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Síntese e estudo cinético de potenciais pró-fármacos para aplicação em MDEPT
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Nesta dissertação vários pró-fármacos de triazenos anti tumorais foram sintetizados e avaliados, de forma a compreender a sua potencial aplicação na estratégia MDEPT (Melanocyte-Directed Enzyme Prodrug Therapy). Esta é uma das estratégias possíveis para o tratamento do melanoma maligno, um dos tipos de cancro da pele mais agressivos.
Os pró-fármacos sintetizados 18a-f, 19a,b e 20a,b são constituídos por um agente alquilante, um derivado de monometiltriazeno 21 (MMT), ligado a um transportador (ácido 5-(4-hidroxifenil)pentanóico, N-(terc-Butoxicarbonil)-L-tirosina e cloridrato de tirosina, respetivamente) através de uma ligação amida. Foram usadas duas metodologias diferentes, que envolveram a ativação do grupo ácido carboxílico da metade transportadora. A metodologia onde se usou como agente ativante um derivado da triazina mostrou ser mais eficiente (rendimentos de 20 e 26 %) em relação à metodologia com a diciclohexilcarbodiimida (DCC) (8 a 18 %).
Para melhorar as propriedades de solubilidade e de estabilidade, os pró-fármacos 18a e 18d foram incorporados em lipossomas. Foram usados diferentes tipos de formulações – fosfatidilcolina (PC), – mistura de PC com fosfatidilglicerol (PG) (7:3), – dimiristoilfosfatidilcolina (DMPC) e – mistura de DMPC com dimiristoilfosfatidilglicerol (DMPG) (7:3). A estabilidade dos pró-fármacos incorporados foi testada a partir da formulação com melhores parâmetros de incorporação.
Dos pró-fármacos sintetizados foram avaliados cineticamente os pró-fármacos 18 e 19. Estes mostraram ser estáveis quimicamente em tampão fosfato salino a pH fisiológico (48 ≤ t1/2(h) ≤ 182, e alguns estáveis durante 2 semanas). A maioria apresentou uma hidrólise lenta no plasma humano (8 ≤ t1/2(h) ≤ 229). Apenas os pró-fármacos 18 mostraram ser bons substratos para o enzima tirosinase (0,7 ≤ t1/2(min) ≤ 6) com libertação do agente citotóxico (MMT). A estabilidade dos pró-fármacos incorporados aumentou consideravelmente em relação aos pró-fármacos livres (aumento do tempo de meia-vida) nos diversos meios.
Este trabalho permitiu-nos concluir que os pró-fármacos de triazenos 18 são promissores para aplicação na terapêutica MDEPT, uma vez que apresentam grande estabilidade em condições fisiológicas, uma excelente afinidade para a tirosinase, com libertação do respetivo agente citotóxico MMT.
In this thesis, a new serie of anti tumor triazene prodrugs was synthesized and evaluated concerning their potencial application in MDEPT (Melanocyte-Directed Enzyme Prodrug Therapy). This is one of the possibility strategies of malignant melanoma, a type of aggressive skin cancer. Prodrugs 18a-f, 19a,b and 20a,b have an alkylating agent, the monomethyltriazene derivate 21 (MMT), linked to a transporter (5-(4-hydroxyphenyl)pentanoic acid, N-(tert-Butoxycarbonyl)-L-tirosine, hydrochloride tyrosine, respectively) by an amide linkage. In the synthesis of prodrugs 18, the amide-bond formation was carried out by two different methodologies, which involved carboxylic acid activation. The 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) methodology was more efficient (20 and 26 %, yields) than dicyclohexylcarbodiimide (DCC) methodology (8 to 18 %). In order to improve some solubility and stability properties, prodrugs 18a and 18d were incorporated in liposomes. Four types of formulations were used – phosphatidylcholine (PC), – a misture of PC and phosphatidylglycerol (PG) (7:3), – dimyristoylphosphatidylcholine (DMPC) and – a misture of DMPC and dimyristoyl phosphatidylglycerol (DMPG) (7:3). The stability of the two incorporated prodrugs was tested from the formulation with the best parameter values for incorporation. Prodrugs 18 and 19 showed to be chemically stable in isotonic phosphate buffer at physiologic pH (48 ≤ t1/2(h) ≤ 182, and some were even stable for 2 weeks). Most prodrugs showed to be slowly hydrolyzed in human plasma (8 ≤ t1/2(h) ≤ 229). Only prodrugs 18 (5-(4-hydroxyphenyl)pentanoic acid derivatives) revealed to be good tyrosinase substrates (0.7 ≤ t1/2(min) ≤ 6) with release of citotoxic agent (MMT). The stability of incorporated prodrugs increase compared to free prodrugs. In conclusion, triazene prodrugs 18 are promising compounds for application in MDEPT strategy, as they have a good stability, an excellent tyrosinase affinity and an efficient release of citotoxic agent MMT.
In this thesis, a new serie of anti tumor triazene prodrugs was synthesized and evaluated concerning their potencial application in MDEPT (Melanocyte-Directed Enzyme Prodrug Therapy). This is one of the possibility strategies of malignant melanoma, a type of aggressive skin cancer. Prodrugs 18a-f, 19a,b and 20a,b have an alkylating agent, the monomethyltriazene derivate 21 (MMT), linked to a transporter (5-(4-hydroxyphenyl)pentanoic acid, N-(tert-Butoxycarbonyl)-L-tirosine, hydrochloride tyrosine, respectively) by an amide linkage. In the synthesis of prodrugs 18, the amide-bond formation was carried out by two different methodologies, which involved carboxylic acid activation. The 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) methodology was more efficient (20 and 26 %, yields) than dicyclohexylcarbodiimide (DCC) methodology (8 to 18 %). In order to improve some solubility and stability properties, prodrugs 18a and 18d were incorporated in liposomes. Four types of formulations were used – phosphatidylcholine (PC), – a misture of PC and phosphatidylglycerol (PG) (7:3), – dimyristoylphosphatidylcholine (DMPC) and – a misture of DMPC and dimyristoyl phosphatidylglycerol (DMPG) (7:3). The stability of the two incorporated prodrugs was tested from the formulation with the best parameter values for incorporation. Prodrugs 18 and 19 showed to be chemically stable in isotonic phosphate buffer at physiologic pH (48 ≤ t1/2(h) ≤ 182, and some were even stable for 2 weeks). Most prodrugs showed to be slowly hydrolyzed in human plasma (8 ≤ t1/2(h) ≤ 229). Only prodrugs 18 (5-(4-hydroxyphenyl)pentanoic acid derivatives) revealed to be good tyrosinase substrates (0.7 ≤ t1/2(min) ≤ 6) with release of citotoxic agent (MMT). The stability of incorporated prodrugs increase compared to free prodrugs. In conclusion, triazene prodrugs 18 are promising compounds for application in MDEPT strategy, as they have a good stability, an excellent tyrosinase affinity and an efficient release of citotoxic agent MMT.
Descrição
Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2012
Palavras-chave
MDEPT Tirosinase Triazenos Pró-fármacos Lipossomas Teses de mestrado - 2012