Design e síntese de derivados da retronecina e avaliação da atividade in vivo no modelo de Caenorhabditis elegans para a doença de alzheimer

Abstract

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease and the most common form of dementia in the elderly worldwide. Pathological characteristics of AD include amyloid beta (Aβ) plaques, neurofibrillary tangles comprising hyperphosphorylated tau protein, decline of cholinergic function, oxidative stress and inflammation. The Aβ peptide is the most studied target, since the appearance of the senile plaques is the trigger for the other processes or acts by feedback in them. The current treatment of AD is based on inhibitors of the enzyme acetylcholinesterase and memantine, an N-methyl-D-aspartate receptor antagonist, however both treatments are palliative because they do not impair the natural course of the disease, hence the new approaches are based on multi-target drugs. Among natural products alkaloids are the most outstanding as source of new drugs, including the anticholinesterasic agents used in the treatment of AD are alkaloids or their semisynthetic derivatives. The aim of this study is to develop new molecules by associating the pyrrolizidine alkaloid nucleus with L-aminoacid and to evaluate their activity in C. elegans an in vivo alternative model with targets related to AD. Because a virtual screening of condensed amino acids to retronecine (RTN) was performed, and the molecules showed affinity for the enzyme acetylcholinesterase (AChE) were selected for synthesis and biological evaluation. monocrotaline (MCT), the starting molecule for the synthesis of the derivatives, was isolated from the seeds of Crotalaria retusa by maceration in methanol followed by acid-base extraction, subsequently the same was hydrolyzed with barium hydroxide and the pyrrolizidine nucleus, retronecine (RTN), was obtained by column isolation of basic alumina. RTN was halogenated through reaction with thionyl chloride at C-9 to favor nucleophilic substitution reactions at this position. Biological assays were performed on the already wellestablished model for DA the strain CL2006 of Caenorhabditis elegans, which expresses the human β-amyloid (βA) peptide in the muscle in a constitutive way, which leads to progressive paralysis. The molecules were tested at 3 concentrations 10, 50 and 100 μmol. The animals were synchronized by the NaClO method and on the fourth day were submitted to a temperature of 35ºC, which accelerates the phenotype of paralysis. The worms were analyzed every hour by touching with the platinum loop, and those that did not move the posterior region of the body were quantified as paralyzed. Memantine, which is the drug of choice for advanced disease, was used as standard. Memantine prolonged the paralysis in the animal, which corroborates with the literature. RTN at the concentration of 100 μmol slightly reduced the paralysis in the animals. The results show that RTN and RTNCl are non-toxic as their starting product, monocrotaline, which is an indication that molecular modifications may be useful for reducing toxicity and broadening the fields of biological activity for pyrrolizidine alkaloids. From the spectra of the reactional products, structures were proposed for the obtained products.