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Desenvolvimento e produção de um medicamento: da abordagem da síntese laboratorial ao processo industrial
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O trabalho realizado no âmbito deste mestrado em Química Tecnológica foi composto por duas vertentes, por um lado o desenvolvimento de novos complexos metálicos para potencial aplicação como agentes antitumorais, realizado na Faculdade de Ciências da Universidade de Lisboa, por outro, o acompanhamento de todo o processo de produção industrial de um fármaco já em uso clínico, na empresa Iberfar, Indústria Farmacêutica SA. A procura de novos fármacos que combatam o cancro e colmatem as ineficiências dos fármacos existentes em uso clínico é de extrema importância. Após a síntese e identificação dos compostos promissores, estes passam por um longo e complicado processo onde são sujeitos a vários estudos como a reprodutibilidade das sínteses, estudos de estabilidade, citotoxicidade, estudos de interacção com proteínas e DNA e estudos de distribuição celular, para identificar os seus modos de acção. Se os compostos cumprirem todos os requisitos necessários poderão a chegar aos estudos pré-clínicos e clínicos, levando este processo anos até atingir o mercado. Os fármacos produzidos na indústria farmacêutica já concluíram com sucesso todos os testes necessários e, por isso, já se encontram no mercado. Neste processo para garantir a qualidade do fármaco todas as etapas da produção, desde a chegada da matéria-prima ao embalamento do fármaco, são sujeitas a diversas análises. A parte do trabalho realizada na Faculdade de Ciências da Universidade de Lisboa, com a duração de aproximadamente oito meses, teve como objectivo a síntese e caracterização de novos complexos binucleares de Ru(II)-Cu(I), Cu(I)-Cu(I) e Ru(II)-Ru(II) com vista à sua aplicação como potenciais agentes anticancerígenos. No presente trabalho foram sintetizados dois complexos mononucleares de cobre de fórmula geral [Cu(mTPPMSNa)2L][BF4] (onde L = 2,3-bis(2-piridil)pirazina (dpp) ou CH3CN). Foram, ainda, sintetizados três novos complexos binucleares inorgânicos de Cu(I)-Cu(I) de formula geral [(P)Cu(μ-dpp)Cu(P)][BF4]2 (onde P = 2PPh3, 2mTPPMSNa ou dppe). Para além da síntese de complexos binucleares de cobre, obtiveram-se mais quatro complexos binucleares organometálicos, três de Ru(II)-Cu(I) de fórmula geral [Cu(P)(μ-dpp)RuCp(PPh3)][CF3SO3][BF4] (onde P = 2PPh3, 2mTPPMSNa ou dppe) e um complexo de Ru(II)-Ru(II) ([(PPh3)CpRu(μ-dpp)Ru(PPh3)][CF3SO3]2). Todos os complexos foram caracterizados por várias técnicas espectroscópicas como FT-IR, UV-vis, 1H RMN, 31P RMN, 13C RMN, e pelas técnicas bidimensionais de RMN cosy, HMBC e HSQC. Para um dos complexos, foram realizadas ainda experiências de RMN a baixas temperaturas (entre 10ºC e -40ºC) e estudos computacionais de teoria do funcional da densidade (cálculos DFT) que nos permitiram comprovar a existência de isómeros. As análises elementares vieram a confirmar algumas estruturas propostas. A actividade citotóxica do complexo [Cu(dppe)(μ-dpp)RuCp(PPh3)][CF3SO3][BF4] (8) foi avaliada nas linhas celulares cancerígenas humanas do carcinoma do ovário (A2780 e A2780cisR), do adenocarcinoma da mama (MCF7) e ainda na linha celular não tumoral humana embrionária do rim (HEK293). A actividade citotóxica dos restantes complexos sintetizados foi avaliada na linha celular cancerígena do adenocarcinoma da mama (MDA-MB-231). De modo a obter alguma informação acerca dos possíveis modos de acção dos complexos foi realizado um estudo de produção de espécies de oxigénio reactivas (ROS) ao complexo [Cu(dppe)(μ-dpp)RuCp(PPh3)][CF3SO3][BF4] (8). O estágio de um mês realizado na empresa Iberfar, Indústria Farmacêutica SA, possibilitou o acompanhamento de todo o processo industrial da produção de um fármaco, desde a recepção da matéria-prima até ao embalamento do produto final.
The work carried out under this master´s degree in Technological Chemistry was composed of two parts, on one side the development of new metallic complexes for potential use as antitumor agents, performed at Faculdade de Ciências da Universidade de Lisboa, on the other side the accompaniment of the entire process of a drug industrial production already in clinical use, at Iberfar, Indústria Farmacêutica SA company. The search of new drugs for cancer treatment that overcomes the inefficiencies of the existing drugs in clinical use is of the utmost importance. After the synthesis and identification of promising compounds, they undergo a long and complicated process, where they are subjected to several studies, such as the synthesis reproducibility, cytotoxicity, stability studies, DNA and proteins interaction studies and cellular distribution studies to clarify their mode of action. If the compounds comply all the necessary requirements, they will be able to enter in pre-clinical and clinical trials, a process that take years to reach the market. In the pharmaceutical industry, the drugs have already accomplishment with success all tests necessary, and are already in the market. In this process to guarantee the quality of the drug every stages of the production, since the arrival of raw material to the final product packaging, are subjected to diverse analysis. The work carried out at Faculdade de Ciências da Universidade de Lisboa, with duration of approximately eight months, aimed the synthesis of new binuclear complexes of Ru(II)-Cu(I), Cu(I)-Cu(I) and Ru(II)-Ru(II), for application as potential anticancer agents. In the present work, two mononuclear copper(I) complexes of general formula [Cu(mTPPMSNa)2L][BF4] were synthetized (where L = 2,3-bis(2-pyridyl)pyrazine (dpp) or CH3CN). Also three inorganic binuclear Cu(I)-Cu(I) complexes of general formula [(P)Cu(μ-dpp)Cu(P)][BF4]2 were synthetized (where P = 2PPh3, 2mTPPMSNa or dppe). In addition to the synthesis of binuclear copper complexes, another four organometallic complexes were obtained, three Ru(II)-Cu(I) complexes of general formula [Cu(P)(μ-dpp)RuCp(PPh3)][CF3SO3][BF4] (where P = 2PPh3, 2mTPPMSNa or dppe) and one Ru(II)-Ru(II) complex ([(PPh3)CpRu(μ-dpp)Ru(PPh3)][CF3SO3]2); All the complexes were characterized by several spectroscopy techniques such as FT-IR, UV-vis, 1H RMN, 31P RMN, 1C RMN, and two-dimensional NMR techniques (cosy, HMBC e HSQC). For one complex it was necessary perform NMR experiments at low temperatures (between 10ºC to -40ºC) that together with computational studies of density functional theory (DFT calculations) showed the presence of isomers in the complex. Elemental analyses have confirmed some of the proposed structures. The cytotoxic activity of the [Cu(dppe)(μ-dpp)RuCp(PPh3)][CF3SO3][BF4] (8) complex was evaluated in ovarian carcinoma (A2780, A2780cisR) and breast adenocarcinoma (MCF7) human cancer cell lines, and in an non-tumoral human embryonic kidney cell line (HEK293). The cytotoxic activity of the remaining synthetized complexes was evaluated in breast adenocarcinoma (MDA-MB-231) human cancer cell line. In order to get some information about the mode of action of the [Cu(dppe)(μ-dpp)RuCp(PPh3)][CF3SO3][BF4] (8) complex a study of the production of reactive oxygen species (ROS) was performed. During the month in Iberfar, Indústria Farmacêutica SA., it was possible follow the entire industrial process of producing drugs, since the reception of raw material to the packaging of the final product.
The work carried out under this master´s degree in Technological Chemistry was composed of two parts, on one side the development of new metallic complexes for potential use as antitumor agents, performed at Faculdade de Ciências da Universidade de Lisboa, on the other side the accompaniment of the entire process of a drug industrial production already in clinical use, at Iberfar, Indústria Farmacêutica SA company. The search of new drugs for cancer treatment that overcomes the inefficiencies of the existing drugs in clinical use is of the utmost importance. After the synthesis and identification of promising compounds, they undergo a long and complicated process, where they are subjected to several studies, such as the synthesis reproducibility, cytotoxicity, stability studies, DNA and proteins interaction studies and cellular distribution studies to clarify their mode of action. If the compounds comply all the necessary requirements, they will be able to enter in pre-clinical and clinical trials, a process that take years to reach the market. In the pharmaceutical industry, the drugs have already accomplishment with success all tests necessary, and are already in the market. In this process to guarantee the quality of the drug every stages of the production, since the arrival of raw material to the final product packaging, are subjected to diverse analysis. The work carried out at Faculdade de Ciências da Universidade de Lisboa, with duration of approximately eight months, aimed the synthesis of new binuclear complexes of Ru(II)-Cu(I), Cu(I)-Cu(I) and Ru(II)-Ru(II), for application as potential anticancer agents. In the present work, two mononuclear copper(I) complexes of general formula [Cu(mTPPMSNa)2L][BF4] were synthetized (where L = 2,3-bis(2-pyridyl)pyrazine (dpp) or CH3CN). Also three inorganic binuclear Cu(I)-Cu(I) complexes of general formula [(P)Cu(μ-dpp)Cu(P)][BF4]2 were synthetized (where P = 2PPh3, 2mTPPMSNa or dppe). In addition to the synthesis of binuclear copper complexes, another four organometallic complexes were obtained, three Ru(II)-Cu(I) complexes of general formula [Cu(P)(μ-dpp)RuCp(PPh3)][CF3SO3][BF4] (where P = 2PPh3, 2mTPPMSNa or dppe) and one Ru(II)-Ru(II) complex ([(PPh3)CpRu(μ-dpp)Ru(PPh3)][CF3SO3]2); All the complexes were characterized by several spectroscopy techniques such as FT-IR, UV-vis, 1H RMN, 31P RMN, 1C RMN, and two-dimensional NMR techniques (cosy, HMBC e HSQC). For one complex it was necessary perform NMR experiments at low temperatures (between 10ºC to -40ºC) that together with computational studies of density functional theory (DFT calculations) showed the presence of isomers in the complex. Elemental analyses have confirmed some of the proposed structures. The cytotoxic activity of the [Cu(dppe)(μ-dpp)RuCp(PPh3)][CF3SO3][BF4] (8) complex was evaluated in ovarian carcinoma (A2780, A2780cisR) and breast adenocarcinoma (MCF7) human cancer cell lines, and in an non-tumoral human embryonic kidney cell line (HEK293). The cytotoxic activity of the remaining synthetized complexes was evaluated in breast adenocarcinoma (MDA-MB-231) human cancer cell line. In order to get some information about the mode of action of the [Cu(dppe)(μ-dpp)RuCp(PPh3)][CF3SO3][BF4] (8) complex a study of the production of reactive oxygen species (ROS) was performed. During the month in Iberfar, Indústria Farmacêutica SA., it was possible follow the entire industrial process of producing drugs, since the reception of raw material to the packaging of the final product.
Descrição
Tese de mestrado, Química Tecnológica, Universidade de Lisboa, Faculdade de Ciências, 2017
Palavras-chave
Ruténio Complexos binucleares Fármaco Cobre Cancro Teses de mestrado - 2017