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3-hydroxy-V-coumarin hydrolysis study through fluorometry and LC-MS analysis
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Abstract(s)
As cumarinas são uma classe de lactonas presentes na natureza, com uma estrutura resultante
da fusão de um anel benzénico com uma α-pirona. Estes compostos exibem uma ampla
variedade de propriedades terapêuticas, tendo despertado o interesse da indústria farmacêutica
nos últimos anos. A sua estrutura e caraterísticas físico-químicas possibilitam a formação de
inúmeros derivados sintéticos que funcionam como scaffolds privilegiados, capazes de atingir
objetivos terapêuticos específicos em diversas doenças. As cumarinas π-expandidas revelam
características espectrofotométricas específicas, tais como maior deslocamento de Stokes e
atributos colorimétricos. Estas características atraíram os investigadores, o que levou ao
desenvolvimento da 3-hidroxi-V-cumarina, cujo metabolismo e potencial terapêutico nunca
foram estudados. A identificação e caracterização de metabolitos nas fases iniciais do
desenvolvimento de fármacos é crucial para avaliar os seus efeitos farmacológicos e
toxicológicos, o que auxilia na exclusão precoce de candidatos tóxicos ou não efetivos. Por
isso, este trabalho de investigação teve como propósito o estudo da hidrólise da 3-hidroxi-V cumarina e dos seus metabolitos.
Para verificar a variação da quantidade da 3-hidroxi-V-cumarina na presença de citosol foi
utilizado o método espectrofluorimétrico, uma vez que este composto apresenta fluorescência.
A cromatografia líquida acoplada à espectrometria de massa, foi a técnica analítica eleita para
corroborar os resultados obtidos por fluorimetria e para detetar e identificar os metabolitos
resultantes da hidrólise da 3-hidroxi-V-cumarina, uma vez que apresenta elevada sensibilidade
e seletividade. Foi também investigada a influência de parâmetros físicos nomeadamente o pH
na hidrólise da 3-hidroxi-V-cumarina. Este projeto incluiu o estudo da cinética desta reação de
hidrólise com a determinação dos parâmetros cinéticos na presença de citosol hepático humano,
bem como a comparação das atividades enzimáticas entre diferentes espécies.
Este projeto compreendeu a fase inicial do estudo do metabolismo da 3-hidroxi-V-cumarina,
com foco na reação de hidrólise que se previa ocorrer tendo em conta a estrutura química do
composto. Estudos posteriores de metabolismo serão conduzidos, nomeadamente estudos das
reações de fase II, de forma a complementar a informação obtida neste trabalho.
Coumarins are a naturally occurring class of lactones with a fused benzene ring and α-pyrone structure. These compounds display a wide variety of therapeutic properties, which has sparked interest in the pharmaceutical industry in recent years. Its structure and physicochemical characteristics enable the formation of numerous synthetic derivatives that function as privileged scaffolds, capable of targeting specific therapeutic objectives in several diseases. Chromeno[3,4-c]chromene-6,7-diones are π-expanded coumarins that reveal specific spectrophotometric characteristics, such as larger Stokes shift and colorimetric attributes. These characteristics attracted researchers and led to the development of 3-hydroxy-V-coumarin, whose metabolism and therapeutic potential have never been studied. Identifying and characterizing metabolites in the initial drug development phases is crucial to evaluating their pharmacological and toxicological effects. This helps in the early exclusion of candidates that may be toxic or ineffective. Thus, this research work studied the hydrolysis of 3-hydroxy-V coumarin and its metabolites. To assess the variation in the quantity of 3-hydroxy-V-coumarin in the presence of cytosol, the spectrofluorimetric method was employed, as this compound exhibits fluorescence. Liquid chromatography coupled with mass spectrometry was the analytical method chosen to corroborate the findings from fluorometry and to detect and identify the metabolites resulting from the hydrolysis of 3-hydroxy-V-coumarin, as it presents high sensitivity and selectivity. The influence of physical parameters on the hydrolysis of 3-hydroxy-V-coumarin, specifically pH, was also investigated. Additionally, this project included a study of the hydrolysis reaction kinetics with the determination of kinetic parameters in the presence of human liver cytosol, as well as a comparison of enzymatic activity among different species. This project comprised the initial phase of investigating the metabolism of 3-hydroxy-V coumarin, with a focus on the hydrolysis reaction expected based on the chemical structure of this compound. Subsequently, further metabolism studies will be conducted, including the examination of phase II reactions to complement the information obtained through this project.
Coumarins are a naturally occurring class of lactones with a fused benzene ring and α-pyrone structure. These compounds display a wide variety of therapeutic properties, which has sparked interest in the pharmaceutical industry in recent years. Its structure and physicochemical characteristics enable the formation of numerous synthetic derivatives that function as privileged scaffolds, capable of targeting specific therapeutic objectives in several diseases. Chromeno[3,4-c]chromene-6,7-diones are π-expanded coumarins that reveal specific spectrophotometric characteristics, such as larger Stokes shift and colorimetric attributes. These characteristics attracted researchers and led to the development of 3-hydroxy-V-coumarin, whose metabolism and therapeutic potential have never been studied. Identifying and characterizing metabolites in the initial drug development phases is crucial to evaluating their pharmacological and toxicological effects. This helps in the early exclusion of candidates that may be toxic or ineffective. Thus, this research work studied the hydrolysis of 3-hydroxy-V coumarin and its metabolites. To assess the variation in the quantity of 3-hydroxy-V-coumarin in the presence of cytosol, the spectrofluorimetric method was employed, as this compound exhibits fluorescence. Liquid chromatography coupled with mass spectrometry was the analytical method chosen to corroborate the findings from fluorometry and to detect and identify the metabolites resulting from the hydrolysis of 3-hydroxy-V-coumarin, as it presents high sensitivity and selectivity. The influence of physical parameters on the hydrolysis of 3-hydroxy-V-coumarin, specifically pH, was also investigated. Additionally, this project included a study of the hydrolysis reaction kinetics with the determination of kinetic parameters in the presence of human liver cytosol, as well as a comparison of enzymatic activity among different species. This project comprised the initial phase of investigating the metabolism of 3-hydroxy-V coumarin, with a focus on the hydrolysis reaction expected based on the chemical structure of this compound. Subsequently, further metabolism studies will be conducted, including the examination of phase II reactions to complement the information obtained through this project.
Description
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2023, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
3-hydroxy-V-coumarin Hydrolysis Fluorometry Liquid Chromatography Mass spectrometry Mestrado Integrado - 2023