit. Structure-activity studies leading to the discovery of RWJ-53308.

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 (significantly 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.

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases.

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca2+]i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca2+]i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca2+]i studies. The kinetic profile for [Ca2+]i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca2+]i in guinea pigs, had no effect in rat, and increased [Ca2+]i in all other species. Staurosporine inhibited SFLLRN-induced [Ca2+]i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca2+]i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.

Antiplatelet and antithrombotic activity of RWJ-53308, a novel orally active glycoprotein IIb/IIIa antagonist.

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.

Positive inotropic and hemodynamic properties of flosequinan, a new vasodilator, and a sulfone metabolite.

Flosequinan, a new orally active vasodilator, and its sulfone metabolite were evaluated for inotropic activity in isolated ferret papillary muscles and pentobarbital anesthetized open-chest dogs. In vitro, flosequinan and its sulfone derivative increased tension development in a concentration-dependent manner (1-50 microM) in electrically stimulated papillary muscles pretreated with the beta-adrenergic blocking agent atenolol (2 microM). Peak increases in tension of 75 +/- 17%, and 111 +/- 46% with potencies (EC50) of 15 and 10 microM were observed for flosequinan and its metabolite, respectively. In vivo, flosequinan increased left ventricular dP/dtmax (74 +/- 12%) and right ventricular contractile force (CF) (104 +/- 10%) after administration of 1.875 mg/kg, i.v. Inotropic activity was dose-dependent and remained elevated for at least 60 min postinfusion. Flosequinan also increased heart rate (HR) (14 +/- 2%) and reduced mean arterial pressure (-9 +/- 3%). The i.v. potency of flosequinan (ED50 = 0.45 mg/kg) and its metabolite (ED50 = 0.38 mg/kg) were similar to that of the inotropic vasodilator amrinone (ED50 = 0.38 mg/kg). Inotropic activity was not significantly altered by pretreatment with propranolol (0.5 mg/kg) and atropine (1.0 mg/kg), further supporting the in vitro data indicating that flosequinan can directly stimulate myocardial contractility independent of beta-adrenergic receptor activation. Additional hemodynamic studies were conducted in an acute heart failure model produced by an overdose of propranolol. Flosequinan (2 mg/kg, i.v.) increased cardiac output (CO) (50 +/- 9%) and stroke volume (SV) (29 +/- 8%) while reducing total peripheral vascular resistance (TPR) (-36 +/- 4%), right atrial pressure (-62 +/- 5%), and left ventricular end-diastolic pressure (LVEDP) (-41 +/- 2%).(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular profile of RWJ 29009, a new potassium channel activator, in anesthetized and conscious dogs.

RWJ-29009, (6S)-trans(-)-1-(6,7-dihydro-6-hydroxy-5,5-dimethyl-2-nitro-5 H-thieno[3,2-b]pyran-7-yl)-2-piperidinone, is a structurally novel and extremely potent potassium channel activator that may be useful for treatment of hypertension and ischemic heart disease. We assessed the cardiovascular profile of RWJ 29009 in anesthetized and conscious dogs. RWJ 29009 (0.1-2 micrograms/kg intravenously, i.v.) dose-relatedly increased coronary blood flow (CBF) and decreased arterial pressure in anesthetized dogs. Total peripheral resistance and coronary vascular resistance were concurrently reduced without significant changes in heart rate (HR) or cardiac output (CO). Left ventricular (LV) dP/dtmax and myocardial contractile force were decreased only at the highest dose of 10 micrograms/kg. Cromakalim (3-100 micrograms/kg), although much less potent, had a qualitatively similar profile. Glyburide pretreatment (5 mg/kg i.v.) shifted the dose response of RWJ 29009 for increasing CBF and decreasing arterial pressure to the right. The dose responses of cromakalim were similarly shifted to the right, whereas the effects of nifedipine on CBF and arterial pressure were not affected by glyburide. RWJ 29009 (0.3 and 1 microgram/kg) had no effect on myocardial O2 consumption (MVO2) except for a transient increase immediately after administration of 1 microgram/kg. MVO2 returned to control 15 min after dosing, although CBF remained significantly increased. In conscious dogs, RWJ 29009 (0.3-10 micrograms/kg, i.v. and orally, p.o.) produced dose-related increases in CBF and decreases in arterial pressure similar to those produced in anesthetized dogs, except that HR was increased concurrently. The i.v. and p.o. potency of RWJ 29009 were comparable, indicating high oral bioavailability. Thus, RWJ 29009 is an extremely potent coronary and peripheral vasodilator with a cardiovascular profile similar to that of other potassium channel activators. Like those of other potassium channel activators, its mechanism of action appears to involve activation of ATP-regulated potassium channels.

Novel thieno[2,3-b]- and [3,4-b]pyrans as potassium channel openers. Thiophene systems--XVII.

The syntheses and antihypertensive activity of the thieno[3,4-b]pyran and thieno[2,3-b]pyran isosteres of the potassium channel opener (PCO) RWJ 26629 (+/- 2a) are reported. While the unsubstituted thiophene derivatives were active at 20 mg/kg, introduction of a strong electron withdrawing group in the 2-position of the thieno[3,2-b] series increased potency. Similar substitution on the thieno[3,4-b] series significantly lowered potency. Compounds 26 and 30 are approximately 5-fold more potent than the prototypic PCO cromakalim (+/- 1).

Administration of a potent antagonist of protease-activated receptor-1 (PAR-1) attenuates vascular restenosis following balloon angioplasty in rats.

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.