RESUMEN
A hit to lead process to identify reversible, orally available ADP receptor (P2Y12) antagonists lead compounds is described. High throughput screening afforded 1. Optimization of 1, using parallel synthesis methods, a methyl scan to identify promising regions for optimization, and exploratory SAR on these regions, provided 22 and 23. Compound 23 is an orally available, competitive reversible antagonist (KBâ¯=â¯94â¯nM for inhibition of ADP-induced platelet aggregation). It exhibits high metabolic stability in human, rat and dog liver microsomes and is orally absorbed. Although plasma level after oral dosing of 22 and 23 to rats is low, reasonable levels were achieved to merit extensive lead optimization of this structural class.
Asunto(s)
Fluorenos/farmacología , Receptores Purinérgicos P2Y12/metabolismo , Administración Oral , Animales , Perros , Relación Dosis-Respuesta a Droga , Fluorenos/administración & dosificación , Fluorenos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Microsomas Hepáticos/metabolismo , Estructura Molecular , Agregación Plaquetaria/efectos de los fármacos , Ratas , Relación Estructura-ActividadRESUMEN
We report here the design and synthesis of a novel series of benzylamines that are potent and selective inhibitors of uPA with promising oral availability in rat. Further evaluation of one representative (ZK824859) of the new structural class showed that this compound lowered clinical scores when dosed in either acute or chronic mouse EAE models, suggesting that uPA inhibitors of this type could be useful for the treatment of multiple sclerosis.
Asunto(s)
Bencilaminas/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Inhibidores de Serina Proteinasa/uso terapéutico , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Animales , Bencilaminas/síntesis química , Bencilaminas/química , Bencilaminas/farmacocinética , Sitios de Unión , Femenino , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Ratas , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacocinética , Relación Estructura-Actividad , Activador de Plasminógeno de Tipo Uroquinasa/químicaRESUMEN
In this Letter we report the synthesis and evaluation of a series of non-amidine inhibitors of Urokinase Plasminogen Activator (uPA). Starting from compound 1, a significant change provided compounds in which the amidine, binding in the S1 pocket, was replaced with a primary amine. Further modifications led to the identification of potent, selective, and orally bioavailable uPA inhibitors.
Asunto(s)
Bencilaminas/química , Inhibidores de Serina Proteinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Administración Oral , Amidinas/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Ratas , Inhibidores de Serina Proteinasa/administración & dosificación , Inhibidores de Serina Proteinasa/síntesis química , Relación Estructura-Actividad , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Antiplatelet drugs are used to prevent aberrant platelet activation in pathophysiologic conditions such as myocardial infarction and ischemic stroke. The key role that ADP plays in this process has led to the development of antiplatelet drugs that target the P2Y12 receptor. The aim of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of the novel P2Y12 receptor antagonists, BX 667 and BX 048. BX 667 blocks ADP-induced platelet aggregation in human, dog and rat blood (IC50=97, 317 and 3000 nM respectively). BX 667 had nominal effects on collagen-induced aggregation and weakly inhibited arachidonic acid-induced aggregation. BX 667 has an active metabolite, BX 048, that also potently inhibits ADP-induced aggregation (IC50=290 nM) in human blood. BX 667 was shown to have high oral bioavailability in both dog and rat unlike BX 048. Administration of BX 667 resulted in a rapid and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition was directly proportional to circulating plasma levels. This report describes the PK/PD properties of BX 667 showing that it has the properties required for a potential antiplatelet therapeutic agent.
Asunto(s)
Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Cetoácidos/farmacocinética , Inhibidores de Agregación Plaquetaria/farmacocinética , Antagonistas del Receptor Purinérgico P2 , Quinolinas/farmacocinética , Receptores Purinérgicos P2/metabolismo , Animales , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Masculino , Modelos Biológicos , Agregación Plaquetaria/efectos de los fármacos , Unión Proteica , Ratas , Receptores Purinérgicos P2Y12 , Especificidad de la EspecieRESUMEN
ADP plays a key role in platelet aggregation which has led to the development of antiplatelet drugs that target the P2Y12 receptor. The aim of this study was to characterize the effects of two novel P2Y12 receptor antagonists, BX 667 and its active metabolite BX 048, on platelets. BX 667 and BX 048 block the binding of 2MeSADP to platelets and antagonize ADP-induced platelet aggregation in human, dog and rat washed platelets. Both compounds were shown to be reversible inhibitors of platelet aggregation. BX 048 prevents the decrease in cAMP induced by treatment of platelets with ADP. The specificity of BX 667 and BX 048 was demonstrated against cell lines expressing P2Y1 and P2Y6 as well as against a panel of receptors and enzymes. Taken all together these data show that both BX 048 and BX 667 are potent P2Y12 antagonists with high specificity which, in the accompanying paper are demonstrated to behave predictably in vivo.
Asunto(s)
Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Cetoácidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Antagonistas del Receptor Purinérgico P2 , Quinolinas/farmacología , Receptores Purinérgicos P2/metabolismo , Adenosina Difosfato/química , Animales , Calcio/metabolismo , Perros , Evaluación Preclínica de Medicamentos , Humanos , Técnicas In Vitro , Ligandos , Modelos Biológicos , Agregación Plaquetaria/efectos de los fármacos , Unión Proteica , Ratas , Receptores Purinérgicos P2Y12 , Especificidad de la EspecieRESUMEN
We have discovered a novel small-molecule (3-phosphinoylpropionic acid) inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa), BX 528, which had an IC (50) of 2 nM in an enzymatic assay and 50 nM in an in-vitro clot lysis assay, with 3,500- to 35,000-fold selectivity against other carboxypeptidases, such as CPN, CPZ and CPD, and 5- and 12-fold selectivity against CPE (CPH) and CPB, respectively. At 10 micro M, BX 528 had no significant activity (<50% inhibition or antagonism) in a panel of 137 enzymes and receptors. It had no effects on blood coagulation and platelet aggregation up to 300 and 10 micro M, respectively. The plasma half-life following intravenous administration was 0.85 hours in rats and 4.5 hours in dogs. No significant metabolism was detected in human, dog or rabbit hepatic microsomes, and no significant inhibition of cytochrome P450 3A4 and 2D6 up to 30 micro M. No cytotoxic or cell proliferative effects were found in three hepatic and renal cell lines up to 300 micro M and no mutagenic activity was seen in the Ames II screen. There were no significant hemodynamic effects in rats and dogs up to 100 and 30 mg/kg with peak plasma drug concentrations of approximately 1,000 and 300 micro M, respectively. In an in-vivo complement activation model in guinea pigs, BX 528 showed minimal inhibition of plasma CPN activity up to 60 mg/kg with peak plasma concentrations up to 250 micro M. Thus, these data demonstrate that BX 528 is a novel, potent, selective and safe TAFIa inhibitor.
Asunto(s)
Carboxipeptidasa B2/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacocinética , Animales , Coagulación Sanguínea/efectos de los fármacos , Carboxipeptidasas/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cobayas , Semivida , Humanos , Concentración 50 Inhibidora , Microsomas Hepáticos/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Propionatos/farmacocinética , Propionatos/farmacología , Conejos , Ratas , Especificidad por SustratoRESUMEN
Irreversible platelet inhibitors, such as aspirin and clopidogrel, have limited anti-thrombotic efficacy in the clinic due to their bleeding risk. We have developed an orally active reversible P2Y(12) receptor antagonist, BX 667. The aim of this study was to determine if the reversible antagonist BX 667 had a greater therapeutic index than the irreversible P2Y(12) receptor antagonist clopidogrel. Since BX 667 is rapidly converted to its active metabolite BX 048 in rats, we first injected BX 048 intravenously (iv) in a rat arterial venous (A-V) shunt model of thrombosis. BX 048 dose- and concentration-dependently attenuated thrombosis. When administered orally, BX 667 and clopidogrel had similar efficacy, but BX 667 caused less bleeding than clopidogrel. In a rat model of a platelet-rich thrombus induced by vessel injury with FeCl(2), both BX 667 and clopidogrel exhibited higher levels of thrombus inhibition after oral administration compared to their potency in the A-V shunt model. Again, BX 667 caused less bleeding than clopidogrel. In a dog cyclic flow model, iv injection of either BX 667 or clopidogrel dose-dependently reduced thrombus formation with lower bleeding for BX 667 than clopidogrel. Inhibition of thrombosis was highly correlated with inhibition of ADP-induced platelet aggregation in these animal models. In dogs pre-treated with aspirin, BX 667 maintained its wider therapeutic index, measured by inhibition of platelet aggregation over bleeding, compared to the aspirin-clopidogrel combination. These data demonstrate that the reversible P2Y(12) receptor antagonist, BX 667, has a wider therapeutic index than clopidogrel in experimental models of thrombosis.
Asunto(s)
Proteínas de la Membrana/antagonistas & inhibidores , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2 , Trombosis/prevención & control , Animales , Derivación Arteriovenosa Quirúrgica , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Clopidogrel , Modelos Animales de Enfermedad , Perros , Técnicas In Vitro , Masculino , Estructura Molecular , Inhibidores de Agregación Plaquetaria/sangre , Inhibidores de Agregación Plaquetaria/química , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2 , Receptores Purinérgicos P2Y12 , Ticlopidina/análogos & derivados , Ticlopidina/farmacologíaRESUMEN
Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.
Asunto(s)
Citocinas/metabolismo , Iones/química , Quelantes del Hierro/química , Quelantes del Hierro/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Espectrometría de Masas , Estructura Molecular , FN-kappa B/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Heat-shock protein-90 is an attractive target for anticancer drugs, as heat-shock protein-90 blockers such as the ansamycin 17-(allylamino)-17-demethoxygeldanamycin greatly reduce the expression of many signaling molecules that are disregulated in cancer cells and are key drivers of tumor growth and metastasis. While 17-(allylamino)-17-demethoxygeldanamycin has shown promise in clinical trials, this compound class has significant template-related drawbacks. In this paper, we describe a new, potent non-ansamycin small-molecule inhibitor of heat-shock protein-90, BX-2819, containing resorcinol and triazolothione rings. Structural studies demonstrate binding of BX-2819 to the ADP/ATP-binding pocket of heat-shock protein-90. The compound blocked expression of heat-shock protein-90 client proteins in cancer cell lines and inhibited cell growth with a potency similar to 17-(allylamino)-17-demethoxygeldanamycin. In a panel of four cancer cell lines, BX-2819 blocked growth with an average IC(50) value of 32 nM (range of 7-72 nM). Efficacy studies demonstrated that treatment with BX-2819 significantly inhibited the growth of NCI-N87 and HT-29 tumors in nude mice, consistent with pharmacodynamic studies showing inhibition of heat-shock protein-90 client protein expression in tumors for greater than 16 h after dosing. These data support further studies to assess the potential of BX-2819 and related analogs for the treatment of cancer.
Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Triazoles/farmacología , Animales , Benzoquinonas/química , Benzoquinonas/farmacología , Línea Celular Tumoral , Simulación por Computador , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Proteínas HSP90 de Choque Térmico/metabolismo , Células HT29 , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacología , Ratones , Ratones Desnudos , Trasplante Heterólogo , Triazoles/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This article reports the crystal structures of inhibitors of the functional form of thrombin-activatable fibrinolysis inhibitor (TAFIa). In vivo experiments indicate that selective inhibitors of TAFIa would be useful in the treatment of heart attacks. Since TAFIa rapidly degrades in solution, the homologous protein porcine pancreatic carboxypeptidase B (pp-CpB) was used in these crystallography studies. Both TAFIa and pp-CpB are zinc-based exopeptidases that are specific for basic residues. The final development candidate, BX 528, is a potent inhibitor of TAFIa (2 nM) and has almost no measurable effect on the major selectivity target, carboxypeptidase N. BX 528 was designed to mimic the tripeptide Phe-Val-Lys. A sulfonamide replaces the Phe-Val amide bond and a phosphinate connects the Val and Lys groups. The phosphinate also chelates the active-site zinc. The electrostatic interactions with the protein mimic those of the natural substrate. The primary amine in BX 528 forms a salt bridge to Asp255 at the base of the S1' pocket. The carboxylic acid interacts with Arg145 and the sulfonamide is hydrogen bonded to Arg71. Isopropyl and phenyl groups replace the side chains of Val and Phe, respectively. A series of structures are presented here that illustrate the evolution of BX 528 from thiol-based inhibitors that mimic a free C-terminal arginine. The first step in development was the replacement of the thiol with a phosphinate. This caused a precipitous drop in binding affinity. Potency was reclaimed by extending the inhibitors into the downstream binding sites for the natural substrate.
Asunto(s)
Carboxipeptidasa B/metabolismo , Péptidos/química , Péptidos/metabolismo , Sitios de Unión , Carboxipeptidasa B/química , Carboxipeptidasa B2/química , Cristalografía por Rayos X/métodos , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Estructura MolecularRESUMEN
A novel series of cyclic potent, selective, small molecule, thiol-based inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) and the crystal structures of TAFIa inhibitors bound to porcine pancreatic carboxypeptidase B are described. Three series of cyclic arginine and lysine mimetic inhibitors vary significantly in their selectivity against other human basic carboxypeptidases, carboxypeptidase N and carboxypeptidase B. (-)2a displays TAFIa IC50 = 3 nM and 600-fold selectivity against CPN. Inhibition of TAFIa with (rac)2a resulted in dose dependent acceleration of human plasma clot lysis in vitro and was efficacious as an adjunct to tPA in an in vivo rabbit jugular vein thrombolysis model.
Asunto(s)
Ácido 3-Mercaptopropiónico/farmacología , Carboxipeptidasa B2/antagonistas & inhibidores , Fibrinolíticos/síntesis química , Animales , Arginina , Carboxipeptidasa B/antagonistas & inhibidores , Cristalografía por Rayos X , Fibrinolíticos/farmacocinética , Fibrinolíticos/farmacología , Humanos , Lisina , Lisina Carboxipeptidasa/antagonistas & inhibidores , Imitación Molecular , Péptidos Cíclicos , Conejos , Relación Estructura-Actividad , Especificidad por Sustrato , PorcinosRESUMEN
HTS screening identified 1 with micromolar inhibitory activity against PDK1. Optimization of 1 afforded 4i (BX-517) which has single-digit nanomolar activity against PDK1 and excellent selectivity against PKA.
Asunto(s)
Indoles/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Diseño de Fármacos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis químicaRESUMEN
Based on the lead compound BX-517, a series of C-4' substituted indolinones have been synthesized and evaluated for PDK1 inhibition. Modification at C-4' of the pyrrole afforded potent compounds (7b and 7d) with improved solubility and ADME properties. In this letter, we describe the synthesis, selectivity profile, and pharmacokinetic data of selected compounds.
Asunto(s)
Indoles/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Urea/análogos & derivados , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Línea Celular Tumoral , Humanos , Indoles/farmacología , Inhibidores de Proteínas Quinasas/química , Urea/química , Urea/farmacologíaRESUMEN
This paper presents the crystal structure of porcine pancreatic carboxypeptidase B (pp-CpB) in complex with a variety of thiol-based inhibitors that were developed as antagonists of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing thrombolytic agents for the treatment of acute myocardial infarction, one of the most prevalent forms of heart attacks. Unfortunately, activated TAFIa rapidly degrades in solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are zinc-based exopeptidases, and the proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the thiol group chelates the active site zinc, the carboxylic acid forms a salt bridge to Arg145, and the guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various carboxypeptidases.
Asunto(s)
Carboxipeptidasa B/antagonistas & inhibidores , Inhibidores de Proteasas/química , Compuestos de Sulfhidrilo/química , Animales , Carboxipeptidasa B/metabolismo , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Páncreas/enzimología , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Conformación Proteica , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Sulfhidrilo/farmacología , PorcinosRESUMEN
The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.