Novel indolylindazolylmaleimides as inhibitors of protein kinase C-beta: synthesis, biological activity, and cardiovascular safety.

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.

3-(7-Azaindolyl)-4-arylmaleimides as potent, selective inhibitors of glycogen synthase kinase-3.

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.

Macrocyclic bisindolylmaleimides as inhibitors of protein kinase C and glycogen synthase kinase-3.

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.

High-affinity thrombin receptor (PAR-1) ligands: a new generation of indole-based peptide mimetic antagonists with a basic amine at the C-terminus.

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.

Blockade of the thrombin receptor protease-activated receptor-1 with a small-molecule antagonist prevents thrombus formation and vascular occlusion in nonhuman primates.

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.