In vitro, antithrombotic and bleeding time studies of BMS-654457, a small-molecule, reversible and direct inhibitor of factor XIa.

BMS-654457 ((+) 3'-(6-carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-quinolin-2-yl)-4-carbamoyl-5'-(3-methyl-butyrylamino)-biphenyl-2-carboxylic acid) is a small-molecule factor XIa (FXIa) inhibitor. We evaluated the in vitro properties of BMS-654457 and its in vivo activities in rabbit models of electrolytic-induced carotid arterial thrombosis and cuticle bleeding time (BT). Kinetic studies conducted in vitro with a chromogenic substrate demonstrated that BMS-654457 is a reversible and competitive inhibitor for FXIa. BMS-654457 increased activated partial thromboplastin time (aPTT) without changing prothrombin time. It was equipotent in prolonging the plasma aPTT in human and rabbit, and less potent in rat and dog. It did not alter platelet aggregation to ADP, arachidonic acid and collagen. In vivo, BMS-654457 or vehicle was given IV prior to initiation of thrombosis or cuticle transection. Preservation of integrated carotid blood flow over 90 min (iCBF, % control) was used as a marker of antithrombotic efficacy. BMS-654457 at 0.37 mg/kg + 0.27 mg/kg/h produced almost 90 % preservation of iCBF compared to its vehicle (87 ± 10 and 16 ± 3 %, respectively, n = 6 per group) and increased BT by 1.2 ± 0.04-fold (P < 0.05). At a higher dose (1.1 mg/kg + 0.8 mg/kg/h), BMS-654457 increased BT by 1.33 ± 0.08-fold. This compares favorably to equivalent antithrombotic doses of reference anticoagulants (warfarin and dabigatran) and antiplatelet agents (clopidogrel and prasugrel) which produced four- to six-fold BT increases in the same model. In summary, BMS-654457 was effective in the prevention of arterial thrombosis in rabbits with limited effects on BT. This study supports inhibition of FXIa, with a small-molecule, reversible and direct inhibitor as a promising antithrombotic therapy with a wide therapeutic window.

Identification of 1-{2-[4-chloro-1'-(2,2-dimethylpropyl)-7-hydroxy-1,2-dihydrospiro[indole-3,4'-piperidine]-1-yl]phenyl}-3-{5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl}urea, a potent, efficacious and orally bioavailable P2Y(1) antagonist as an antiplatelet agent.

Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y1 receptor. Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spiropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in the identification of compound 3e, a potent, orally bioavailable P2Y1 antagonist with a suitable PK profile in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved bleeding risk profile compared to the P2Y12 antagonist clopidogrel in rat efficacy/bleeding models.

Discovery of Potent and Selective Quinoxaline-Based Protease-Activated Receptor 4 (PAR4) Antagonists for the Prevention of Arterial Thrombosis.

PAR4 is a promising antithrombotic target with potential for separation of efficacy from bleeding risk relative to current antiplatelet therapies. In an effort to discover a novel PAR4 antagonist chemotype, a quinoxaline-based HTS hit 3 with low µM potency was identified. Optimization of the HTS hit through the use of positional SAR scanning and the design of conformationally constrained cores led to the discovery of a quinoxaline-benzothiazole series as potent and selective PAR4 antagonists. The lead compound 48, possessing a 2 nM IC50 against PAR4 activation by γ-thrombin in platelet-rich plasma (PRP) and greater than 2500-fold selectivity versus PAR1, demonstrated robust antithrombotic efficacy and minimal bleeding in the cynomolgus monkey models.

Discovery of nonbenzamidine factor VIIa inhibitors using a biaryl acid scaffold.

In this Letter, we describe the synthesis of several nonamidine analogs of biaryl acid factor VIIa inhibitor 1 containing weakly basic or nonbasic P1 groups. 2-Aminoisoquinoline was found to be an excellent surrogate for the benzamidine group (compound 2) wherein potent inhibition of factor VIIa is maintained relative to most other related serine proteases. In an unanticipated result, the m-benzamide P1 (compounds 21a and 21b) proved to be a viable benzamidine replacement, albeit with a 20-40 fold loss in potency against factor VIIa.

Discovery of diarylurea P2Y(1) antagonists with improved aqueous solubility.

Preclinical data suggests that P2Y1 antagonists, such as diarylurea compound 1, may provide antithrombotic efficacy similar to P2Y12 antagonists and may have the potential of providing reduced bleeding liabilities. This manuscript describes a series of diarylureas bearing solublizing amine side chains as potent P2Y1 antagonists. Among them, compounds 2l and 3h had improved aqueous solubility and maintained antiplatelet activity compared with compound 1. Compound 2l was moderately efficacious in both rat and rabbit thrombosis models and had a moderate prolongation of bleeding time in rats similar to that of compound 1.

A platelet target for venous thrombosis? P2Y1 deletion or antagonism protects mice from vena cava thrombosis.

A role for platelets in the pathogenesis of venous thrombosis was suggested by clinical and preclinical studies. However, examination of the platelet receptor, P2Y1, in this area has been limited. The goal of the current study was to examine effects of P2Y1 deletion, or selective antagonism with MRS2500, in oxidative venous thrombosis in mice. The P2Y12 antagonist, clopidogrel, was included as a reference agent. Anesthetized C57BL/6 or genetically modified mice underwent 3.5 or 5 % FeCl(3)-induced vena cava thrombosis. Pharmacokinetic properties of MRS2500 were defined for dose selection. Platelet aggregation and renal or tail bleeding times (BT) were measured to put antithrombotic effects into perspective. P2Y1 deletion significantly reduced (p < 0.001) venous thrombus weight by 74 % in 3.5 % FeCl(3) injury compared to P2Y1(+/+) littermates. MRS2500 (2 mg/kg, i.v.) significantly decreased (p < 0.001) thrombus weight 64 % in C57BL/6 mice. In the more severe 5 % FeCl(3)-induced injury model, thrombus weight significantly (p < 0.001) decreased 68 % in P2Y1(-/-) mice versus P2Y1(+/+) mice, and MRS2500 (2 mg/kg) was also beneficial (54 % decrease, p < 0.01). Renal BT doubled in P2Y1(-/-) versus P2Y1(+/+) mice, and increased threefold with MRS2500 compared to vehicle. Tail BT was markedly prolonged in P2Y1(-/-) mice (7.9X) and in C57BL/6 mice given MRS2500. The current study demonstrates that P2Y1 deletion or antagonism significantly reduced venous thrombosis in mice, suggesting that P2Y1 receptors play a role in the pathogenesis of venous thrombosis, at least in this species. However as with many antithrombotic agents the benefit comes at the potential price of an increase in provoked bleeding times.

Inhibition of factor XIa as a new approach to anticoagulation.

The dose-limiting issue with available anticoagulant therapies is bleeding. Is there an approach that could provide antithrombotic protection with reduced bleeding? One hypothesis is that targeting proteases upstream from the common pathway provides a reduction in thrombin sufficient to impede occlusive thrombosis yet allows enough thrombin generation to support hemostasis. The impairment of intrinsic coagulation by selective inhibition of factor XI (FXI) leaves the extrinsic and common pathways of coagulation intact, making FXI a drug target. This concept is supported by the observation that human deficiency in FXI results in a mild bleeding disorder compared with other coagulation factor deficiencies, and that elevated levels of FXI are a risk factor for thromboembolic disease. Moreover, FXI knockout mice have reduced thrombosis with little effect on hemostasis. The results from genetic models have been supported by studies using neutralizing antibodies, peptide inhibitors, and small-molecule inhibitors. These agents impede thrombosis without affecting bleeding time in a variety of experimental animals, including primates. Together, these data strongly support FXIa inhibition as a viable method to increase the ratio of benefit to risk in an antithrombotic drug.

Arylsulfonamidopiperidone derivatives as a novel class of factor Xa inhibitors.

The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and selective FXa inhibitors is reported. The arylsulfonamide-valerolactam scaffold was derived based on the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC(50) of 7 nM and EC(2×PT) of 1.7 µM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide-valerolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4 pockets.

A small-molecule factor XIa inhibitor produces antithrombotic efficacy with minimal bleeding time prolongation in rabbits.

BMS-262084 is a 4-carboxy-2-azetidinone-containing irreversible inhibitor of FXIa, which is selective over other coagulation proteases. We evaluated the in vitro and in vivo properties of BMS-262084 in rabbits. Studies were conducted in arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT), electrolytic-mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. BMS-262084 was infused IV from 1 h before thrombus induction or cuticle transection to the end of the experiment. In vitro, BMS-262084 prolonged activated partial thromboplastin time (aPTT) with EC(2x) (concentration required to double aPTT) of 10.6 µM in rabbit plasma, and did not prolong prothrombin time (PT), thrombin time (TT) and HepTest. In vivo, BMS-262084 produced dose-dependent antithrombotic effects in rabbits with antithrombotic ED(50) (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT of 0.4, 0.7 and 1.5 mg/kg/h IV, respectively. BMS-262084 increased ex vivo aPTT dose-dependently without changes in PT and TT. The antithrombotic effect of BMS-262084 was significantly correlated with its ex vivo aPTT, supporting the use of ex vivo aPTT as a pharmacodynamic biomarker. BMS-262084 did not alter ex vivo rabbit platelet aggregation to ADP and collagen. BT (fold-increase) determined at 3 and 10 mg/kg/h of BMS-262084 were 1.17 ± 0.04 and 1.52 ± 0.07*, respectively (*P < 0.05 vs. control). This study demonstrated that BMS-262084 prevented experimental thrombosis at doses with low BT effects in rabbits, and suggests that a small molecule FXIa inhibitor may represent a promising antithrombotic therapy.

Identification of a Hydroxypyrimidinone Compound (21) as a Potent APJ Receptor Agonist for the Potential Treatment of Heart Failure.

This paper describes our continued efforts in the area of small-molecule apelin receptor agonists. Recently disclosed compound 2 showed an acceptable metabolic stability but demonstrated monodemethylation of the dimethoxyphenyl group to generate atropisomer metabolites in vitro. In this article, we extended the structure-activity relationship at the C2 position that led to the identification of potent pyrazole analogues with excellent metabolic stability. Due to the increased polarity at C2, the permeability for these compounds decreased. Further adjustment of the polarity by replacing the N1 2,6-dimethoxyphenyl group with a 2,6-diethylphenyl group and reoptimization for the potency of the C5 pyrroloamides resulted in potent compounds with improved permeability. Compound 21 displayed excellent pharmacokinetic profiles in rat, monkey, and dog models and robust pharmacodynamic efficacy in the rodent heart failure model. Compound 21 also showed an acceptable safety profile in preclinical toxicology studies and was selected as a backup development candidate for the program.

Identification of 6-Hydroxypyrimidin-4(1H)-one-3-carboxamides as Potent and Orally Active APJ Receptor Agonists.

The apelin receptor (APJ) is a significant regulator of cardiovascular function and is involved in heart failure and other cardiovascular diseases. (Pyr1)apelin-13 is one of the endogenous agonists of the APJ receptor. Administration of (Pyr1)apelin-13 increases cardiac output in preclinical models and humans. Recently we disclosed clinical lead BMS-986224 (1), a C3 oxadiazole pyridinone APJ receptor agonist with robust pharmacodynamic effects similar to (Pyr1)apelin-13 in an acute rat pressure-volume loop model. Herein we describe the structure-activity relationship of the carboxamides as oxadiazole bioisosteres at C3 of the pyridinone core and C5 of the respective pyrimidinone core. This study led to the identification of structurally differentiated 6-hydroxypyrimidin-4(1H)-one-3-carboxamide 14a with pharmacodynamic effects comparable to those of compound 1.

Discovery of a Hydroxypyridinone APJ Receptor Agonist as a Clinical Candidate.

Apelin-13 is an endogenous peptidic agonist of the apelin receptor (APJ) receptor with the potential for improving cardiac function in heart failure patients. However, the low plasma stability of apelin-13 necessitates continuous intravenous infusion for therapeutic use. There are several approaches to increase the stability of apelin-13 including attachment of pharmacokinetic enhancing groups, stabilized peptides, and Fc-fusion approaches. We sought a small-molecule APJ receptor agonist approach to target a compound with a pharmacokinetic profile amenable for chronic oral administration. This manuscript describes sequential optimization of the pyrimidinone series, leading to pyridinone 14, with in vitro potency equivalent to the endogenous ligand apelin-13 and with an excellent oral bioavailability and PK profile in multiple preclinical species. Compound 14 exhibited robust pharmacodynamic effects similar to apelin-13 in an acute rat pressure-volume loop model and was advanced as a clinical candidate.

Effect of the direct factor Xa inhibitor apixaban in rat models of thrombosis and hemostasis.

Apixaban is an oral, direct, and highly selective factor Xa inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. Apixaban was evaluated in rat thrombosis and hemostasis models. Thrombosis was produced in the carotid artery by FeCl2 application, in the vena cava by either FeCl2 application or tissue factor injection, and in an arterial-venous shunt. Hemostasis was assessed using cuticle, renal cortex, and mesenteric artery bleeding times. Intravenous apixaban infusions of 0.1, 0.3, 1, and 3 mg/kg per hour increased the ex vivo prothrombin time to 1.24, 1.93, 2.75, and 3.98 times control, respectively. The 0.3, 1, and 3-mg/kg per hour doses inhibited thrombosis in all models. Concentrations for 50% thrombus reduction ranged from 1.84 to 7.57 microM. The 3-mg/kg per hour dose increased cuticle, renal, and mesenteric bleeding times to 1.92, 2.13, and 2.98 times control, respectively. Lower doses had variable (1 mg/kg per hour) or no effect (0.1, 0.3 mg/kg per hour) on hemostasis. Heparin's prolongation of renal and cuticle bleeding time was twice that of apixaban when administered at a dose that approximated apixaban (3 mg/kg per hour) efficacy in arterial thrombosis. In summary, apixaban was effective in a broad range of thrombosis models at doses producing modest increases in multiple bleeding time models.

Clopidogrel versus prasugrel in rabbits. Effects on thrombosis, haemostasis, platelet function and response variability.

The new P2Y(12) antagonist prasugrel produces greater inhibition of ADP-induced platelet aggregation (IPA) and reduction of thrombotic events in patients versus approved doses of clopidogrel, but increases major bleeding. We examined whether IPA level or P2Y(12) receptor occupancy (RO) could be optimized to better balance the efficacy and bleeding effects of these thienopyridines and reduce the response variability in rabbits. Rabbits were given three daily oral doses of clopidogrel (0.3-30 mg/kg/d), prasugrel (0.03-10 mg/kg/d) or vehicle (n = 6-40/group). Electrically-induced carotid artery thrombosis (AT, % thrombus weight reduction), cuticle bleeding time (BT, fold-increase over control), IPA to 20 microM ADP (% inhibition of peak light transmission) and RO (% inhibition of [(33)P]-2MeS-ADP binding to P2Y(1)-blocked platelets) were determined 2-3 hours after the last dose. ED(50) (doses for half-maximal effect, mg/kg/d) of AT, BT, IPA and RO were 1.6, 6.7, 1.9 and 1.4 for clopidogrel vs. 1.2, 1.9, 0.5 and 0.2 for prasugrel. IPA of 30-40% for both compounds produced the optimal balances of efficacy (AT: 50-60%) and BT of about 2-fold with significant RO of 70-80%. IPA of 50-60% achieved higher efficacy (AT: 60-80%), but with increased BT of five- to six-fold and >90% RO. Box-plot suggests no significant difference in the IPA and RO response variability between both compounds. Clopidogrel was 1.3-7 times less potent than prasugrel in rabbits, depending upon which biomarker was studied. The ratio of efficacy: bleeding was most favorable at a moderate IPA of 30% to 40%. Both compounds had similar IPA and RO response variability.

Cyanoguanidine-based lactam derivatives as a novel class of orally bioavailable factor Xa inhibitors.

The N,N'-disubstituted cyanoguanidine is an excellent bioisostere of the thiourea and ketene aminal functional groups. We report the design and synthesis of a novel class of cyanoguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The SAR studies led to the discovery of compound 4 (BMS-269223, K(i)=6.5nM, EC(2xPT)=32muM) as a selective, orally bioavailable FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models. The X-ray crystal structure of 4 bound in FXa is presented and key ligand-protein interactions are discussed.

Aroylguanidine-based factor Xa inhibitors: the discovery of BMS-344577.

We report the design and synthesis of a novel class of N,N'-disubstituted aroylguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The structure-activity relationships (SAR) investigation led to the discovery of the nicotinoyl guanidine 22 as a potent FXa inhibitor (FXa IC(50)=4 nM, EC(2xPT)=7 microM). However, the potent CYP3A4 inhibition activity (IC(50)=0.3 microM) of 22 precluded its further development. Detailed analysis of the X-ray crystal structure of compound 22 bound to FXa indicated that the substituent at the 6-position of the nicotinoyl group of 22 would be solvent-exposed, suggesting that efforts to attenuate the unwanted CYP activity could focus at this position without affecting FXa potency significantly. Further SAR studies on the 6-substituted nicotinoyl guanidines resulted in the discovery of 6-(dimethylcarbamoyl) nicotinoyl guanidine 36 (BMS-344577, IC(50)=9 nM, EC(2xPT)=2.5 microM), which was found to be a selective, orally efficacious FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models.

Biphenyl Acid Derivatives as APJ Receptor Agonists.

The APJ receptor and its endogenous peptidic ligand apelin have been implicated as important modulators of cardiovascular function, and APJ receptor agonists may be beneficial in the treatment of heart failure. In this article, we describe the discovery of a series of biphenyl acid derivatives as potent APJ receptor agonists. Following the identification of initial high-throughput screen lead 2, successive optimization led to the discovery of lead compound 15a. Compound 15a demonstrated comparable in vitro potency to apelin-13, the endogenous peptidic ligand for the APJ receptor. In vivo, compound 15a demonstrated a dose-dependent improvement in the cardiac output in male Sprague Dawley rats with no significant changes in either mean arterial blood pressure or heart rate, consistent with the hemodynamic profile of apelin-13 in an acute pressure volume loop model.

Identification and characterization of Bacillus anthracis spores by multiparameter flow cytometry.

In response to the need for methods that can rapidly detect potentially virulent Bacillus anthracis spores, we developed a two-color flow cytometric assay capable of simultaneously identifying B. anthracis spores and the presence of spore-associated protective antigen, a virulence marker for strains harboring the pXO1 plasmid.

Prediction of the therapeutic index of marketed anti-coagulants and anti-platelet agents by guinea pig models of thrombosis and hemostasis.

INTRODUCTION: Animal models of thrombosis and hemostasis are critical for target validation in pharmaceutical research. Guinea pig haemostatic mechanisms, such as the platelet thrombin receptor repertoire, resemble those of humans. Measuring the performance characteristics of marketed antithrombotic drugs in guinea pig models is a key to predicting therapeutic indices of new agents. The goal of the current study was to benchmark representative marketed drugs in thrombosis and hemostasis models in guinea pigs. METHODS: Effects of the cyclooxygenase inhibitor, aspirin, the P2Y(12) antagonist, clopidogrel, the glycoprotein IIb/IIIa inhibitor, tirofiban, and the direct thrombin inhibitors, argatroban and hirudin, were evaluated in this study. Antithrombotic agents were tested in FeCl(3)-induced carotid artery thrombosis and arterio-venous shunt thrombosis models. Haemostatic effects of drugs were evaluated in cuticle and renal bleeding models. Ex vivo measurements of platelet function and coagulation inhibition were performed to benchmark preclinical doses of each agent to those used clinically. RESULTS: The overall rank-order of potency in thrombosis models based on per cent of vessels occluded, average carotid blood flow, and thrombus weight was aspirin=argatroban=tirofiban

Surrounding land cover types as predictors of palustrine wetland vegetation quality in conterminous USA.

The loss of wetland habitats and their often-unique biological communities is a major environmental concern. We examined vegetation data obtained from 380 wetlands sampled in a statistical survey of wetlands in the USA. Our goal was to identify which surrounding land cover types best predict two indices of vegetation quality in wetlands at the regional scale. We considered palustrine wetlands in four regions (Coastal Plains, North Central East, Interior Plains, and West) in which the dominant vegetation was emergent, forested, or scrub-shrub. For each wetland, we calculated weighted proportions of eight land cover types surrounding the area in which vegetation was assessed, in four zones radiating from the edge of the assessment area to 2km. Using Akaike's Information Criterion, we determined the best 1-, 2- and 3-predictor models of the two indices, using the weighted proportions of the land cover types as potential predictors. Mean values of the two indices were generally higher in the North Central East and Coastal Plains than the other regions for forested and emergent wetlands. In nearly all cases, the best predictors of the indices were not the dominant surrounding land cover types. Overall, proportions of forest (positive effect) and agriculture (negative effect) surrounding the assessment area were the best predictors of the two indices. One or both of these variables were included as predictors in 65 of the 72 models supported by the data. Wetlands surrounding the assessment area had a positive effect on the indices, and ranked third (33%) among the predictors included in supported models. Development had a negative effect on the indices and was included in only 28% of supported models. These results can be used to develop regional management plans for wetlands, such as creating forest buffers around wetlands, or to conserve zones between wetlands to increase habitat connectivity.