Planejamento, síntese e avaliação anticâncer de derivados de lapachol e umbeliferona

Submitted by Tallita Machado (tallita.machado@yahoo.com.br) on 2019-10-10T19:46:37Z No. of bitstreams: 3 Tallita Machado_dissertação.pdf: 3608252 bytes, checksum: 5b9831fd9bfd3b10e631a1919a80d141 (MD5) Ata de defesa_Tallita Machado.pdf: 1059402 bytes, checksum: 3a50ce738da3477fba6fb7d630bf9e7b (MD5) CartaEncaminhamentoAutodepósito.pdf: 120527 bytes, checksum: 7d8c79abd31829611b51b88290cef61b (MD5)Approved for entry into archive by MARIA CLARA RAMOS (ppgcf@ufam.edu.br) on 2019-10-11T12:37:04Z (GMT) No. of bitstreams: 3 Tallita Machado_dissertação.pdf: 3608252 bytes, checksum: 5b9831fd9bfd3b10e631a1919a80d141 (MD5) Ata de defesa_Tallita Machado.pdf: 1059402 bytes, checksum: 3a50ce738da3477fba6fb7d630bf9e7b (MD5) CartaEncaminhamentoAutodepósito.pdf: 120527 bytes, checksum: 7d8c79abd31829611b51b88290cef61b (MD5)Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2019-10-11T17:47:00Z (GMT) No. of bitstreams: 3 Tallita Machado_dissertação.pdf: 3608252 bytes, checksum: 5b9831fd9bfd3b10e631a1919a80d141 (MD5) Ata de defesa_Tallita Machado.pdf: 1059402 bytes, checksum: 3a50ce738da3477fba6fb7d630bf9e7b (MD5) CartaEncaminhamentoAutodepósito.pdf: 120527 bytes, checksum: 7d8c79abd31829611b51b88290cef61b (MD5)Made available in DSpace on 2019-10-11T17:47:00Z (GMT). No. of bitstreams: 3 Tallita Machado_dissertação.pdf: 3608252 bytes, checksum: 5b9831fd9bfd3b10e631a1919a80d141 (MD5) Ata de defesa_Tallita Machado.pdf: 1059402 bytes, checksum: 3a50ce738da3477fba6fb7d630bf9e7b (MD5) CartaEncaminhamentoAutodepósito.pdf: 120527 bytes, checksum: 7d8c79abd31829611b51b88290cef61b (MD5) Previous issue date: 2018-04-25CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior92992117129Cancer is the second cause of death in the world and is considered a public health problem. Cancer is the second leading cause of death in the world and is considered a public health problem. Due to this scenario, the search for substances that possess anticancer potential from compounds of natural or semi-synthetic origin is increasing, as well as the use of in silico methods in order to predict pharmacokinetic, toxicological and biological activity of molecules for rational planning of drugs. The objective of this research was to plan, synthesize and evaluate the anticancer potential of lapachol and umbelliferone derivatives, associating in silico and in vitro methods in order to potentiate the chance of finding bioactive compounds. 25 umbelliferone derivatives and 25 lapachol derivatives were designed, which were evaluated by molecular anchoring using the Autodock vina® software having topoisomerase I and II enzymes with biological targets, as well as their molecular, toxicological and pharmacokinetic properties evaluated by through Molinspiration®, Osiris® and PreADMET® software. The selected derivatives were synthesized and confirmed by NMR analysis and mass spectrometry and evaluated in vitro for their anticancer potential by cytotoxicity, genotoxicity analysis that was evaluated by the alkaline pH comet assay, which detects any damage caused to the DNA, of neutral pH, specific for breaks of the double tape and damage caused to the DNA by topoisomerase inhibitors I and II, as well as the evaluation of the gene expression of these enzymes. After the in silico analysis, the derivatives LP04, LP017, LP18, LP20, LP23, LP24, LP25, UMB22, UMB24 and UMB25 were selected because they presented a better complementarity profile with the active site of topoisomerase I and II, as well as interactions with critical amino acid residues characteristic of the inhibitors of these enzymes. In addition, they presented good molecular profiles, pharmacokinetics and low toxicological effect, characteristics desired in the planning of drugs. The cytotoxicity evaluation was performed by the Alamar Blue method, the derivatives LP04, LP17, LP18, LP20 and LP25 presented cytotoxic activity in the breast cancer (MCF-7) and colorectal (HCT116) strains, being selected for the subsequent tests, exhibiting an IC 50 in 72 hours for the MCF7 strain ranging from 2.41 to 8.20 μM and from 5.40 to 18.68 μM in the HCT116 strain, all derivatives having better activity when compared to the starting molecule . Both the umbelliferone and the starter molecules showed cytotoxic activity greater than 20 μM, and did not present an anticancer potential. Through the analysis of the damage index, it was observed that the derivatives presented significant damage (p <0.05) in alkaline pH, neutral pH and in the analysis of induction of DNA damage by topoisomerase inhibitors, presenting a higher index of neutral comet, which ranged from 37.6 ± 0.76 to 83.0 ± 3.5 in the HCT116 line and from 40.6 ± 2.7 to 105.3 ± 2.0 in the MCF7 strain suggesting that to act as inhibitors of topoisomerase II. In the analysis of topoisomerase gene expression, only the activity of LP25, which showed a reduction in the expression of the topoisomerase I, II alpha and II beta genes at the concentration of 7.5 μM and 15 μM, could be evaluated. Thus, the derivatives showed anticancer potential and more specific studies were necessary to evaluate topoisomerase inhibition directly as well as to evaluate other pathways of inhibition and mechanisms of death of these derivatives.O câncer é segunda causa de morte no mundo sendo considerado um problema de saúde pública. Devido a este panorama, a pesquisa por substâncias que possuam potencial anticâncer é crescente, bem como utilização de métodos in silico com o intuito de predizer propriedades farmacocinéticas, toxicológicas e atividade biológica de moléculas para um planejamento racional de fármacos. Objetivou-se nesta pesquisa, planejar, sintetizar e avaliar o potencial anticâncer de derivados do lapachol e da umbeliferona, associando métodos in silico e in vitro com o intuito de potencializar a chance de se encontrar compostos bioativos. Foram planejados 25 derivados da umbeliferona e 25 derivados do lapachol, os quais foram avaliados por meio da ancoragem molecular utilizando o software Autodock vina® tendo as enzimas topoisomerase I e II como alvos biológicos, bem como tiveram suas propriedades moleculares, toxicológicas e farmacocinéticas avaliadas por meio dos softwares Molinspiration®, Osiris® e PreADMET®. Os derivados selecionados foram sintetizados e tiveram sua estrutura confirmada por análise de RMN e espectrometria de massas e avaliados in vitro quanto ao seu potencial anticâncer por meio da análise de citotoxicidade pelo método do Alamar Blue, genotoxicidade que foi avaliada pelo ensaio do cometa de pH alcalino, que detecta qualquer dano causado ao DNA, e de pH neutro, específico para quebras da fita dupla e dano causado ao DNA por inibidores da topoisomerase I e II, bem como a avalição da expressão gênica destas enzimas. Após a realização das análises in silico, foram selecionados os derivados LP04, LP017, LP18, LP20, LP23, LP24, LP25, UMB22, UMB24 e UMB25 por apresentarem melhor perfil de complementariedade com o sítio ativo da topoisomerase I e II, bem como interações com resíduos de aminoácidos críticos característicos dos inibidores destas enzimas. Além disso, apresentaram bons perfis moleculares, farmacocinéticos e baixo efeito toxicológico, características desejadas no planejamento de fármacos. Os derivados LP04, LP17, LP18, LP20 e LP25 apresentaram atividade citotóxica nas linhagens de câncer de mama (MCF-7) e colorretal (HCT116), exibindo uma CI50 em 72 horas para a linhagem MCF7 que variou de 2,41 a 8,20 μM, e de 5,40 a 18,68 μM na linhagem HCT116, apresentando maior atividade quando comparados à molécula de partida. Tanto os derivados da umbeliferona quanto sua molécula de partida apresentou atividade citotóxica maior que 20 μM. Através da análise do índice de dano, observou-se que os derivados apresentaram danos significativos (p