Determinação da atividade anti-Mycobacterium tuberculosis e citotoxicidade de produtos naturais e sintéticos

Abstract

RESUMO: A tuberculose (TB) é uma doença infecciosa que vem causando enorme morbidade e mortalidade em todo o mundo. O regime terapêutico usado atualmente, apesar de promover a cura na maioria dos casos, possui muitas desvantagens como o tempo prolongado e a elevada toxicidade levando ao abandono pelos pacientes e com isso o surgimento de Mycobacterium tuberculosis multidroga resistente (MDR). O objetivo deste estudo foi avaliar a atividade anti-M. tuberculosis (H37Rv e isolados clínicos MDR) por Resazurin Microtiter Assay Plate (REMA) e a citotoxicidade em macrófagos J774A.1, utilizando brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio (MTT), de produtos naturais e sintéticos. Os resultados obtidos permitiram a elaboração de um artigo e dois manuscritos. No primeiro artigo intitulado "Anti-Mycobacterium tuberculosis activity of Calophyllum brasiliense extracts obtained by supercritical fluid extraction and conventional techniques" foram isoladas seis substâncias majoritárias (-)-mammea A/BB (1), (-)-mammea B/BB (2), mammea B/BB cyclo D (3), ponnalide (4), mammea A/BA cyclo D (5) e amentoflavone (6) das folhas de Calophyllum brasiliense, na qual, a substância (-)-mammea A/BB (1) foi a que apresentou melhor atividade contra M. tuberculosis H37Rv com concentração inibitória mínima (CIM) de 31,25 ?g/mL e um índice de seletividade (IS) de 1,06. Nesse artigo foram também comparados os extratos preparados por métodos convencionais e por extração por fluido supercrítico utilizando CO2 (SC-CO2), no qual, foi observado que a extração por SC-CO2 foi a mais promissora para a obtenção da substância 1. No primeiro manuscrito, intitulado "Synthesis and structure-activity relationship of natural and synthetic coumarin derivatives against Mycobacterium tuberculosis," foram sintetizados derivados da cumarina natural (-)-mammea A/BB (1a-h) e realizado o estudo da relação estrutura-atividade (REA), na qual, a substância 1g foi a mais promissora em M. tuberculosis H37Rv (CIM = 62,5 ?g/mL) com IS de 0,32 e em isolados clínicos (MDR-TB) com CIM de 31,2 ?g/mL. Em seguida, foi realizado um estudo da REA com 13 cumarinas sintéticas (2-14), das quais, as cumarinas 5, 6, 12 e 14 foram as que obtiveram melhores resultados em M. tuberculosis H37Rv e em isolados clínicos (MDR-TB) apresentando CIM entre 915,6 e 125,0 ?g/mL e IS variando de 1,07 a 4,27. No segundo manuscrito intitulado "Synthesis and activity of chalcone derivatives in Mycobacterium tuberculosis" foram estudadas a REA de 22 chalconas sintéticas (1-22), sendo que as chalconas 2 e 6 foram as que apresentaram melhor ação contra M. tuberculosis H37Rv com valores de CIM de 15,6 ?g/mL (IS = 1,9) e 7,8 ?g/mL (IS = 2,9), respectivamente. Diante dos resultados obtidos nesses três trabalhos torna-se necessária a continuação dos ensaios pré-clínicos in vitro com os derivados das cumarinas e chalconas mais promissoras, além da síntese de novos derivados destas classes de substâncias com a realização do estudo da REA, a fim de se encontrar potenciais candidatos a fármacos anti-TB que sejam mais eficazes e tenham baixa toxicidade

ABSTRACT: Tuberculosis (TB) is an infectious disease, which has caused enormous morbidity and mortality worldwide. The currently used therapeutic regimen despite promote healing in most cases has many disadvantages with prolonged and high toxicity leading to abandonment by patients and increasing Mycobacterium tuberculosis multidrug-resistant tuberculosis (MDR). The aim of this study was to evaluate anti-M. tuberculosis activity (H37Rv and clinical isolates MDR), by resazurin microtiter assay plate (REMA), and the cytotoxicity using J774A.1 macrophages by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT), of natural and synthetic products. The results allowed the preparation of an article and two manuscripts. The first article entitled "Anti- Mycobacterium tuberculosis activity of Calophyllum brasiliense extracts obtained by supercritical fluid extraction and conventional techniques" six major compounds were isolated (-)-mammea A/BB (1), (-)-mammea B/BB (2), mammea B/BB cyclo D (3), ponnalide (4), mammea A/BA cyclo D (5) e amentoflavone (6) from leaves of Calophyllum brasiliense, in which the compound (-)- mammea A/BB (1) was the one who showed the best activity against M. tuberculosis H37Rv with a minimum inhibitory concentration (MIC) of 31.25 ?g/mL and a selectivity index (SI) of 1.06. In this article we were also compared to extracts obtained by conventional methods and extraction using supercritical fluid using CO2 (SC-CO2) in which it was observed that the extraction SC-CO2 was the most promising for obtaining the compound 1. The first manuscript entitled "Synthesis and structure-activity relationship of natural and synthetic coumarin derivatives against Mycobacterium tuberculosis" were synthesized natural coumarin derivatives (-)-mammea A/BB (1a-h) and conducted the study of the structure-activity relationship (SAR), in which the compound 1g was the most promising against M. tuberculosis H37Rv (MIC = 62.5 ?g/mL with an SI of 0.32 and clinical isolates (MDR-TB) with a MIC of 31.2 ?g/mL. Next, a study was conducted SAR of 13 synthetic coumarins (2-14), in which the coumarins 5, 6, 12 and 14 were those that best results obtained in M. tuberculosis H37Rv and clinical isolates (MDR-TB) with a MIC between 15.6 and 125.0 ug/mL and IS ranging from 1.07 to 4.27. In the second manuscript entitled "Synthesis and 11 activity of chalcone derivatives in Mycobacterium tuberculosis" were studied the SAR of 22 synthetic chalcones (1-22), and chalcones 2 and 6 showed the best activity against M. tuberculosis H37Rv with a MIC values of 15.6 ?g/mL (IS = 1.9) 7.8 ?g/mL (IS = 2.9), respectively. Considering these results obtained in these three works become necessary the continuation of pre-clinical in vitro assays with derivatives of the most promising coumarins and chalcones and synthesis of new derivatives of these classes of compounds with the SAR study in order to meet potential candidates for anti-TB drugs that are more effective and have low toxicity