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.

Dissecting the activation of thienopyridines by cytochromes P450 using a pharmacodynamic assay in vitro.

The thienopyridine antiplatelet drugs, such as ticlopidine, clopidogrel, and prasugrel, require activation by cytochromes P450 in vivo to effectively block platelet aggregation. The study of the metabolic activation of these compounds has been hampered by the lability and reactivity of the ring-opened active metabolite (AM) and by the numerous metabolites that can be formed in such a transformation. We have developed a novel method whereby platelets are incubated with the cytochrome P450 and the thienopyridine of interest for various amounts of time, and the effects on ADP-driven platelet aggregation are directly examined. In this way, the platelet is used as a biosensor for detection of the AM. Using this method, cytochromes P450 capable of converting clopidogrel, prasugrel, and 2-oxo-clopidogrel to metabolites that inhibit ADP-induced platelet aggregation were identified as well as which cytochromes P450 were capable of catalyzing partial reactions (e.g., conversion of 2-oxo-clopidogrel to the AM). These studies show that, in vitro, CYP3A4/5, 2C19, and 2B6 are individually capable of converting clopidogrel and prasugrel to the AM and that the cytochrome P450 preference for these two thienopyridines is very similar.

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.

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.

Design, structure-activity relationships, X-ray crystal structure, and energetic contributions of a critical P1 pharmacophore: 3-chloroindole-7-yl-based factor Xa inhibitors.

An indole-based P1 moiety was incorporated into a previously established factor Xa inhibitor series. The indole group was designed to hydrogen-bond with the carbonyl of Gly218, while its 3-methyl or 3-chloro substituent was intended to interact with Tyr228. These interactions were subsequently observed in the X-ray crystal structure of compound 18. SAR studies led to the identification of compound 20 as the most potent FXa inhibitor in this series (IC(50) = 2.4 nM, EC(2xPT) = 1.2 microM). An in-depth energetic analysis suggests that the increased binding energy of 3-chloroindole-versus 3-methylindole-containing compounds in this series is due primarily to (a) the more hydrophobic nature of chloro- versus methyl-containing compounds and (b) an increased interaction of 3-chloroindole versus 3-methylindole with Gly218 backbone. The stronger hydrophobicity of chloro- versus methyl-substituted aromatics may partly explain the general preference for chloro- versus methyl-substituted P1 groups in FXa, which extends beyond the current series.

Synthesis and evaluation of acylguanidine FXa inhibitors.

A series of acylguanidine derivatives were prepared and investigated as inhibitors of Factor Xa (FXa). These compounds were made by guanidine acylation with carboxylic acids using carbonyl diimidazole (CDI) as the coupling reagent. Conditions for the rapid synthesis and purification of these compounds are described along with their ability to inhibit FXa. The best FXa inhibitor is 1 with a FXa IC(50) of 6 nM.

Amino(methyl) pyrrolidines as novel scaffolds for factor Xa inhibitors.

The design and synthesis of a novel class of amino(methyl) pyrrolidine-based sulfonamides as potent and selective FXa inhibitors is reported. The amino(methyl) pyrrolidine scaffolds were designed based on the proposed bioisosterism to the piperazine core in known FXa inhibitors. The SAR study led to compound 15 as the most potent FXa inhibitor in this series, with an IC(50) of 5.5 nM and PT EC(2x) of 1.7 microM. The proposed binding models show that the pyrrolidine cores are in van der Waals contact with the enzyme surface, and the flexibility of amino(methyl) pyrrolidines allows the two nitrogen atoms to anchor both the P1 and P4 groups to fit similarly in the S1 and S4 pockets.

Antithrombotic and hemostatic effects of a small molecule factor XIa inhibitor in rats.

The effect of inhibiting activated blood coagulation factor XIa was determined in rat models of thrombosis and hemostasis. BMS-262084 is an irreversible and selective small molecule inhibitor of factor XIa with an IC(50) of 2.8 nM against human factor XIa. BMS-262084 doubled the activated thromboplastin time in human and rat plasma at 0.14 and 2.2 microM, respectively. Consistent with factor XIa inhibition, the prothrombin time was unaffected at up to 100 microM. BMS-262084 administered as an intravenous loading plus sustaining infusion was effective against FeCl(2)-induced thrombosis in both the vena cava and carotid artery. Maximum thrombus weight reductions of 97 and 73%, respectively (P<0.05), were achieved at a pretreatment dose of 12 mg/kg+12 mg/kg/h which increased the ex vivo activated thromboplastin time to 3.0 times control. This dose level also arrested growth of venous and arterial thrombi when administered after partial thrombus formation. BMS-262084 was most potent in FeCl(2)-induced venous thrombosis, decreasing thrombus weight 38% (P<0.05) at a threshold dose of 0.2 mg/kg+0.2 mg/kg/h. In contrast, doses of up to 24 mg/kg+24 mg/kg/h had no effect on either tissue factor-induced venous thrombosis or the ex vivo prothrombin time. Doses of up to 24 mg/kg+24 mg/kg/h also did not significantly prolong bleeding time provoked by either puncture of small mesenteric blood vessels, template incision of the renal cortex, or cuticle incision. These results demonstrate that pharmacologic inhibition of factor XIa achieves antithrombotic efficacy with minimal effects on provoked bleeding.

Development and validation of a platelet calcium flux assay using a fluorescent imaging plate reader.

Calcium signaling in platelets is an important physiological response to various aggregation stimuli. Loading platelets with various fluorescent dyes and measuring the change in calcium concentration using a spectrofluorometer has been the traditional approach to studying calcium signaling. This method suffers from the need for large platelet samples and a decrease in total fluorescence signal with time due to photobleaching. Therefore, it is rarely used to measure the quantitative effect of an agonist or antagonist on calcium signaling. Adaptation of these measurements to a fluorescent imaging plate reader (FLIPR) format allows the sample size to be reduced by 5- to 10-fold, and the microplate format allows a significant increase in throughput. Addition of the agonists to all wells simultaneously serves to normalize the total response. This article describes the first use of a FLIPR to study the calcium flux in human platelets. The IC(50) values showed a linear correlation with the K(i) for receptor binding in washed platelets. The generality of the methodology was shown by measuring EC(50) values for agonists and IC(50) values for antagonists of the platelet G protein-coupled receptor P2Y(1) and for the ion channel P2X(1).

Ketene aminal-based lactam derivatives as a novel class of orally active FXa inhibitors.

N,N'-Disubstituted ketene aminals are good bioisosteres of thiourea functional groups. We report the design and synthesis of a novel class of ketene aminal-based lactam derivatives as potent and orally active FXa inhibitors.

Biphenylsulfonamide endothelin receptor antagonists. 4. Discovery of N-[[2'-[[(4,5-dimethyl-3-isoxazolyl)amino]sulfonyl]-4-(2-oxazolyl)[1,1'-biphenyl]- 2-yl]methyl]-N,3,3-trimethylbutanamide (BMS-207940), a highly potent and orally active ET(A) selective antagonist.

We have previously disclosed the selective ET(A) receptor antagonist N-(3,4-dimethyl-5-isoxazolyl)-4'-(2-oxazolyl)[1,1'-biphenyl]-2-sulfonamide (1, BMS-193884) as a clinical development candidate. Additional SAR studies at the 2'-position of 1 led to the identification of several analogues with improved binding affinity as well as selectivity for the ET(A) receptor. Following the discovery that a 3-amino-isoxazole group displays significantly improved metabolic stability in comparison to its 5-regioisomer, the 3-amino-isoxazole group was combined with the optimal 2'-substituent leading to 16a (BMS-207940). Compound 16a is an extremely potent (ET(A) K(i) = 10 pM) and selective (80,000-fold for ET(A) vs ET(B)) antagonist. It is also 150-fold more potent and >6-fold more selective than 1. The bioavailability of 16a was 100% in rats and the systemic clearance and volume of distribution are higher than that of 1. In rats, intravenous 16a blocks big ET pressor responses with 30-fold greater potency than 1. After oral dosing at 3 micromol/kg, 16a displays enhanced duration relative to 1.

Biphenylsulfonamide endothelin receptor antagonists. Part 3: structure-activity relationship of 4'-heterocyclic biphenylsulfonamides.

A number of 4'-heterocyclic biphenylsulfonamide derivatives, formally derived from BMS-193884 (1) by replacing the oxazole ring with other heterocyclic rings, are potent and selective endothelin A (ET(A)) receptor antagonists. Among the analogues examined, the pyrimidine derivative 18 is the most potent (K(i)=0.9 nM) and selective for the ET(A) receptor, approximately equivalent to 1.