ABSTRACT
The design and synthesis of a new class of nonpeptide direct thrombin inhibitors, built on the structure of 1-(pyridin-4-yl)piperidine-4-carboxamide, are described. Starting from a strongly basic 1-amidinopiperidine derivative (6) showing poor thrombin (fIIa) and factor Xa (fXa) inhibition activities, anti-fIIa activity and artificial membrane permeability were considerably improved by optimizing the basic P1 and the X-substituted phenyl P4 binding moieties. Structure-activity relationship studies, usefully complemented with molecular modeling results, led us to identify compound 13b, which showed excellent fIIa inhibition (Ki = 6 nM), weak anti-Xa activity (Ki = 5.64 µM), and remarkable selectivity over other serine proteases (e.g., trypsin). Compound 13b showed in vitro anticoagulant activity in the low micromolar range and significant membrane permeability. In mice (ex vivo), 13b demonstrated anticoagulant effects at 2 h after oral dosing (100 mg·kg(-1)), with a significant 43% prolongation of the activated partial thromboplastin time (aPTT), over controls (P < 0.05).
Subject(s)
Anticoagulants/pharmacology , Blood Coagulation/drug effects , Enzyme Inhibitors/pharmacology , Factor X/antagonists & inhibitors , Piperidines/pharmacology , Pyrrolidines/pharmacology , Thrombin/antagonists & inhibitors , Animals , Anticoagulants/chemical synthesis , Anticoagulants/chemistry , Blood Coagulation Tests , Cattle , Crystallography, X-Ray , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Male , Mice , Models, Molecular , Molecular Dynamics Simulation , Molecular Structure , Piperidines/chemical synthesis , Piperidines/chemistry , Pyrrolidines/chemical synthesis , Pyrrolidines/chemistry , Serine Proteases/metabolism , Structure-Activity Relationship , Thrombin/metabolismABSTRACT
A number of C-3 spirocyclic 2-benzazepine analogs of α-phenyl-N-tert-butyl nitrone (PBN) were synthesized and tested for their activity in protecting rat brain mitochondria and dopaminergic (DA) neurons against 6-hydroxydopamine (6-OHDA), a toxin inducing destruction of the DA nigro-striatal pathway in rodent models of Parkinson's disease. The newly synthesized nitrone derivatives were firstly investigated for their activity in decreasing the level of hydroxyl radicals generated during 6-OHDA oxidation, and inhibit lipid peroxidation (TBARS assay) and protein carbonyl content (PCC) in rat brain mitochondria. Most of the studied 2-benzazepine nitrones showed inhibitory potencies in both TBARS and PCC assays at least two magnitude orders higher than that of PBN. The data obtained usefully complemented the known structure-activity relationships. In particular, 5 and 10, bearing C-3 spiro cyclopentyl and tetrahydropyranyl moieties, respectively, at 8 µM concentration proved to be significantly more effective than PBN in protecting cultured DA neurons exposed to 6-OHDA, which alone causes about 45% cell loss in 24 h. In addition, we found that 5 inhibited butyrylcholinesterase with an IC(50) value of 16.8 µM, which would enhance its potential as neuroprotective agent in Alzheimer's neurodegeneration. These findings extend the utility of benzazepine-based PBN analogs in the treatment of age-related free radical-mediated disorders.
Subject(s)
Benzazepines/pharmacology , Brain/drug effects , Dopaminergic Neurons/drug effects , Neuroprotective Agents/pharmacology , Nitrogen Oxides/pharmacology , Oxidative Stress/drug effects , Animals , Benzazepines/chemistry , Brain/metabolism , Brain/pathology , Cell Count , Cells, Cultured , Cholinesterase Inhibitors/pharmacology , Cyclic N-Oxides/pharmacology , Disease Models, Animal , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Inhibitory Concentration 50 , Lipid Peroxidation/drug effects , Mitochondria/metabolism , Molecular Structure , Neuroprotective Agents/chemistry , Nitrogen Oxides/chemistry , Oxidopamine , Parkinson Disease, Secondary/chemically induced , Parkinson Disease, Secondary/drug therapy , Parkinson Disease, Secondary/metabolism , Parkinson Disease, Secondary/pathology , Primary Cell Culture , Protein Carbonylation/drug effects , Structure-Activity RelationshipABSTRACT
New chloro-substituted biarylmethoxyphenyl piperidine-4-carboxamides were synthesized and assayed in vitro as inhibitors of the blood coagulation enzymes factor Xa (fXa) and thrombin. An investigation of effects of the amidine and isopropyl groups attached at the piperidine nitrogen and 5-(halogenoaryl)isoxazol-3-yl groups as biaryl substituents led us to identify new compounds which proved to be selective fXa inhibitors, with inhibition constants in the low nanomolar range. The most potent compound 21e, that incorporates 2-Cl-thiophen-5-yl group as the P1 motif and 1-isopropylpiperidine P4 group, inhibited fXa with K(i) value of 0.3nM and very high selectivity over thrombin and some other tested serine proteases, achieving moderate levels of anticoagulant activity in the low micromolar range, as assessed by the prothrombin time clotting assay (PT(2)=3.30µM). Based on reliable docking simulations, molecular modeling provided a rationale for interpreting structure-activity relationships. The predicted binding modes highlighted the structural requirements for addressing the subsites S1 and S4 of the fXa enzyme.
