Advances in the synthesis and application of quinones

Chapter 1 presents a brief overview of sustainable methodologies in chemical synthesis, together with the role of quinones. In Chapter 2 the biological activity of twelve 5-aminoindolequinones against NQO2, an underexplored enzyme, was assessed and three new 5-amino indolequinone derivatives displayed good inhibitory profile against this enzyme. Besides, the UV-visible absorbance curves and the nucleophilic trapping of the activated electrophilic iminium intermediate following reductive activation of the indolequinone scaffold were effectively carried out for two substrates. These experiments were used in order to gather evidence that elimination of the leaving group, required for the activity of these compounds, would happen. The interaction of 5-aminoindolequinone 37 with NQO2 was analysed by mass spectrometry and the formation of the adduct 37/FAD suggests a mechanism-based inhibition of the enzyme. In Chapter 3 the experiments were performed to advance towards a more sustainable synthesis of 5-aminoindolequinones. A range of reaction conditions, such as time, metal loading, solvent, and concentration of the solution were adjusted throughout the synthetic sequence but the main focus was on the optimisation of the conditions for the reaction between 2-bromo-5-methoxy-1,4-benzoquinone 156 and enamine 158 through a copper(II)-mediated process in the presence of a more environmentally benign solvent system in which water was used as the main solvent. There are no precedents in the literature for the synthesis of these derivatives in water. At the end of the synthetic sequence, sixteen 5-amino substituted indolequinones were prepared. In Chapter 4 the experiments were designed to develop a more sustainable synthesis of halogenated phenols. A range of reaction conditions were optimised in the reactivity of quinone diazides towards nucleophilic substitution. The concentration of the solution was increased from 0.5 to 1 M, the catalyst loading was reduced from 10 to 0.5 mol% and the reaction time decreased from 1 h to approximately 10 min. Thirteen halogenated phenols were successfully prepared, typically in low to moderate yields. Finally, Chapter 5 contains details of all experimental procedures, and describes the chemical elimination reductive studies, examined in Chapter 2, the synthesis of 5-aminoindolequinones and their precursors, discussed in Chapter 3, and the synthesis of halogenated phenols and corresponding precursors, quinone diazides, analysed in Chapter 4

Síntese de análogos de epigalocatequina-3-galato

Dissertação de mestrado em Química MedicinalO principal objetivo desta tese de mestrado consistiu no desenvolvimento de procedimentos experimentais que permitissem a síntese de derivados de epigalocatequina galato para serem sujeitos a testes in vitro contra a linha celular HCT116 do carcinoma humano colo-rectal. Vários derivados de produtos naturais foram sintetizados recorrendo a procedimentos, simples e diretos, baseados em reações sequenciais. O processo global foi alcançado através do uso dos produtos formados em cada reação como reagente de partida, na reação que lhe sucedeu. O trabalho experimental teve início com a síntese de derivados de calconas, com rendimentos de moderados a bons. As reações decorreram em meio alcalino, através de um processo eco-friendly que fez uso de condições reacionais idênticas às usadas na reação de condensação aldólica típica: o uso de hidróxido de sódio e de solventes inócuos, como água e etanol. De um modo geral, as reações foram realizadas à temperatura ambiente. As calconas obtidas foram, posteriormente, oxidadas com peróxido de hidrogénio e catálise básica (um procedimento baseado na reação de Algar Flynn Oyamada), à temperatura ambiente e/ou com aquecimento. Os flavonóis foram isolados com rendimentos de moderados a bons. Na última etapa desta abordagem sintética concertada, fez-se a reação dos flavonóis com derivados de ácido benzóico, usando DCC e DMAP. Esta reação representa uma esterificação de Steglich, decorreu à temperatura ambiente, durante vários dias, e permitiu a formação dos ésteres derivados da quercetina com um rendimento moderado. A análise farmacológica estrutura-atividade foi realizada na linha celular HCT116 do carcinoma humano colo-rectal e mostrou uma atividade anticancerígena mais efetiva dos derivados sintéticos quando comparados com a atividade da quercetina, um flavonol natural, que foi usada como um controlo positivo. De um modo geral, os derivados halogenados (quer as calconas como os flavonóis) mostraram apresentar um maior potencial contra o carcinoma humano colo-rectal por comparação com os derivados metilados/metoxilados. Os derivados de calcona e flavonol mais ativos induziram a progressão do ciclo celular nas fases S e G2/M, através de análise por citometria de fluxo, e revelaram um aumento do número de células na fase sub-G1. A inibição do ciclo celular e a indução da apoptose foram confirmadas por western blot e condensação nuclear.The main aim of this Master’s thesis was the development of experimental procedures to the synthesis of epigallocatechin gallate derivatives to be tested in vitro against the human HCT116 colorectal cancer cell line. Several derivatives were synthesized, applying simple and straightforward procedures based on sequential reactions. The complete process was achieved using the products formed as the reagents in the succeeding reaction. Experimental work started with the synthesis of chalcone derivatives, isolared in moderate to good yield. The experiments were performed in basic media, by an eco-friendly approach which applies the same reaction conditions as in Aldol condensation: the use of innocuous solvents such as water and ethanol. In general, these reactions were performed at room temperature. The compounds were oxidized by hydrogen peroxide with base catalysis (a procedure based on the Algar Flynn Oyamada reaction), at room temperature and/or with heating, and afforded the flavonols also in a moderate to good yields. The reaction of flavonols with benzoic acid derivatives, using DCC and DMAP proceeded at room temperature and allowed the isolation of quercetin esters derivatives, in moderated yield. This reaction represents a Steglich esterification. Structure-activity pharmacological evaluation was performed with the human colorectal carcinoma cell line HCT116 and revealed a more effective anticancer activity of the synthetic chalcones and flavonols when compared to the natural compound, quercetin which was used as positive control. In general, halogenated derivatives (both chalcones and flavonols) proved to have more potential against colorectal cancer when compared with the corresponding methylated/methoxylated analogues. The most active calcone and flavonol derivatives induced cell cycle arrest at S and G2/M phases, by flow citometry analysis, and revealed an increase at sub-G1 phase, characteristic of apoptosis. Cell cycle inhibition and apoptosis induction were confirmed by Western blot and nuclear condensation

Advances in the synthesis and application of quinones

Chapter 1 presents a brief overview of sustainable methodologies in chemical synthesis, together with the role of quinones. In Chapter 2 the biological activity of twelve 5-aminoindolequinones against NQO2, an underexplored enzyme, was assessed and three new 5-amino indolequinone derivatives displayed good inhibitory profile against this enzyme. Besides, the UV-visible absorbance curves and the nucleophilic trapping of the activated electrophilic iminium intermediate following reductive activation of the indolequinone scaffold were effectively carried out for two substrates. These experiments were used in order to gather evidence that elimination of the leaving group, required for the activity of these compounds, would happen. The interaction of 5-aminoindolequinone 37 with NQO2 was analysed by mass spectrometry and the formation of the adduct 37/FAD suggests a mechanism-based inhibition of the enzyme. In Chapter 3 the experiments were performed to advance towards a more sustainable synthesis of 5-aminoindolequinones. A range of reaction conditions, such as time, metal loading, solvent, and concentration of the solution were adjusted throughout the synthetic sequence but the main focus was on the optimisation of the conditions for the reaction between 2-bromo-5-methoxy-1,4-benzoquinone 156 and enamine 158 through a copper(II)-mediated process in the presence of a more environmentally benign solvent system in which water was used as the main solvent. There are no precedents in the literature for the synthesis of these derivatives in water. At the end of the synthetic sequence, sixteen 5-amino substituted indolequinones were prepared. In Chapter 4 the experiments were designed to develop a more sustainable synthesis of halogenated phenols. A range of reaction conditions were optimised in the reactivity of quinone diazides towards nucleophilic substitution. The concentration of the solution was increased from 0.5 to 1 M, the catalyst loading was reduced from 10 to 0.5 mol% and the reaction time decreased from 1 h to approximately 10 min. Thirteen halogenated phenols were successfully prepared, typically in low to moderate yields. Finally, Chapter 5 contains details of all experimental procedures, and describes the chemical elimination reductive studies, examined in Chapter 2, the synthesis of 5-aminoindolequinones and their precursors, discussed in Chapter 3, and the synthesis of halogenated phenols and corresponding precursors, quinone diazides, analysed in Chapter 4

Exploitation of new chalcones and 4H-chromenes as agents for cancer treatment

Chalcone and chromene derivatives were synthesized in good yield through simple and effective reactions using innocuous solvents such as water and ethanol and high yielding aldol condensations. Generally, the reactions were performed at room temperature, leading to the isolation of highly pure compounds. These compounds were tested on breast cancer cells (MCF-7 and Hs578T) and breast non-neoplastic cells (MCF-10A). After determination of IC50 value, specific assays were performed to analyze the potential of these compounds, and those bearing halogenated substituents presented enhanced activity comparing to methoxyl or methyl groups. More specifically, the bromine atom was often present in the bioactive molecules that proceeded to the final assays and showed to be promising candidates for further studies. The selected chromene acted as a cell migration inhibitory agent and triggered regulated cell death associated with G2/M cell-arrest and microtubule destabilization. For chalcones, the results suggest an anti-proliferative activity. Also, results for combination-therapy potentiated the antitumor effect of doxorubicin and reduced cytotoxicity in MCF-10A cells.We acknowledge the financial support from University of Minho, Fundaçao para a Ci ~ encia e a Tecnologia and FEDER- ^ COMPETE for financial support through Centro de Química (UID/ QUI/00686/2013 and UID/QUI/0686/2016), and for the postdoctoral grants awarded to Marta Costa (SFRH/BPD/79609/2011) and Belem Sampaio-Marques (SFRH/BPD/90533/2012), PhD grants of Filipa Santos (SFRH/BD/87139/2012) and Olívia Pontes (SFRH/BD/128850/ 2017). The confocal microscope Olympus FV1000 acquired under the financial support of PPBI-POCI-01-0145-FEDER-022122. The NMR spectrometer Bruker Avance III 400 is part of the National NMR Network (RNRMN) and was purchased within the framework of the National Program for Scientific Re-equipment, contract REDE/1517/RMN/2005 with funds from POCI 2010 (FEDER) and FCT. This work was also developed under the project NORTE-01-0145- FEDER-000013, by the Northern Portugal Regional Operational Program (NORTE 2020), through the European Regional Development Fund (FEDER) and the Competitiveness Factors Operational Program (COMPETE) and by National funds, through the Foundation for Science and Technology (ref. POCI-01-0145-FEDER007038)