ABSTRACT
An accumulating body of evidence incriminates Rho kinase (ROCK) in the pathogenesis of pulmonary hypertension (PH). The therapeutic efficacy of azaindole-1, a novel highly selective and orally active ROCK inhibitor, has not yet been investigated in PH. This study aimed to investigate the effects of azaindole-1 on 1) acute hypoxic pulmonary vasoconstriction (HPV), 2) proliferation of pulmonary arterial smooth muscle cells (PASMCs) and 3) animal models of PH. Azaindole-1 significantly inhibited HPV in isolated, ventilated and buffer-perfused murine lungs and proliferation of primary rat PASMCs in vitro. Azaindole-1 was administered orally from 21 to 35 days after monocrotaline (MCT) injection in rats and hypoxic exposure in mice. Azaindole-1 (10 and 30 mg per kg body weight per day in rats and mice, respectively) significantly improved haemodynamics and right ventricular hypertrophy. Moreover, the medial wall thickness and muscularisation of peripheral pulmonary arteries were ameliorated. Azaindole-1 treatment resulted in a decreased immunoreactivity for phospho-myosin phosphatase target subunit 1 and proliferating cell nuclear antigen in pulmonary vessels of MCT-injected rats, suggesting an impaired ROCK activity and reduced proliferating cells. Azaindole-1 provided therapeutic benefit in experimental PH, and this may be attributable to its potent vasorelaxant and antiproliferative effects. Azaindole-1 may offer a useful approach for treatment of PH.
Subject(s)
Azabicyclo Compounds/therapeutic use , Hypertension, Pulmonary/drug therapy , Indoles/therapeutic use , Animals , Cell Proliferation , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Hemodynamics , Lung/pathology , Male , Mice , Rats , Rats, Sprague-Dawley , Telemetry/methods , Tetrazolium Salts/pharmacology , Thiazoles/pharmacology , Thymidine/chemistry , Treatment Outcome , rho-Associated Kinases/antagonists & inhibitorsABSTRACT
BACKGROUND AND PURPOSE: Rho-kinase (ROCK) has been implicated in the pathophysiology of altered vasoregulation leading to hypertension. Here we describe the pharmacological characterization of a potent, highly selective and orally active ROCK inhibitor, the derivative of a class of azaindoles, azaindole 1 (6-chloro-N4-{3,5-difluoro-4-[(3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]-phenyl}pyrimidine-2,4-diamine). EXPERIMENTAL APPROACH: Pharmacological characterization of azaindole 1 was performed with human recombinant ROCK in vitro. Vasodilator activity was determined using isolated vessels in vitro and different animal models in vivo. KEY RESULTS: This compound inhibited the ROCK-1 and ROCK-2 isoenzymes with IC50 s of 0.6 and 1.1 nM in an ATP-competitive manner. Although ATP-competitive, azaindole 1 was inactive against 89 kinases (IC50>10 microM) and showed only weak activity against an additional 21 different kinases (IC50=1-10 microM). Only the kinases TRK und FLT3 were inhibited by azaindole 1 in the sub-micromolar range, albeit with IC50 values of 252 and 303 nM, respectively. In vivo, azaindole 1 lowered blood pressure dose-dependently after i.v. administration in anaesthetized normotensive rats. In conscious normotensive and spontaneously hypertensive rats azaindole 1 induced a dose-dependent decrease in blood pressure after oral administration without inducing a significant reflex increase in heart rate. In anaesthetized dogs, azaindole 1 induced vasodilatation with a moderately elevated heart rate. CONCLUSIONS AND IMPLICATIONS: Azaindole 1 is representative of a new class of selective and potent ROCK inhibitors and is a valuable tool for the elucidation of the role of ROCK in the cardiovascular system.
Subject(s)
Cardiovascular System/drug effects , Diamines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , rho-Associated Kinases/antagonists & inhibitors , Administration, Oral , Animals , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Binding Sites/drug effects , Blood Pressure/drug effects , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Computer Simulation , Death-Associated Protein Kinases , Dogs , Dose-Response Relationship, Drug , Female , Humans , Injections, Intravenous , Male , Mice , Models, Animal , Models, Molecular , Organ Culture Techniques , Phosphorylation , Polymerase Chain Reaction/methods , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Rabbits , Rats , Rats, Inbred SHR , Rats, Wistar , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Time Factors , Vasodilator Agents/administration & dosage , Vasodilator Agents/chemistry , Vasodilator Agents/pharmacology , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolismABSTRACT
The synthesis of cephalotaxine ring analogues 10 was achieved by two successive intramolecular palladium-catalyzed reactions of 12 via 11, namely an allylic amination and a Heck reaction. The substrates 12 were obtained by alkylation of primary amines 13 with tosylates 14.
