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Actividade antibacteriana dos constituintes químicos da planta medicinal africana Cleistochlamys kirkii
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O trabalho apresentado nesta dissertação teve como objectivo principal a obtenção de compostos com actividade antibacteriana (Staphylococcus aureus) a partir da planta medicinal africana C. kirkii (Annonaceae). Nesse sentido utilizaram-se duas abordagens: i) isolamento dos constituintes maioritários de uma fracção do extracto de metanol; e ii) derivatização molecular de um composto isolado em grande quantidade.
A partir de uma fracção, resultante do fracionamento bioguiado do extracto de metanol de C. kirkii, foram isolados, por técnicas cromatográficas, um novo composto com o esqueleto do ciclo-hexeno, denominado cleistinol (2.2), um novo alcalóide aporfínico, a cleistonina (2.7), e quatro derivados do heptano, três dos quais são novos compostos, o (R)-(E)-melodorinol (2.4), o (R)-(E)-acetilmelodorinol (2.5) e o kirkiinolato (2.6), e umheptano nunca isolado anteriormente de fontes naturais, o (R)-(Z)-melodorinol (2.3). Foi ainda isolada a dichamanetina (2.1), uma flavanona já descrita na literatura.
Por derivatização molecular da dichamanetina (2.1), isolada em grande quantidade e utilizando vários derivados de aminas, foram obtidas nove iminas, nomeadamente: uma oxima (2.8), quatro hidrazonas (2.9 a 2.12), três carbo-hidrazidas (2.13 a 2.15) e uma semicarbazona (2.16). Preparou-se também um derivado tri-acetilado da dichamanetina (2.17).
As estruturas químicas dos compostos foram estabelecidas com base nas suas características físicas ([α]22D) e espectroscópicas (ressonância magnética unidimensional – 1H-RMN e 13C-RMN – e bidimensional - 1H-1H-COSY, HMQC, HMBC e NOESY) e por comparação com dados descritos na literatura.
A actividade antibacteriana dos compostos foi avaliada em estirpes de Staphyloccocus aureus, nomeadamente numa estirpe sensível (ATCC 6538), numa estirpe resistente à meticilina (ATCC 43300) e ainda na estirpe CIP 106414 meticilina resistente com resistência intermédia à vancomicina. Entre os compostos isolados, a dichamanetina (2.1) demonstrou ser um potente agente antibacteriano em todas as estirpes (CMI = 0,49 μg mL-1). O composto 2.5 apresentou valores de CMI = 15,63 e 62,5 μg mL-1, na estirpe sensível e na ertirpe resistente CIP 106414, respectivamente. Os compostos 2.3 e 2.4 apresentaram um valor de CMI = 62,5 μg mL- na estirpe sensível. O composto 2.3 exibiu também CMI = 62,5 μg mL-1 na estirpe resistente CIP 106414.
Relativamente aos derivados obtidos a partir da dichamanetina, os valores mais baixos de CMI verificaram-se para os compostos 2.8, 2.9 e 2.11 em todas as estirpes do Staphylococcus aureus (CMI = 0,98 a 1,95 μg mL-1). As hidrazonas 2.10 e 2.13 exibiram valores de CMI entre 7,81 e 31,25 μg mL-1 para todas as estirpes. De salientar ainda a actividade das carbo-hidrazidas 2.14 e 2.16 nas estirpes ATCC6538 e CIP106414 (CMI = 3,9 e 15,63 μg mL-1, respectivamente). No entanto, nenhum dos derivados de imina revelou ser mais potente que a própria dichamanetina, evidenciando a importância do grupo carbonilo em C4 para actividade antibacteriana, assim como a presença dos grupos hidroxilo livres, uma vez que a sua acetilação levou a uma perda da actividade.
Posteriormente foi avaliada a interacção entre os compostos 2.1 a 2.17 e alguns antibióticos de referência. Um total de oito compostos, três isolados (2.3, 2.4 e 2.5) e cinco sintetizados por derivatização (2.9, 2.10, 2.12, 2.14 e 2.15) foram capazes de exercer um efeito modulador na actividade dos três antibióticos ensaiados (amoxicilina, ofloxacina e vancomicina) nas mesmas estirpes utilizadas para avaliar a actividade antibacteriana. De salientar o composto 2.12, um composto inactivo, que se revelou um forte efeito modulador na actividade dos três antibióticos aqui testados, em todas as estirpes usadas.
The main goal of this dissertation was to find compounds with antibacterial activity (S. aureus) from the African medicinal plant C. kirkii (Annonaceae). Accordingly, two approaches were used: i) the isolation of major constituents from a fraction of the methanol extract; and ii) molecular derivatization of a compound isolated in large amount. From a fraction resulting from the bioguided fractionation of the methanol extract of C. kirkii were isolated, by chromatographic techniques, a new compound with a cyclohexene scaffold, named cleistinol (2.2), an aporfinic alkaloid, cleistonine (2.7), and four heptane derivatives, three of which are new compounds: (R)-(E)-melodorinol (2.4), (R)-(E)-acetilmelodorinol (2.5) and kirkiinolato (2.6); and one was never isolated before from natural sources, (R)-(Z)-melodorinol (2.3). Dichamanetine (2.1), a flavanone already described in the literature, was also isolated. By molecular derivatization of dichamanetine (2.1), isolated in large amount, and using several amine derivatives, were obtained nine imines, namely: an oxime (2.8), four hydrazones (2.9 – 2.12), three carbohydrazides (2.13 – 2.15) and a semicarbazone (2.16). A tri-acetylated derivative of dichamanetine (2.17) was also prepared. The chemical structures of compounds were elucidated based on their physical ( [α]22D), and spectroscopic data ( IV, one-dimensional nuclear magnetic resonance– 1H-NMR e 13C-NMR – and bidimensional – 1H-1H-COSY, HMQC, HMBC e NOESY) and by comparison with data described in the literature. The antibacterial activity of compounds (2.1 – 2.17) was evaluated in susceptible (ATCC 6538), methicillin-resistant (ATCC 43300), and methicillin-resistant and vancomycin-intermediate (CIP 106414) Staphylococcus aureus strains. Among the isolated compounds, dichamanetine (2.1) showed to be a potent antibacterial agent across all strains (MIC = 0.49 μg mL-1). Compound 2.5 exhibited MIC values of 15.63 and 62.5 μg mL-1, in S. aureus ATCC 6538 and CIP 106414, respectively. For compounds 2.3 and 2.4 was observed a MIC = 62.5 μg mL-1 in the susceptible strain. Compound 2.3 also showed a MIC = 62.5 μg mL-1 in the resistant strain CIP 106414. Concerning dichamanetine derivatives, the lowest MIC values were observed for compounds 2.8, 2.9 and 2.11 (0.98 to 1.95 μg mL-1) in all strains of S. aureus. Hydrazones 2.10 and 2.13 exhibited MIC values between 7.81 and 31.25 μg mL-1 in all strains. Similarly, carbohydrazide derivatives 2.14 and 2.16 showed high antibacterial activities in ATCC 6538 and CIP 106414 strains, with MIC values of 3.9 and 15.63 μg mL-1, respectively. However, none of the imine derivatives was more potent than dichamanetine itself, providing evidence for the importance of the carbonyl group at C4 for the antibacterial activity as well as the free hydroxyl groups, since their acetylation led to a lose of the activity. The interaction between compounds (2.1 – 2.17) and the antibiotics amoxicillin, ofloxacin and vancomycin was also evaluated in the same strains. A total of eight compounds, three isolated (2.3, 2.4 and 2.5) and five derivatives (2.9, 2.10, 2.12, 2.14 e 2.15) were able to modulate the activity the three antibiotics, in all S. aureus strains assayed. Compound 2.12, without antibacterial activity, revealed the strongest modulatory effect on the antibacterial activity of the three tested antibiotics, in all strains.
The main goal of this dissertation was to find compounds with antibacterial activity (S. aureus) from the African medicinal plant C. kirkii (Annonaceae). Accordingly, two approaches were used: i) the isolation of major constituents from a fraction of the methanol extract; and ii) molecular derivatization of a compound isolated in large amount. From a fraction resulting from the bioguided fractionation of the methanol extract of C. kirkii were isolated, by chromatographic techniques, a new compound with a cyclohexene scaffold, named cleistinol (2.2), an aporfinic alkaloid, cleistonine (2.7), and four heptane derivatives, three of which are new compounds: (R)-(E)-melodorinol (2.4), (R)-(E)-acetilmelodorinol (2.5) and kirkiinolato (2.6); and one was never isolated before from natural sources, (R)-(Z)-melodorinol (2.3). Dichamanetine (2.1), a flavanone already described in the literature, was also isolated. By molecular derivatization of dichamanetine (2.1), isolated in large amount, and using several amine derivatives, were obtained nine imines, namely: an oxime (2.8), four hydrazones (2.9 – 2.12), three carbohydrazides (2.13 – 2.15) and a semicarbazone (2.16). A tri-acetylated derivative of dichamanetine (2.17) was also prepared. The chemical structures of compounds were elucidated based on their physical ( [α]22D), and spectroscopic data ( IV, one-dimensional nuclear magnetic resonance– 1H-NMR e 13C-NMR – and bidimensional – 1H-1H-COSY, HMQC, HMBC e NOESY) and by comparison with data described in the literature. The antibacterial activity of compounds (2.1 – 2.17) was evaluated in susceptible (ATCC 6538), methicillin-resistant (ATCC 43300), and methicillin-resistant and vancomycin-intermediate (CIP 106414) Staphylococcus aureus strains. Among the isolated compounds, dichamanetine (2.1) showed to be a potent antibacterial agent across all strains (MIC = 0.49 μg mL-1). Compound 2.5 exhibited MIC values of 15.63 and 62.5 μg mL-1, in S. aureus ATCC 6538 and CIP 106414, respectively. For compounds 2.3 and 2.4 was observed a MIC = 62.5 μg mL-1 in the susceptible strain. Compound 2.3 also showed a MIC = 62.5 μg mL-1 in the resistant strain CIP 106414. Concerning dichamanetine derivatives, the lowest MIC values were observed for compounds 2.8, 2.9 and 2.11 (0.98 to 1.95 μg mL-1) in all strains of S. aureus. Hydrazones 2.10 and 2.13 exhibited MIC values between 7.81 and 31.25 μg mL-1 in all strains. Similarly, carbohydrazide derivatives 2.14 and 2.16 showed high antibacterial activities in ATCC 6538 and CIP 106414 strains, with MIC values of 3.9 and 15.63 μg mL-1, respectively. However, none of the imine derivatives was more potent than dichamanetine itself, providing evidence for the importance of the carbonyl group at C4 for the antibacterial activity as well as the free hydroxyl groups, since their acetylation led to a lose of the activity. The interaction between compounds (2.1 – 2.17) and the antibiotics amoxicillin, ofloxacin and vancomycin was also evaluated in the same strains. A total of eight compounds, three isolated (2.3, 2.4 and 2.5) and five derivatives (2.9, 2.10, 2.12, 2.14 e 2.15) were able to modulate the activity the three antibiotics, in all S. aureus strains assayed. Compound 2.12, without antibacterial activity, revealed the strongest modulatory effect on the antibacterial activity of the three tested antibiotics, in all strains.
Descrição
Tese de mestrado, Química Farmacêutica e Terapêutica, Universidade de Lisboa, Faculdade de Farmácia, 2014
Palavras-chave
Cleistochlamys kirkii Staphylococcus aureus Dichamanetina Alcalóide aporfínico Derivados do heptano Derivados do ciclo-hexeno Derivados da dichamanetina Teses de mestrado - 2014