RESUMO
We have explored a series of spirocyclic piperidine amide derivatives (5) as tryptase inhibitors. Thus, 4 (JNJ-27390467) was identified as a potent, selective tryptase inhibitor with oral efficacy in two animal models of airway inflammation (sheep and guinea pig asthma models). An X-ray co-crystal structure of 4 x tryptase revealed a hydrophobic pocket in the enzyme's active site, which is induced by the phenylethynyl group and is comprised of amino acid residues from two different monomers of the tetrameric protein.
Assuntos
Asma/tratamento farmacológico , Hipersensibilidade Respiratória/tratamento farmacológico , Inibidores de Serina Proteinase/farmacologia , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Triptases/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Inibidores das Enzimas do Citocromo P-450 , Modelos Animais de Doenças , Cães , Cobaias , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Ratos , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacocinética , Ovinos , Espectrometria de Massas por Ionização por Electrospray , Compostos de Espiro/farmacocinética , Tripsina/metabolismo , Triptases/metabolismoAssuntos
Plaquetas/efeitos dos fármacos , Nucleosídeos/síntese química , Antagonistas do Receptor Purinérgico P2 , Tetrazóis/síntese química , Cálcio/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Humanos , Nucleosídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12 , Transdução de Sinais , Tetrazóis/farmacologiaRESUMO
We have continued to explore spirobenzazepines as vasopressin receptor antagonists to follow up on RWJ-339489 (2), which had advanced into preclinical development. Further structural modifications were pursued to find a suitable backup compound for human clinical studies. Thus, we identified carboxylic acid derivative 3 (RWJ-676070; JNJ-17158063) as a potent, balanced vasopressin V(1a)/V(2) receptor antagonist with favorable properties for clinical development. Compound 3 is currently undergoing human clinical investigation.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Benzazepinas/química , Compostos de Espiro/química , Animais , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/química , Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/farmacocinética , Benzazepinas/administração & dosagem , Benzazepinas/farmacocinética , Benzazepinas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Masculino , Ratos , Ratos Long-Evans , Receptores de Vasopressinas/metabolismo , Receptores de Vasopressinas/fisiologia , Compostos de Espiro/administração & dosagem , Compostos de Espiro/metabolismo , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Vasopressinas/metabolismoRESUMO
Novel bis(indolyl)maleimide pyridinophanes 3, 9a, 9b, 10a, 10b, and 11 were prepared by cobalt-mediated [2+2+2] cycloaddition of an appropriate alpha,omega-diyne with an N,N-dialkylcyanamide. These macrocyclic heterophanes were found to be potent, selective inhibitors of glycogen synthase kinase-3beta. An X-ray structure of a co-crystal of GSK-3beta and 3 (IC(50)=3nM) depicts the hydrogen bonding and hydrophobic interactions in the ATP-binding pocket of this serine/threonine protein kinase.
Assuntos
Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Piridinas/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Quinase 3 da Glicogênio Sintase/química , Glicogênio Sintase Quinase 3 beta , Interações Hidrofóbicas e Hidrofílicas , Maleimidas/química , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Piridinas/química , Relação Estrutura-AtividadeRESUMO
Thrombin inhibitors are potentially useful in medicine for their anticoagulant and antithrombotic effects. We synthesized and evaluated diverse heterocycle-activated ketones based on the d-Phe-Pro-Arg, and related thrombin active-site recognition motifs, as candidate inhibitors. The peptide-based alpha-ketoheterocycles were typically prepared by either an imidate or a Weinreb amide route (Schemes 1 and 2), the latter of which proved to be more general. Test compounds were generally assayed for inhibition of human alpha-thrombin and bovine trypsin. From a structure-based design standpoint, the heterocycle allows one to explore and adjust interactions within the S1' subsite of thrombin. The preferred alpha-ketoheterocycle is a pi-rich 2-substituted azole with at least two heteroatoms proximal to the carbon bearing the keto group, and a preferred thrombin inhibitor is 2-ketobenzothiazole 3, with a potent K(i) value of 0.2 nM and ca. 15-fold selectivity over trypsin. 2-Ketobenzothiazole 13 exhibited exceedingly potent thrombin inhibition (K(i) = 0.000 65 nM; slow tight binding). Several alpha-ketoheterocycles had thrombin K(i) values in the range 0.1-400 nM. The "Arg" unit in the alpha-ketoheterocycles can be sensitive to stereomutation under mildy basic conditions. For example, 2-ketothiazoles 4 and 59 readily epimerize at pH 7.4, although they are fairly stable stereochemically at pH 3-4; thus, suitable conditions had to be selected for the enzymatic assays. Lead d-Phe-Pro-Arg 2-benzothiazoles 3, 4, and 68 displayed good selectivity for thrombin over other key coagulation enzymes (e.g., factor Xa, plasmin, protein Ca, uPA, tPA, and streptokinase); however, their selectivity for thrombin over trypsin was modest (<25-fold). Compounds 3, 4, and 68 exhibited potent in vitro antithrombotic activity as measured by inhibition of gel-filtered platelet aggregation induced by alpha-thrombin (IC(50) = 30-40 nM). They also proved to be potent anticoagulant/antithrombotic agents in vivo on intravenous administration, as determined in the canine arteriovenous shunt (ED(50) = 0.45-0.65 mg/kg) and the rabbit deep vein thrombosis (ED(50) = 0.1-0.4 mg/kg) models. Intravenous administration of 3, and several analogues, to guinea pigs caused hypotension and electrocardiogram abnormalities. Such cardiovascular side effects were also observed with some nonguanidine inhibitors and inhibitors having recognition motifs other than d-Phe-Pro-Arg. 2-Benzothiazolecarboxylates 4 and 68 exhibited significantly diminished cardiovascular side effects, and benzothiazolecarboxylic acid 4 had the best profile with respect to therapeutic index. The X-ray crystal structures of the ternary complexes 3-thrombin-hirugen and 4-thrombin-hirugen depict novel interactions in the S(1)' region, with the benzothiazole ring forming a hydrogen bond with His-57 and an aromatic stacking interaction with Trp-60D of thrombin's insertion loop. The benzothiazole ring of 3 displaces the Lys-60F side chain into a U-shaped gauche conformation, whereas the benzothiazole carboxylate of 4 forms a salt bridge with the side chain of Lys-60F such that it adopts an extended anti conformation. Since 3 has a 10-fold greater affinity for thrombin than does 4, any increase in binding energy resulting from this salt bridge is apparently offset by perturbations across the enzyme (viz. Figure 4). The increased affinity and selectivity of 2-ketobenzothiazole inhibitors, such as 3, may be primarily due to the aromatic stacking interaction with Trp-60D. However, energy contour calculations with the computer program GRID also indicate a favorable interaction between the benzothiazole sulfur atom and a hydrophobic patch on the surface of thrombin.
Assuntos
Anticoagulantes/síntese química , Fibrinolíticos/síntese química , Cetonas/síntese química , Oligopeptídeos/síntese química , Tiazóis/síntese química , Trombina/antagonistas & inibidores , Animais , Anticoagulantes/química , Anticoagulantes/farmacologia , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Cães , Desenho de Fármacos , Eletrocardiografia/efeitos dos fármacos , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Cobaias , Humanos , Concentração de Íons de Hidrogênio , Hipotensão/induzido quimicamente , Técnicas In Vitro , Cetonas/química , Cetonas/farmacologia , Modelos Moleculares , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Coelhos , Estereoisomerismo , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacologia , Trombina/química , Tripsina/química , Trombose Venosa/prevenção & controleRESUMO
We investigated the formation of macrocycles from alpha,omega-diynes in cobalt-mediated co-cyclotrimerization reactions. Long-chain alpha,omega-diynes underwent metal-mediated [2 + 2 + 2] cycloadditions with nitriles, cyanamides, or isocyanates in the presence of CpCo(CO)2 (Cp = cyclopentadienide) to yield pyridine-containing macrocycles, i.e., meta- and para-pyridinophanes, such as 5m/5p, 35m/35p, and 41m/41p. The regioselectivity of these reactions was affected by the length and type of linker unit between the alkyne groups, as well as by certain stereoelectronic factors. An analogous alpha,omega-cyano-alkyne, 28, combined with an alkyne to yield two isomeric meta-pyridinophanes, such as 5m and 29m, and an ortho cycloadduct (benzannulation product), such as 29o. We developed a reaction protocol for these cobalt-based [2 + 2 + 2] cycloadditions that involves markedly improved conditions such that this process offers a convenient, flexible synthetic approach to macrocyclic pyridine-containing compounds. For example, diyne 6 reacted with p-tolunitrile in 1,4-dioxane to give 7p and 7m (7:1 ratio) in 87% yield at a moderate temperature of ca. 100 degrees C in 24 h without photoirradiation or syringe-pump addition. Isocyanates were also effective reactants, as exemplified by the formation of 44p almost exclusively (44p:44m > 50:1) in 64% yield from diyne 8 and 2-phenylethylisocyanate. By using this improved protocol we were able to co-cyclotrimerize long-chain alpha,omega-diynes with alkynes in certain cases to demonstrate a successful macrocyclic variant of the Vollhardt reaction. For instance, diyne 6 reacted with dipropylacetylene to give paracyclophane 57p and benzannulene 57o (2:1 ratio) in 29% yield.
RESUMO
Novel indolylindazolylmaleimides were synthesized and examined for kinase inhibition. We identified low-nanomolar inhibitors of PKC-beta with good to excellent selectivity vs other PKC isozymes and GSK-3beta. In a cell-based functional assay, 8f and 8i effectively blocked IL-8 release induced by PKC-betaII (IC(50) = 20-25 nM). In cardiovascular safety assessment, representative lead compounds bound to the hERG channel with high affinity, potently inhibited ion current in a patch-clamp experiment, and caused a dose-dependent increase of QT(c) in guinea pigs.
Assuntos
Indazóis/síntese química , Indóis/síntese química , Maleimidas/síntese química , Proteína Quinase C/antagonistas & inibidores , Animais , Linhagem Celular , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/química , Glicogênio Sintase Quinase 3 beta , Cobaias , Humanos , Indazóis/farmacologia , Indazóis/toxicidade , Indóis/farmacologia , Indóis/toxicidade , Interleucina-8/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Síndrome do QT Longo/induzido quimicamente , Maleimidas/farmacologia , Maleimidas/toxicidade , Modelos Moleculares , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Proteína Quinase C/química , Proteína Quinase C beta , Relação Estrutura-AtividadeRESUMO
CpCo(CO)2-mediated cyclotrimerisation of bis-alkynes and cyanamides provides multisubstituted 2-aminopyridines, including macrocyclic products, such as 22 (50% yield).
Assuntos
Alcinos/síntese química , Cobalto/química , Cianamida/síntese química , Compostos Organometálicos/química , Alcinos/química , Cianamida/química , Ciclização , Estrutura Molecular , Compostos Organometálicos/síntese químicaRESUMO
Two approaches were developed to synthesize the novel 7-azaindolyl-heteroarylmaleimides. The first approach was based upon the palladium-catalyzed Suzuki cross-coupling or Stille cross-coupling of 2-chloro-maleimide 5 with various arylboronic acids or arylstannanes. The second approach was based upon the condensation of ethyl 7-azaindolyl-3-glyoxylate 12 with various acetamides. The hydroxypropyl-substituted 7-azaindolylmaleimide template was first used to screen different heteroaryls attached to the maleimide. Replacement of hydroxypropyl with different chain lengths and different functional groups were studied next. Many compounds synthesized were demonstrated to have high potency at GSK-3beta, good GS activity in HEK293 cells and good to excellent metabolic stability in human liver microsomes. Three representative compounds (21, 33, and 34) were demonstrated to have good selectivity against a panel of 80 kinase assays. Among them, compound 33 exhibited very weak inhibitions at the other 79 kinase assays, and behaved as a highly selective GSK-3beta inhibitor.
Assuntos
Compostos Aza/síntese química , Compostos Aza/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Maleimidas/química , Maleimidas/farmacologia , Animais , Compostos Aza/química , Linhagem Celular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Maleimidas/síntese química , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Coelhos , Especificidade por SubstratoRESUMO
A novel series of acyclic 3-(7-azaindolyl)-4-(aryl/heteroaryl)maleimides was synthesized and evaluated for activity against GSK-3beta and selectivity versus PKC-betaII, as well as a broad panel of protein kinases. Compounds 14 and 17c potently inhibited GSK-3beta (IC(50)=7 and 26 nM, respectively) and exhibited excellent selectivity over PKC-betaII (325 and >385-fold, respectively). Compound 17c was also highly selective against 68 other protein kinases. In a cell-based functional assay, both 14 and 17c effectively increased glycogen synthase activity by inhibiting GSK-3beta.
Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Maleimidas/química , Inibidores de Proteínas Quinases/química , Linhagem Celular , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Maleimidas/farmacologia , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Protease activated receptor-1 (PAR-1) is a key mediator of the cellular actions of alpha-thrombin. Thus, antagonism of this unique G-protein coupled receptor with a small molecule represents a means of selectively inhibiting thrombin's cellular actions without inhibiting its proteolytic activity. RWJ-58259 (alphaS)-N-[(1S)-3-amino-1-[[(phenylmethyl)- amino]carbonyl]propyl]-alpha-[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indazol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide) is a potent and selective inhibitor of PAR-1 identified as part of a synthetic chemistry program based upon a de novo design approach. RWJ-58259 inhibited thrombin-induced platelet aggregation in human platelets with an IC50 of 0.37 microM without inhibiting thrombin's proteolytic activity or aggregation induced by other agonists. RWJ-58259 was not effective in guinea pig models of thrombosis. This reflected the presence of a second thrombin-sensitive receptor system in guinea pigs (PAR-3/4) and the selectivity of RWJ-58259 for PAR-1. However, RWJ-58259 was effective in a non-human primate model of thrombosis. Because human platelets have a PAR expression profile similar to the non-human primate, PAR-1 antagonism has the potential to be antithrombotic in humans. RWJ-58259 also inhibited thrombin-induced intracellular calcium signaling and proliferation in rat vascular smooth muscle cells. Perivascular application of RWJ-58259 in vivo significantly inhibited arterial injury-induced stenosis in a rat model of balloon angioplasty. These preclinical results suggest a potential clinical utility of RWJ-58259 for treatment of thrombotic disorders and vascular injury associated with acute coronary interventions and atherosclerosis. Given the potential role of PAR-1 in thrombin's actions in other cell types and disease states, RWJ-58259 provides a means for assessing additional clinical utilities of PAR-1 antagonism in disease conditions such as inflammation, cancer and neurodegeneration.
Assuntos
Indazóis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Receptor PAR-1/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/farmacologia , Animais , Disponibilidade Biológica , Meia-Vida , Humanos , Indazóis/química , Indazóis/farmacocinética , Músculo Liso Vascular/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Ureia/química , Ureia/farmacocinéticaRESUMO
[reaction: see text] Cobalt-mediated [2 + 2 + 2] cycloaddition of alpha,omega-diynes and isocyanates provides a direct approach to macrocyclic 2-oxopyridinophanes. This macrocyclization process, which proceeded most efficiently with aliphatic isocyanates, was conveniently performed at a moderate temperature (85 degrees C) without irradiation or syringe-pump addition.
RESUMO
Efficient methods were developed to synthesize a novel series of macrocyclic bisindolylmaleimides containing linkers with multiple heteroatoms. Potent inhibitors (single digit nanomolar IC(50)) for PKC-beta and GSK-3beta were identified, and compounds showed good selectivity over PKC-alpha, -gamma, -delta, -epsilon, and -zeta. Representative compound 5a also had high selectivity in a screening panel of 10 other protein kinases. In cell-based functional assays, several compounds effectively blocked interleukin-8 release induced by PKC-betaII and increased glycogen synthase activity by inhibiting GSK-3beta.
Assuntos
Indóis/síntese química , Maleimidas/síntese química , Proteína Quinase C/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular , Ciclização , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Humanos , Indóis/farmacologia , Concentração Inibidora 50 , Isoenzimas/síntese química , Isoenzimas/farmacologia , Maleimidas/farmacologia , Proteína Quinase C beta , Relação Estrutura-AtividadeRESUMO
A new generation of indole-based peptide mimetics, bearing a basic amine at the C-terminus, was developed by the agency of two complementary, multistep, trityl resin-based approaches. Thus, we obtained several high-affinity thrombin receptor (PAR-1) ligands, such as 32 and 34. Compounds 32 and 34 were found to bind to PAR-1 with excellent affinity (IC(50)=25 and 35 nM, respectively) and to effectively block platelet aggregation induced by SFLLRN-NH(2) (TRAP-6) and alpha-thrombin.
Assuntos
Indóis/síntese química , Indóis/farmacologia , Receptores de Trombina/efeitos dos fármacos , Ureia/análogos & derivados , Aminas/química , Hemostáticos/antagonistas & inibidores , Hemostáticos/farmacologia , Humanos , Técnicas In Vitro , Indazóis/química , Ligantes , Mimetismo Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Relação Estrutura-Atividade , Trombina/antagonistas & inibidores , Trombina/farmacologia , Ureia/químicaRESUMO
Although it is well recognized that human platelet responses to alpha-thrombin are mediated by the protease-activated receptors PAR-1 and PAR-4, their role and relative importance in platelet-dependent human disease has not yet been elucidated. Because the expression profile of PARs in platelets from nonprimates differs from humans, we used cynomolgus monkeys to evaluate the role of PAR-1 in thrombosis. Based on reverse transcription-polymerase chain reaction, PAR expression in platelets from cynomolgus monkeys consisted primarily of PAR-1 and PAR-4, thereby mirroring the profile of human platelets. We probed the role of PAR-1 in a primate model of vascular injury-induced thrombosis with the selective PAR-1 antagonist (alpha S)-N-[(1S)-3-amino-1-[[(phenylmethyl)amino]carbonyl]propyl]-alpha-[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indazol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide (RWJ-58259). After pretreatment with RWJ-58259 or vehicle, both carotid arteries of anesthetized monkeys were electrolytically injured and blood flow was monitored for 60 min. Time to occlusion was significantly extended after RWJ-58259 administration (27 +/- 3 to 53 +/- 8 min; p < 0.048). Vessels from three of the five treated animals remained patent. Ex vivo platelet aggregation measurements indicated complete PAR-1 inhibition, as well as an operational PAR-4 response. Immunohistochemical staining of mural thrombi with antibodies to the platelet marker CD61 and fibrinogen indicated that RWJ-58259 significantly reduced thrombus platelet deposition. Drug treatment had no effect on key hematological or coagulation parameters. Our results provide direct evidence that PAR-1 is the primary receptor that mediates alpha-thrombin's prothrombotic actions in primates and suggest that PAR-1 antagonists may have potential for the treatment of thrombotic disorders in humans.
Assuntos
Artéria Carótida Primitiva/efeitos dos fármacos , Receptores de Trombina/antagonistas & inibidores , Trombose/prevenção & controle , Ureia/análogos & derivados , Animais , Artéria Carótida Primitiva/patologia , Feminino , Indazóis/química , Indazóis/farmacologia , Indazóis/uso terapêutico , Macaca fascicularis , Masculino , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Receptor PAR-1 , Trombose/tratamento farmacológico , Trombose/patologia , Ureia/química , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
The serine protease thrombin (EC 3.4.21.5) is central to the maintenance of haemostatic balance through its coagulant, anticoagulant and platelet activating properties. In addition, this enzyme affects numerous cellular responses in a wide variety of cells, such as cell proliferation, cytokine and growth factor release, lipid metabolism and tissue remodelling. A family of G-protein-coupled protease-activated receptors (PARs) mediates these cellular actions of thrombin. While thrombin can activate three of the four PAR family members, PAR-1 represents the primary thrombin-responsive receptor in human cells. The expression of PAR-1 in platelets, the vasculature and myocardium, in cells within atherosclerotic plaque and tissues after vascular injury, indicates that this receptor plays an important role during the response to tissue injury and associated inflammatory processes. With the development of PAR-deficient mice and small-molecule antagonists, it is now clear that intervening in processes mediated by PAR-1 presents a new approach to treating a variety of disorders dependent on thrombin generation, including thrombosis and restenosis. The full potential of PAR-1 antagonists has yet to be realised, but the promise of novel therapeutics that modulate receptor function rather than thrombin's proteolytic activity, provides an alternative and, perhaps, more desirable means to dampen the pathological effects of thrombin.
Assuntos
Fibrinolíticos/uso terapêutico , Receptores de Trombina/antagonistas & inibidores , Animais , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Humanos , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/fisiologia , Receptor PAR-1 , Receptores de Trombina/química , Receptores de Trombina/metabolismo , Receptores de Trombina/fisiologia , Trombina/metabolismoRESUMO
Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g. platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-miimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.