Sulfonamidopyrrolidinone factor Xa inhibitors: potency and selectivity enhancements via P-1 and P-4 optimization.

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa) inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxybenzamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a potent fXa inhibitor (K(i) = 2 nM) with selectivity against structurally related serine proteinases (>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in vivo (e.g. rat FeCl(2)-induced carotid artery thrombosis model).

Identification and initial structure-activity relationships of a novel class of nonpeptide inhibitors of blood coagulation factor Xa.

The discovery and some of the basic structure-activity relationships of a series of novel nonpeptide inhibitors of blood coagulation Factor Xa is described. These inhibitors are functionalized beta-alanines, exemplified by 2a. Docking experiments placing 2a in the active site of Factor Xa implied that the most expeditious route to enhancing in vitro potency was to modify the group occupying the S3 site of the enzyme. Increasing the hydrophobic contacts between the inhibitor and the enzyme in this region led to 8, which has served as the prototype for this series. In addition, an enantioselective synthesis of these substituted beta-alanines was also developed.

Low-molecular-weight heparin (fragmin) and thrombin active-site inhibitor (argatroban) compared in experimental arterial and venous thrombosis and bleeding time.

The antithrombotic and bleeding effects of a low-molecular-weight heparin (LMWH, fragmin) and a thrombin active-site inhibitor (argatroban) were determined in anesthetized rats. Occlusive thrombi were produced in the vena cava, either by partial stasis of blood flow or transmural vessel injury, and in the carotid artery by transmural vessel injury. Bleeding time was measured by puncturing small mesenteric arteries. Each drug was tested in multiple intravenous (i.v.) doses and inhibited venous and arterial thrombosis when the activated partial thromboplastin time (APTT) was increased as much as or more than twofold, although greater APTT increases were required with fragmin and against arterial thrombosis. Fragmin and argatroban decreased to an equivalent extent the weight of venous thrombi induced by stasis (> or = 99%) or vessel injury (90 and 96%, respectively). The maximum inhibition of arterial thrombosis was less with fragmin (69%) and argatroban (65%) and required higher doses of each drug relative to venous thrombosis. At doses that were just optimal against arterial thrombosis, bleeding time was increased moderately by fragmin (32%) and was unaffected by argatroban. These studies demonstrate that doses of fragmin and argatroban that exert comparable antithrombotic activity in large arteries and veins have only moderate effects on bleeding time in small arteries.

Sensitivity of experimental venous and arterial thrombosis and bleeding to ancrod-induced defibrinogenation.

The effect of ancrod-induced defibrinogenation on thrombosis and bleeding time was determined in anesthetized rats. Functional plasma fibrinogen levels were reduced 42, 71, 94 and 93% by ancrod doses of 5, 10, 20 and 30 U/kg, respectively, while a 2.5 U/kg dose was without significant effect. Ancrod inhibited vena cava thrombosis induced by partial stasis of blood flow combined with mild vascular injury. Thrombus weight was decreased 85 and 93% by the 10 and 20 U/kg doses, but was unaffected at lower doses. In contrast, ancrod doses of up to 30 U/kg did not significantly decrease carotid artery thrombi formed in response to oxidative transmural vessel injury. Ancrod caused a dose-dependent increase in bleeding time measured by puncturing small mesenteric arteries with a hypodermic needle. The bleeding time increase was approximately 38% in response to the 2.5 and 5 U/kg doses, and 182% in response to the 10 U/kg dose. These studies demonstrate that ancrod-induced reductions in plasma fibrinogen more effectively inhibit venous compared to arterial thrombosis, although these activities require doses that also increase bleeding time in small arteries.

Comparison of thrombin active site and exosite inhibitors and heparin in experimental models of arterial and venous thrombosis and bleeding.

Different pharmacological approaches to thrombin inhibition were compared for their effects on thrombosis and bleeding time in anesthetized rats. Thrombosis was induced in the carotid artery by transmural vessel injury and in the vena cava by partial blood flow stasis combined with mild endothelial disruption. Small mesenteric arteries were punctured with a hypodermic needle to measure the bleeding time. Dose-response relationships were determined with a thrombin active site inhibitor, N-methyl (GYKI 14,766); a thrombin exosite inhibitor, succinyl-Phe-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-Gln (BMS 180,742); and heparin. BMS 180,742 interferes with fibrinogen binding to the thrombin exosite but, unlike GYKI 14,766, it does not block thrombin's catalytic site. The effects on thrombosis and bleeding time were correlated with ex vivo clotting times using the activated partial thromboplastin time for heparin and the thrombin time for GYKI 14,766 and BMS 180,742. Venous thrombosis was inhibited more than 90% by all three inhibitors at doses that either produced threshold increases or had no effect on bleeding and clotting times. Arterial thrombosis was inhibited 82% by GYKI 14,766 and 63% by heparin but it was not inhibited by BMS 180,742. These antithrombotic activities were accompanied by a maximal activated partial thromboplastin time increase and doubling of the bleeding time with heparin and a maximal thrombin time prolongation and 35% increase in bleeding time with GYKI 14,766. These results suggest that thrombin inhibitors, which act at the active site or exosite or through antithrombin III, are equally efficacious against venous thrombosis but active site inhibitors are the most effective against arterial thrombosis.

Superior activity of a thromboxane receptor antagonist as compared with aspirin in rat models of arterial and venous thrombosis.

We determined the effects of aspirin and a novel thromboxane A2/prostaglandin endoperoxide (TP)-receptor antagonist, BMS-180291, on thrombosis and bleeding times in skin and mesenteric arteries. In anesthetized rats, occlusive thrombosis was induced in the carotid artery by topical application of ferrous chloride and in the vena cava by blood flow stasis combined with either infusion of thromboplastin or hypotonic saline. Aspirin (1, 10, and 50 mg/kg) did not reduce arterial or venous thrombus weight significantly. BMS 180,291 (150 micrograms/kg/min) decreased arterial thrombus weight and hypotonic saline-induced caval thrombus weight by 58 and 57%, respectively. BMS-180291 lacked antithrombotic activity at a lower dose (50 micrograms/kg/min) and failed to inhibit thromboplastin-induced caval thrombosis. BMS-180291 (150 micrograms/kg/min) significantly reduced arterial thrombus weight by 40% when plasma epinephrine concentration was increased to 5 ng/ml. BMS-180291 and aspirin produced increases of only < or = 30% in bleeding times. These results demonstrate that BMS-180291 has antithrombotic activity in experimental aspirin-resistant arterial and venous thrombosis. Both aspirin and BMS-180291 have only modest effects on small artery hemostasis in rats.

Comparison of a thromboxane receptor antagonist and aspirin in experimental arterial thrombosis.

The antithrombotic activities of aspirin, the thromboxane (Tx) A2/prostaglandin endoperoxide-receptor (TP-receptor) antagonist, SQ 30,741, and heparin were determined in anesthetized rats. Heparin (3 doses of 50, 300 U/kg), aspirin (1 and 10 mg/kg), SQ 30,741 (1 mg/kg + 1 mg/kg/h), or the combination of SQ 30,741 and aspirin (10 mg/kg) was administered intravenously before inducing occlusive thrombosis with 0.1-mA stimulation of the intimal surface of the carotid artery. Light and electron microscopy revealed the thrombi to be composed predominantly of platelets enmeshed in a fibrin network. Heparin (300 U/kg), SQ 30,741 and SQ 30,741 + aspirin decreased average thrombus weight by 54, 57 and 39%, respectively. These treatments also reduced the incidence of occlusion and improved carotid blood flow during thrombosis. In contrast, aspirin alone (1 and 10 mg/kg) and the lower heparin dose (50 U/kg) did not significantly affect thrombus weight or carotid blood flow. To verify adequate drug dosage, pharmacological activities were characterized ex vivo in separate rats. Aspirin (10 mg/kg) inhibited maximum thromboxane (Tx) B2 production in whole blood by 99 +/- 1% and SQ 30,741 blocked 96% of platelet TP-receptors. Heparin increased the activated partial thromboplastin time (APTT) partially at 50 U/kg (approximately 3-fold) and maximally at 300 U/kg (> 10-fold). These experiments demonstrate the contribution of platelet and coagulation mechanisms to a thrombosis model which is sensitive to a TP-receptor antagonist, but not aspirin.

Effects of antithrombotic drugs in a rat model of aspirin-insensitive arterial thrombosis.

These studies describe experimental conditions where aspirin is less effective than other antiplatelet and anticoagulant drugs in inhibiting acute arterial thrombosis. External electrolytic injury of the rat carotid artery was used to induce occlusive thrombi in 97% of vehicle-treated rats. Thrombi were revealed by light and electron microscopy to be comprised primarily of platelets enmeshed in a fibrin network. The thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (PPACK; 6 mg/kg, i.v.) decreased thrombus weight by 90%. Aspirin alone (1, 10 and 30 mg/kg, i.v.), dipyridamole alone (5 mg/kg i.v.) and aspirin (1 and 10 mg/kg, i.v.) in combination with dipyridamole (5 mg/kg, i.v.) did not inhibit thrombosis. The platelet-activating factor (PAF) antagonist, WEB 2086, (1 mg/kg i.v.) was also ineffective. Other drugs had intermediate activity. Thrombi were decreased 56% by the thromboxane receptor antagonist, BMS 180,291, either alone (5.8 mg/kg i.v.) or in combination with aspirin (10 mg/kg, i.v.). Heparin (900 U/kg, i.v.), warfarin (0.25 mg/kg, p.o. once daily for 3 days) and ticlopidine (200 mg/kg, p.o. once daily for 3 days) reduced thrombus weight by 63, 73 and 43% respectively. Reductions in thrombus weight were always associated with improvements in either average blood flow or vessel patency.

Activity of the short-acting thromboxane receptor antagonist, SQ 30,741, in thrombolytic and vasospastic models in monkeys.

The effects of the thromboxane receptor antagonist SQ 30,741 (1 mg/kg) on reflow after thrombolysis and on vasoconstrictor responses to the thromboxane agonist U-46,619 was determined in cynomolgus monkeys. SQ 30,741 (n = 5) or vehicle (n = 4) was administered to anesthetized monkeys upon reocclusion of a stenotic and electrically injured carotid artery, which had been recanalized successfully with streptokinase and heparin. Once blood flow again decreased to zero the treatment was repeated. SQ 30,741 significantly (P less than .05) enhanced reflow by 113% after the first administration and by 150% after the second administration. The respective times to each reocclusion were greater after SQ30,741 (49 +/- 9 and 61 +/- 23 min; P less than .01 and P less than .05) than with vehicle (10 +/- 3 and 15 +/- 2 min). The potency of SQ 30,741 was demonstrated in other anesthetized monkeys by a 8.5 +/- 1.1-fold (n = 3) shift to the right in the U-46,619 dose-response for renal vasoconstriction. The effect of SQ 30,741 (n = 5) on pre-existing renal vasoconstriction was determined using conscious monkeys in which an individually tailored dose of U-46,619 was chosen to sustain an average 82% reduction in blood flow. An arterial injection of SQ 30,741 rapidly returned flow to base-line values, but this antagonism was limited in duration, and flow again reached the nadir within 46 +/- 7 min of continuous U-46,619 infusion. The abbreviated duration of the biological activity of SQ 30,741 in vivo was consistent with its short plasma T1/2 (9.5 +/- 1.3 min; n = 3) determined in separate unanesthetized monkeys by a radioreceptor assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Protamine-induced pulmonary hypertension in heparinized monkeys and pigs is inhibited by the thromboxane receptor antagonist SQ 30,741.

When protamine reverses heparin anticoagulation a small fraction of patients develops pulmonary hypertension. This response is variably expressed in other species and thromboxane may be one of its mediators. We have compared the pulmonary vascular responses of pigs and monkeys to protamine (3 mg/kg, i.v.) administered 15 min after heparin (300 U/kg, i.v.). The role of thromboxane A2/prostaglandin H2 (TxA2/PGH2)-receptor activation in this response was investigated with the selective TxA2/PGH2-receptor antagonist, SQ 30,741, at a dose (1 mg/kg, i.v.) shown to inhibit U-46,619-induced pulmonary vasoconstriction by greater than or equal to 99%. SQ 30,741 or vehicle (1.5 ml saline) was given 2 min before protamine in Yucatan minipigs (n = 6-7) and African green monkeys (n = 8-9). In saline-treated monkeys and pigs, protamine increased pulmonary vascular resistance (131 +/- 46 and 478 +/- 18%, respectively) primarily by increasing pulmonary artery pressures (54 +/- 19 and 166 +/- 42%, respectively). In pigs only, pulmonary artery flow was also reduced by 33 +/- 9%. These responses peaked within 1 to 3 min and returned to baseline in approximately 5 (monkey) and approximately 15 (pig) min. In monkeys and pigs pretreated with SQ 30,741 the increases in pulmonary vascular resistance (17 +/- 4 and 16 +/- 9%, respectively, p less than 0.05) and pulmonary artery pressure (10 +/- 3 and 16 +/- 9%, respectively, p less than 0.05) were significantly inhibited. SQ 30,741 also accelerated reversal of established hypertension in pigs when given 1 min after protamine. However, transient reductions in circulating monkey leukocytes (approximately 70%) and platelets (approximately 16%) were unaffected by SQ 30,741.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukotrienes cause mesenteric vasoconstriction and hemoconcentration in rats without activating thromboxane receptors.

Thromboxane A2/prostaglandin H2 (TP)-receptor activation has been reported to participate in some of the responses to peptide leukotrienes (LT). We examined the effect of TP-receptor antagonism on LT-induced mesenteric vasoconstriction and hemoconcentration in anesthetized rats. The antagonist used in these studies, SQ 30,741, was shown to have high selectivity and potency for vascular TP-receptors in the rat. Arterial (i.a.) injection of LTC4 and D4 elicited dose-dependent and transient reductions in mesenteric blood flow without changes in arterial blood pressure. These responses were unaffected by a dose of SQ 30,741 which produced approximately 99% inhibition of similar responses to U-46,619. In contrast, LT-induced mesenteric vasoconstriction was inhibited approximately 90% by two LT antagonists, LY 171,883 and SKF 104,353. In other experiments i.v. infusion of LTD4 caused increases in hematocrit and reductions in arterial blood pressure that were not influenced by SQ 30,741. These data suggest that increases in mesenteric vascular resistance and hemoconcentration in response to LTs are not the result of TP-receptor activation.

Effect of thromboxane antagonism on recanalization during streptokinase-induced thrombolysis in anesthetized monkeys.

The effect of a selective thromboxane A2 (TxA2) receptor antagonist, SQ 30,741, on streptokinase-induced thrombolysis was examined in pentobarbital-anesthetized cynomolgus monkeys. The intimal surface of a stenosed carotid artery was stimulated with 100 microA anodal current to produce an occlusive thrombus. After 45 min of zero blood flow, a 1-h intraarterial (i.a.) infusion of streptokinase (680 U/min) was injected proximal to the thrombus. Five minutes before streptokinase (SK) intravenous (i.v.) infusions of heparin (200 U/kg + 120 U/h) and either SQ 30,741 (2 mg/kg + 2 mg/kg/min, n = 8) or vehicle (1 ml/h saline, n = 7) were started and maintained for 3 h. In four monkeys not given streptokinase or heparin, no recanalization was detected and occlusive thrombi were observed after 3 h. All animals receiving streptokinase were recanalized. SQ 30,741 had no effect on return of flow during streptokinase infusion, but increased average reflows during the second (60%, p less than 0.05) and third hours (159%, p less than 0.01). Average blood flows were decreased from the second to third hours with vehicle (p less than 0.001) and remained stable with SQ 30,741. Thromboxane antagonism also increased minimal blood flows during the third hour (438%, p less than 0.01) and decreased the total time reoccluded by 73% (p less than 0.05). However, SQ 30,741 had no effect on the time to recanalization, the maximum reflow, and both number of animals reoccluded and average number of reocclusions. Fibrinogen levels were equivalently diminished (8%, p less than 0.05), and platelet counts were unaffected in both treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Magnitude of thromboxane receptor antagonism necessary for antithrombotic activity in monkeys.

SQ 30741 was characterized as a competitive antagonist of thromboxane receptor-mediated platelet activation in vitro that does not inhibit the activity of enzymes involved in prostaglandin, prostacyclin, and thromboxane biosynthesis. The threshold intravenous dose for antithrombotic activity was measured in anesthetized monkeys as the minimum amount of SQ 30741 required to inhibit thrombotic cyclic blood flow reductions in stenotic renal arteries. Platelet responsiveness was measured ex vivo before and during inhibition of thrombosis by the shape-change response to a thromboxane mimetic, U 46619. The threshold antithrombotic SQ 30741 dose (0.32 +/- 0.04 mg/kg; n = 5) was accompanied by an 8.5 +/- 1.1-fold shift to the right of the U 46619 concentration-effect curve, implying antagonism of 87 +/- 3% of platelet thromboxane receptors. The antithrombotic activity of SQ 30741 persisted for 109 +/- 10 min and was not reversed by indomethacin. However, in two out of seven monkeys SQ 30741 (7 mg/kg iv) did not interrupt the cyclical flow reductions. Vehicle treatment did not impede thrombosis and caused a 1.4 +/- 0.3-fold shift of the U 46619 concentration-effect curve (n = 4). In separate monkeys, SQ 30741 (0.33 mg/kg iv) produced identical dose ratios (8.6 +/- 0.7, n = 8) for inhibition of U 46619-induced mesenteric vasoconstriction. Thus the threshold antithrombotic dose of SQ 30741 caused the same magnitude of antagonism of platelet (ex vivo) and vascular (in vivo) thromboxane receptors.

Effect of thromboxane receptor antagonists on venous thrombosis in rats.

The activities of two structurally unrelated thromboxane/prostaglandin endoperoxide receptor antagonists, SQ 30,741 and BM 13,505, were compared to heparin in a model of venous thrombosis. A combination of blood stasis with osmotic and pressure stress was used to induce thrombus formation in the vena cava of anesthetized rats. Intravenous infusions of SQ 30,741 (500 micrograms/kg/min) and BM 13,505 (50 micrograms/kg/min) produced significant (P less than .01) and equivalent reductions in thrombus mass of 58 and 56%, respectively. Thrombus reduction in response to heparin (50 U/kg) was greater (95%; P less than .001) than in response to the thromboxane antagonists. Either lower doses of SQ 30,741 (50 and 100 micrograms/kg/min) or aspirin (30 and 60 mg/kg) were ineffective in altering thrombus formation. However, a subthreshold dose of SQ 30,741 (100 micrograms/kg/min) increased (P less than .01) the antithrombotic activity obtained with both threshold (0.5 U/kg) and subthreshold (0.3 U/kg) doses of heparin. SQ 30,741 (500 micrograms/kg/min) did not change activated partial thromboplastin times or inhibit platelet loss induced by contact activation in response to kaolin in vivo. This suggests that SQ 30,741 does not interfere with components of the coagulation cascade that are not dependent on platelet factors. The extent of thromboxane antagonism achieved with SQ 30,741 (50 and 500 micrograms/kg/min) and BM 13,505 (50 micrograms/kg/min) was determined from parallel shifts in dose-dependent U-46,619-induced vasoconstriction in vivo (approximately 200- and 1300-fold, respectively, for SQ 30,741 and 200-fold for BM 13,505). These data demonstrate that thromboxane antagonists inhibit venous thrombosis partially, but only at doses producing near complete receptor inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombotic effects of the thromboxane A2 antagonist, SQ 28,668, in vitro and in the coronary circulation in vivo.

SQ 28,668 is a thromboxane receptor antagonist. In whole blood and platelet-rich plasma, micromolar concentrations of SQ 28,668 inhibited aggregation of human and porcine platelets induced by arachidonate and U-46,619, and the secondary phase of epinephrine-induced aggregation of human platelets. SQ 28,668 did not inhibit ADP-induced platelet aggregation and did not influence cAMP concentrations. High concentrations of SQ 28,668 inhibited platelet thromboxane synthase (IC50 = 660 microM), but SQ 28,668 did not inhibit cyclooxygenase or prostacyclin synthetase. In anesthetized pigs with pacing-induced tachycardia, placement of a critical stenosis at a focus of coronary vascular injury initiated a reproducible pattern of coronary blood flow reduction, reflecting thrombus formation at the site of injury. Treatment with SQ 28,668 (1.0 mg/kg, i.v.) abolished reductions in coronary blood flow in five of six pigs for 58 +/- 7 min and slowed the rate of blood flow reduction in the sixth. Before SQ 28,668 treatment, the rate of blood-flow reduction averaged 5.3 +/- 0.7 ml/min per min. Thirty and sixty minutes after treatment, the rates of blood-flow reduction averaged 0.6 +/- 0.6 and 2.1 +/- 1.0 ml/min per min, respectively (P less than 0.05). In control pigs, the rate of blood flow reduction before and 30 min after vehicle treatment averaged 5.7 +/- 2.0 and 5.5 +/- 1.6 ml/min per min, respectively (not significant). These findings indicate that SQ 28,668 is an effective antiplatelet agent in vitro and in vivo.

Comparison of the acute and chronic hemodynamic effects of captopril and guanethidine in spontaneously hypertensive rats.

The present study compared the hemodynamic effects of captopril with those of guanethidine as well as with a combination of these two drugs in conscious male spontaneously hypertensive rats. Acutely, captopril or guanethidine or a combination of the two lowered arterial pressure to similar levels. The reduction in pressure with captopril alone or in combination with guanethidine, was due to a lowering of all organ vascular resistances. Guanethidine alone lowered pressure by reducing cardiac output. Chronically, captopril or guanethidine lowered arterial pressure to similar levels by reducing organ vascular resistances to comparable levels, particularly skeletal muscle, skin and splanchnic organs. Combination of these two drugs had an additive effect on the reduction of arterial pressure and organ vascular resistances. Since, chronically, the addition of guanethidine to captopril therapy enhanced rather than attenuated the hemodynamic changes induced by captopril, these drugs are probably acting by different mechanisms and the mechanisms of action of captopril does not require an intact sympathetic nervous system.

Effects of SQ 14,225, an orally active inhibitor of angiotensin-converting enzyme on blood pressure, heart rate and plasma renin activity of conscious normotensive dogs.

Oral administration of SQ 14,225 (0.03--3 mg/kg) to conscious normotensive dogs caused inhibition of the pressor response to intravenously administered angiotensin I (AI), the duration of which was dose-dependent. All doses of 0.1 mg/kg or greater caused 85--95% inhibition 30 min after administration whereas 0.03 mg/kg produced only a 25% inhibition. Pressor responses to angiotensin II (AII) were not similarly inhibited. Blood pressure was moderately reduced in a dose-related manner and followed the same pattern as inhibition of the AI pressor responses. The maximum change occurred after 1.0 mg/kg with only a more rapid onset occurring after the 3.0 mg/kg dose. Heart rate was not appreciably changed. SQ 14,225 also increased plasma renin activity (PRA), the levels and duration of which were dose-related. These data indicate that SQ 14,225 is an orally effective, potent inhibitor of angiotensin I-converting enzyme (ACE) in dogs. It appears that in mongrel dogs, ACE inhibition results in a slight to moderate reduction in blood pressure and an increase in PRA.