Fluorescence studies on new potential antitumoral 1,3-diarylurea derivatives of thieno[3,2-b]pyridines in solution and in nanoliposomes

This work was supported by Fundação para a Ciência e a Tecnologia (Portugal), QREN and FEDER through CFUM, CQ/UM and Project PTDC/QUI/81238/2006 cofinanced by FCT and program FEDER/COMPETE (FCOMP-01-0124-FEDER-007467)

New 1,3-diarylureas linked by C-C Suzuki coupling to the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate moiety: synthesis and fluorescence studies in solution and in lipid membranes

New six fluorescent 1,3-diarylureas linked by C-C Suzuki coupling to the 6-position of the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate moiety were prepared by reaction of the amino groups on the ortho or meta positions relative to the C-C bond of the Suzuki coupling products, with different para-substituted arylisocyanates (H, OMe, CN), in high to excellent yields. The fluorescence properties of the 1,3-diarylureas in solution and in lipid membranes of egg-yolk phosphatidylcholine (Egg-PC), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG) or dioctadecyldimethylammonium bromide (DODAB), with or without cholesterol (Ch), were studied. The six 1,3-diarylureas have reasonable fluorescence quantum yields in several solvents (between 0.02 and 0.69) and present a moderately solvent sensitive emission, but are not fluorescent in alcohols and water. The compounds bearing the arylurea moiety in the meta position relative to the C-C bond, especially with the OMe and CN substituents, present the better solvatochromic properties. Incorporation of the six compounds in lipid membranes indicates that all the compounds are deeply located in the hydrophobic region of the lipid bilayers, feeling the transition between the rigid gel phase and fluid phases.To the Foundation for the Science and Technology (FCT, Portugal) for inancial support to the NMR portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho). To the FCT and FEDER (European Fund for Regional Development)-COMPETE-QREN-EU for financial support to the Research Centres, CQ/UM [PEst-C/QUI/UI0686/2011 (FCOMP-01-0124-FEDER-022716)] and CFUM [PEst-C/FIS/UI0607/2011 (F-COMP-01-0124-FEDER-022711)], and to the research projects PTDC/QUI/81238/2006 (FCOMP-01-0124-FEDER-007467) (photophysical studies) and PTDC/QUI-QUI/111060/2009 (F-COMP-01-0124-FEDER-015603) (organic synthesis)

A new antitumoral Heteroarylaminothieno[3,2-b]pyridine derivative : its incorporation into liposomes and interaction with proteins monitored by fluorescence

Advance Article. 2014 Oct 16 [Epub ahead of print]The fluorescence properties of the new potent antitumoral methyl 3-amino-6-(benzo[d]thiazol-2-ylamino)thieno[3,2-b]pyridine-2-carboxylate in solution and when encapsulated in several different nanoliposome formulations were investigated. The compound exhibits very reasonable fluorescence quantum yields and a solvent sensitive emission in several polar and non-polar media, despite not being fluorescent in protic solvents. Fluorescence anisotropy measurements of the compound incorporated in liposomes revealed that this thienopyridine derivative can be carried in the hydrophobic region of the lipid membrane. Liposome formulations including this antitumor compound are nanometric in size, with a diameter lower than 130 nm and generally low polydispersity, being promising for future drug delivery developments. The interaction of the compound with bovine serum albumin (BSA) and the multidrug resistance protein MDR1 was monitored by FRET, the compound acting as energy acceptor. It was observed a lower interaction with MDR1 protein than with the native form of BSA, which is an important result regarding applications of this antitumoral drug.Foundation for the Science and Technology (FCT, Portugal), FEDER/COMPETE and QREN for financial support to the Research Centres, CFUM [Strategic Project PEst- C/FIS/UI0607/2013 (FCOMP-01-0124-FEDER-022711) and CQ/UM [Strategic Project PEst-C/QUI/UI0686/2013 (FCOMP-01-0124-FEDER-022716)] and n-STeP Project NORTE-07-0124-FEDER-000039 supported by the Operational Regional Programme of North of Portugal (ON.2). FCT and POPH/QREN/FSE are acknowledged for the Post- Doc. grant of R.C.C. (SFRH/BPD/68344/2010)

Novel highly emissive non proteinogenic amino acids : synthesis of 1,3,4-thiadiazolyl asparagines and evaluation as fluorimetric chemosensors for biologically relevant transition metal cations

Highly emissive heterocyclic asparagine derivatives bearing a 1,3,4-thiadiazolyl unit at the side chain, functionalised with electron donor or acceptor groups, were synthesised and evaluated as amino acid based fluorimetric chemosensors for metal cations such as Cu2+, Zn2+, Co2+ and Ni2+. The results suggest that there is a strong interaction through the donor heteroatoms at the side chain of the various asparagine derivatives, with high sensitivity towards Cu2+ in a ligand-metal complex with 1:2 stoichiometry. Association constants and detection limits for Cu2+ were calculated. The photophysical and metal ion sensing properties of these asparagine derivatives confirm their potential as fluorimetric chemosensors and suggest that they can be suitable for incorporation into chemosensory peptidic frameworks.Fundação para a Ciência e a Tecnologia (FCT) - PTDC/QUI/66250/2006 (FCOMP-01-0124-FEDER-007428

Estudo da interação de novos compostos potencialmente antitumorais com proteínas e membranas biológicas

Dissertação de mestrado em Biofísica e BionanossitemasOs lipossomas de tamanho nanométrico encontram-se entre os desenvolvimentos tecnológicos para entrega de fármacos quimioterapêuticos e, potencialmente, esta técnica poderá superar problemas no tratamento do cancro como a solubilidade dos fármacos, a farmacocinética, a estabilidade e a toxicidade. Neste trabalho, as propriedades fotofísicas de quatro novos compostos fluorescentes potencialmente antitumorais, um derivado de tieno[3,2- b]piridina (composto A3), e três diheteroarilaminas (compostos 474D, 480D e 481D), foram estudadas em solução, quando incorporados em lipossomas e na presença de duas proteínas, a albumina do soro bovino (BSA) e uma proteína membranar do tipo “multidrug resistance” (MDR1). Os compostos apresentam rendimentos quânticos de fluorescência razoáveis (6%≤F≤77%), e exibem um desvio para o vermelho na emissão com o aumento da polaridade do solvente. Contudo, os compostos não fluorescem em solventes próticos. Quando incorporados em lipossomas, todos os compostos apresentam fluorescência razoável, indicando que podem ser transportados na membrana do lipossoma. Em geral, as medidas de anisotropia de fluorescência mostram uma diminuição no seu valor quando os lípidos se encontram acima da temperatura de transição de fase ou quando se adiciona colesterol (Ch), indicando que este fará aumentar a fluidez da membrana. No entanto, no caso da mistura de lípidos DPPC:Ch, a anisotropia aumenta à temperatura mais alta, podendo indicar que ocorre uma relocalização do composto numa região menos fluida ou uma diminuição da fluidez das membranas de DPPC provocada pelo colesterol. Estudou-se a interação entre o composto A3 e as proteínas BSA e MDR1, por transferência de energia não radiativa (FRET) entre os aminoácidos das proteínas (doadores) e o composto (aceitante). Foi possível concluir que, no caso da BSA, a interação é maior quando a proteína se encontra na conformação mais enrolada (forma N). Comparando as duas proteínas, a interação parece ser mais fraca com a MDR1. Estes resultados podem ser importantes para aplicações futuras de libertação controlada destes compostos potencialmente antitumorais, utilizando lipossomas como transportadores. São também promissores para uma futura utilização do composto A3 como fármaco antitumoral, uma vez que, sendo menor a sua interação com a proteína de tipo “multidrug resistance”, não será de esperar que seja amplamente transportado para fora da célula por proteínas desta classe.The nanosized liposomes are amongst the technological developments for delivery of chemotherapeutic drugs and potentially, this technique will overcome some problems in treating cancer, such as solubility, pharmacokinetics, stability and toxicity. In this work, the photophysical properties of four new fluorescent potential antitumor compounds, a thieno[3,2-b]pyridine derivative (compound A3) and three diheteroarylamines (compounds 474D, 480D and 481D), were studied in solution, when incorporated in liposomes, and interacting with two proteins, bovine serum albumin (BSA) and a multidrug resistance membrane protein (MDR1). The compounds exhibit reasonable fluorescence quantum yields (6%≤F≤ 77%) and a red shift in emission with increasing solvent polarity. However, the compounds are not fluorescent in protic solvents. When incorporated in liposomes, all compounds exhibit reasonable fluorescence emission, indicating that they can be transported in the membrane region of the liposome. Fluorescence anisotropy measurements show a decrease in anisotropy when the lipids are above the phase transition temperature and in the presence of cholesterol (Ch), indicating that Ch can promote an increase in membrane fluidity. However, for the DPPC:Ch lipid mixture, the anisotropy is higher at increasing temperature, which may indicate a relocation of compounds in a less fluid region of the membrane or a decrease in fluidity of DPPC membranes induced by cholesterol. The interaction between compound A3 and the proteins BSA and MDR1 was studied by resonance energy transfer (FRET) from the protein amino acids (donors) to the compound (acceptor). It was possible to conclude that, for BSA, the interaction is larger when the protein is in the compact normal conformation (N form). Comparing both proteins, the compound interaction with MDR1 seems to be weaker. These results may be important for future applications of controlled drug delivery of these potential antitumor compounds using liposomes as carriers. The results are also promising for the future use of compound A3 as an antitumor drug, since, as its interaction with multidrug resistance protein is weaker, it is not expected that the compound is widely transported out of the cell by this type of proteins

Desenvolvimento Rural e Reconhecimento: tensões e dilemas envolvendo o Pronaf

Resumo: Este artigo objetiva investigar como o Pronaf é definido e avaliado pelo Estado brasileiro a fim de desconstruir qualquer relação de causalidade entre desenvolvimento e reconhecimento que possa existir nas informações sobre o Programa. A metodologia utilizada constitui-se por revisão de literatura e pela pesquisa de dados oficiais sobre o Pronaf, a qual orientou os questionamentos sobre o volume de recursos disponibilizado para o crédito, bem como sobre a distribuição destes entre os grupos de beneficiários e as regiões do País. Como principais resultados, evidencia-se que apenas a redistribuição dos recursos não é suficiente para o reconhecimento dos agricultores