Design,synthesis And Biological Evaluation Of 3-alkoxyisoxazoles As ?1 Receptor Ligands

Sigma receptors belong to an unique class of receptors with two subtypes: ?1 and ?2.The ?1 receptor is widely distributed in the central nervous system,playing an variety of biological roles in intracellular signal transduction,apoptosis and metabolic regulation,and related to various diseases.At present,the pharmacological activities of ?1 receptor ligands mainly focus on analgesia and treatment of neurodegenerative diseases.Among of them,?1 receptor antagonist E-52862 have entered the clinical phase II research for treatment of cancer pain,neuralgia and postoperative pain.On the base of our preliminary work,this dissertation focused on the rational structure modification of the 3-alkoxyisoxazole derivative 2-2(Ki,?1 = 4.6 nM,Ki,?2 = 1314 nM)which was selected as the hit compound.The compound 2-2 was modified through the preliminary structure-activity analysis,based on the pharmacophore model of ?1 receptor ligands,and the prediction of blood-brain barrier permeability using LogBB and CNS MPO,and relevant activities of its derivatives were tested and evaluated.1.The primary hydrophobic region of compound 2-2 was modified based on the rational drug design principal.A new series of derivatives based on the 3-(2-((S)-tetrahydropyrrole)methoxy)-isoxazole were designed,synthesized and characterized,and their binding affinity of ? receptors were evaluated.The test results showed that most of the derivatives retained high affinity(Ki,?1 < 10 nM)to parent compound 2-2 at ?1 receptor but subdued selectivity over the ?2 receptor(Ki,?2 > 100 nM).2.The secondary hydrophobic region of compound 2-2 was modified by introducing secondary hydrophobic region and replacing the 3-alkyoxy groups with cyclic amines.A new series of derivatives based on the 5-Phenoxymethyl-isoxazole scaffold were designed,synthesized and characterized,and their binding affinity of ?1 receptor were evaluated.The test results showed that the introduction of secondary hydrophobic region could improve binding affinity of ?1 receptor,some of substituents(compounds 2-29~2-32)resulted in compounds with low single-digit nanomolar to subnanomolar Ki values at the ?1 receptor.Piperazine-substituted analogue 2-42 displayed a very similar Ki value to the parent compound 2-2 at ?1 receptor indicated that the basic nitrogen atoms are extremely important for the binding affinity of ?1 receptor.3.Based on the superior pharmacophore combination principal,both of the A and C regions were modified.A new series of derivatives based on the 3,5-disubstituted isoxazole were designed,synthesized and characterized,and their binding affinity of ? receptors were evaluated.The test results showed that all of the derivatives in this series retained similar binding affinity to the ?1 receptor.Among of them,compound 2-52(Ki,?1 = 0.7 nM,Ki,?2 = 667 nM)and compound 2-57(Ki,?1 = 0.9 nM,Ki,?2 = 311 nM)showed increased selectivity over sigma2 receptor when compared with parent compound 2-2.4.All of the derivatives with great binding affinities(Ki,?1 < 10nM)were assessed the binding affinities of at the biogenic a mine transporters(NET,DAT,and SERT).The test results showed the derivatives had excellent selectivity for ?1 receptor over DAT,NAT and SERT.In functional profile test,the selected compound 2-3 and 2-29 were evaluated by phenytoin-induced ?1 receptor binding affinity tests,the results displayed that found to bind less potently to the ?1 receptor in the presence of phenytoin,indicating that they behave as antagonists.In vitro PAMPA-BBB test,compound 2-42 was found to possess the greatest permeability,with a Pe value of 7.36 × 10-6 cm·s-1.The compound 2-29 was also selected for in vivo pharmacokinetic studies in mouse.The data suggest that compound 2-29 was absorbed rapidly following oral ad ministration and readily crosses the BBB.Antinociceptive efficacy of compound 2-29 was assessed using the mouse formalin test.The test results displayed that compound 2-29(80 mg/kg)significantly attenuated paw licking behavior in both phases in mice,while 40 mg/kg of compound 2-29 markedly reduced paw licking in phase II but not in phase I compared to the vehicle group.In rotarod test,compound 2-29 did not significantly alter the time mice remained on the rotarod at both 40 and 80 mg/kg,suggesting no interference with motor coordination.5.Based on our preli minary work in the ?1 receptors drug discovery,we further screened some of the most potent derivatives in a cellular model of neurite outgrowth.Compound 2-28 exhibited neurite outgrowth effects,and the neurite outgrowth efficacy could be antagonized by ?1 receptor antagonist.With compound 2-28 as the hit compound,a new series of derivatives based on N-cyclobutyla minoethoxyisoxazole scaffold were designed,synthesized and characterized,and the their binding affinity of ? receptors were evaluated.Most of the derivatives showed potent binding affinities to the ?1 receptor(Ki < 10 nM)and good selectivity over the ?2 receptor.The majority of the derivatives scored high in the LogBB and CNS MPO appraisal system,indicating their high likelihood in penetrating the blood–brain barrier.A number of these compounds exhibited significant neurite outgrowth efficacy in N1E-115 neuronal cells and displayed excellent selectivity for ?1 receptors over the selected endogenous neurotransmitter transporters,such as DAT,NET and SERT.In summary,a new series of 3-alkoxyisoxazole derivatives were designed,synthesized and characterized as novel compounds,and their binding affinity of ? receptors were evaluated.Among of them,2-29 was identified as the ?1 receptor antagonists with antinociceptive efficacy in the mouse formalin-induced inflammation pain model and compound 3-12 with significant neuritogenesis effects which emerged as a promising selective ?1 receptor ligand that warrants its further evaluation as a potential therapeutic for neurodegenerative diseases.This data and findings would lay a foundation for further drug discovery based on the ?1 receptor.