Compostos de paládio(II) contendo tiossemicarbazonas: síntese, interação com alvos enzimáticos e avaliação do potencial antitumoral
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Data
2020-04-28
Autores
Farias, Renan Lira de
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Universidade Estadual Paulista (Unesp)
Resumo
Prescrições semanais de 2 a 5 mg kg-1 de cisplatina tem oferecido um avanço no tratamento de vários tipos de cânceres geniturinários, sendo as taxas de sobrevida ca. 98%. Contudo, os complexos de Pt(II) se mostram inespecíficos frente às células normais de rápida proliferação, o que limita a dose a ser administrada. Nesta tese de doutorado, foram preparados oito complexos de Pd(II) do tipo [PdCl2(L)PPh3]Cl (família 1 a 4) e [Pd(L)2] (família 5 a 8), onde PPh3 = trifenilfosfina e L = trans-cinamaldeído-N4-R-tiossemicarbazona (R = H, Metila, Etila e Fenila). Os compostos foram caracterizados através das técnicas espectroscópicas de infravermelho com transformada de Fourier, ressonância magnética nuclear de 1H, 13C e 31P. As formulas mínimas e purezas dos compostos foram determinadas através de microanálise CHN%. Dados de condutância molar em DMF exibiram o perfil de eletrólito 1:1 para 1-4, enquanto os complexos 5 a 6 foram não eletrólitos. Os complexos 2, 3 e 6 formaram cristais aptos aos experimentos de difração de raios X por monocristal e obtiveram suas estruturas cristalinas resolvidas. Com o intuito de selecionar compostos líderes, ensaios in vitro de citotoxicidade e efeitos hemolíticos foram conduzidos como sequência de triagem. As respectivas citotoxicidades para todos os compostos foram investigadas pelo método colorimétrico MTT e expressas como CI50, frente às culturas tumorais humanas MCF-7 (adenocarcinoma mamário) e A549 (carcinoma de pulmão). Além disso, os índices de seletividade (IS) foram estimados a partir das razões entre os valores de CI50 frente às culturas celulares não tumorais MRC5 (fibroblasto de pulmão) e LLC-MK2 (rim de macaco Rhesus). Assim, três compostos foram considerados promissores (L3, 3 e 7), entretanto, apenas o complexo 3 exibiu padrões aceitáveis de hemólise, < 9 %. Ensaios de supressão de fluorescência evidenciaram que adições crescentes de 3 promovem decréscimo na banda de emissão correspondente a HSA ( λmax = 333 nm) através de um mecanismo de supressão misto (dinâmico + estático). Os parâmetros termodinâmicos ΔH > 0 e ΔS > 0 indicaram que o processo ocorre por interações predominantemente hidrofóbicas. Outros estudos de ligação com a biomolécula ctDNA foram empregados. O valor da constante de formação aparente (Kb = 6,63 x 103 M-1), em combinação aos experimentos de CD e competição com a sonda Hoechst 33258 sugerem fraca interação do complexo 3 pelo ctDNA. A atividade antitumoral de 3 foi investigada in vivo através do implante de tumor xenográfico de células MCF-7 em camundongos nude BALB/c. Em dose única (i.p) de 5 mg kg-1, 3 exibiu atividade antitumoral capaz de retardar a progressão tumoral entre o 2º e 7º dia de tratamento, com regressão do tumor até o 16º dia de tratamento. É importante ressaltar que a partir do 7º dia de tratamento até o 4º dia após o tratamento, 3 induziu a redução de apenas 7,8 % no peso corporal dos animais. Esses resultados são promissores tendo em vista que a redução está de acordo com padrões éticos. A capacidade de 3, e seus análogos, 1, 2 e 4, em inibir a ação de decatenação do kDNA mediada pela enzima Topoisomerase IIα foi avaliada pela técnica de eletroforese em gel de agarose. Os dados mostraram que apenas 3 e 4 foram capazes de inibir parcialmente o funcionamento da enzima para as concentrações entre 1 a 100 μM, enquanto que 2 demonstra leve atividade a 100 μM. Cálculos in silico sugeriram que 2, 3 e 4 estariam atuando como moduladores alostéricos. Portanto, os resultados dessa tese são promissores e parecem convergir ao arcabouço molecular [Pd(II)(TSC)PR3] (sendo, TSC = tiossemicarbazona bidentada e PR3 = fosfina terciária) como arquitetura privilegiada para o design de novos metalofármacos antitumorais mais seletivos as células tumorais como resultado da afinidade por outro alvo terapêutico que não o DNA.
Weekly, prescriptions ranging at 2-5 mg kg-1 of cisplatin have offered an advance in the treatment of several genitourinary cancers, being survival rates ca. 98%. On the other hand, Pt(II) complexes are no selective in rapidly proliferating normal cells, which limits the dose to be administered. Herein, eight Pd (II) complexes of the type [PdCl2(L)PPh3]Cl (1-4) and [Pd(L)2] (5-8) were prepared (where, PPh3 = triphenylphosphine and L = trans-cinnamaldehyde-N4-R-thiosemicarbazone; R = H, Methyl, Ethyl and Phenyl). The compounds were characterized by using infrared and NMR 1H, 13C and 31P spectroscopic techniques. The minimum formula and purities of them were determined by using CHN% microanalysis. Molar conductivity data (at DMF) showed the electrolyte profile 1:1 to 1-4, while complexes 5-6 were non-electrolytes. Complexes 2, 3 and 6 formed suitable crystals for single-crystal X-ray diffraction experiments and their crystalline structures were solved. To select leading compounds, in vitro cytotoxicity and RBC hemolytic assays were performed out as a screening. For all compounds, the cytotoxicities were investigated by the MTT colourimetric method and expressed as IC50 in human tumour cultures MCF-7 (mammary adenocarcinoma) and A549 (lung carcinoma). Besides, selectivity index (IS) were estimated from the ratios between IC50 values against non-tumour cells MRC5 (lung fibroblast) and LLC-MK2 (Rhesus monkey kidney). Accordingly, three compounds were considered promising (L3, 3 and 7), however, just 3 showed acceptable patterns of hemolysis, e.g., hemolysis < 9 %. Based on fluorescence trials, increasing concentration of 3 promotes a quenching of the emission band corresponding to HSA (λmax = 333 nm) through a mixed suppression mechanism. The thermodynamic parameters ΔH> 0 and ΔS> 0 indicated the process occurs by hydrophobic interactions. Other binding studies to ctDNA were employed out. The value of the binding constant, Kb = 6.63 x 103 M-1 with both CD and competition experiments with the Hoechst 33258 dye suggested a weak affinity of complex 3 for the ctDNA structure. The antitumor activity of 3 was investigated in vivo by tumour xenograft implantation of MCF-7 cells in BALB/c nude mice. Take into account a single dose (i.p) of 5 mg kg-1 day-1, 3 exhibited antitumor activity capable of delaying tumour progression between the 2nd and 7th day of treatment, also tumour regression until the 16th day of treatment. It is important to note that after the 7th day of treatment until the 4th day after treatment, 3 induced 7.8% of body weight loss. These results are promising considering the reduction does not exceed ethical standards. The ability of 3, and its analogues, 1, 2 and 4, to inhibit the kDNA decatenation action mediated by the Topoisomerase IIα enzyme was evaluated using the agarose gel electrophoresis technique. The data showed that only 3 and 4 were able to partially inhibit enzyme function in a concentration ranging at 1-100 μM, whereas 1 was inactive and 2 shows poor activity at 100 μM. In silico calculations suggested that 2, 3 and 4 would be acting as allosteric modulators. Therefore, the results of this thesis are promising and seem to converge to the molecular scaffold [PdII(TSC) PR3] (being, TSC = bidentate thiosemicarbazone and PR3 = tertiary phosphine) as a privileged architecture for the design of new metallodrugs with greater selectivity against tumours cells, as a result of affinity by another therapeutic target than DNA.
Weekly, prescriptions ranging at 2-5 mg kg-1 of cisplatin have offered an advance in the treatment of several genitourinary cancers, being survival rates ca. 98%. On the other hand, Pt(II) complexes are no selective in rapidly proliferating normal cells, which limits the dose to be administered. Herein, eight Pd (II) complexes of the type [PdCl2(L)PPh3]Cl (1-4) and [Pd(L)2] (5-8) were prepared (where, PPh3 = triphenylphosphine and L = trans-cinnamaldehyde-N4-R-thiosemicarbazone; R = H, Methyl, Ethyl and Phenyl). The compounds were characterized by using infrared and NMR 1H, 13C and 31P spectroscopic techniques. The minimum formula and purities of them were determined by using CHN% microanalysis. Molar conductivity data (at DMF) showed the electrolyte profile 1:1 to 1-4, while complexes 5-6 were non-electrolytes. Complexes 2, 3 and 6 formed suitable crystals for single-crystal X-ray diffraction experiments and their crystalline structures were solved. To select leading compounds, in vitro cytotoxicity and RBC hemolytic assays were performed out as a screening. For all compounds, the cytotoxicities were investigated by the MTT colourimetric method and expressed as IC50 in human tumour cultures MCF-7 (mammary adenocarcinoma) and A549 (lung carcinoma). Besides, selectivity index (IS) were estimated from the ratios between IC50 values against non-tumour cells MRC5 (lung fibroblast) and LLC-MK2 (Rhesus monkey kidney). Accordingly, three compounds were considered promising (L3, 3 and 7), however, just 3 showed acceptable patterns of hemolysis, e.g., hemolysis < 9 %. Based on fluorescence trials, increasing concentration of 3 promotes a quenching of the emission band corresponding to HSA (λmax = 333 nm) through a mixed suppression mechanism. The thermodynamic parameters ΔH> 0 and ΔS> 0 indicated the process occurs by hydrophobic interactions. Other binding studies to ctDNA were employed out. The value of the binding constant, Kb = 6.63 x 103 M-1 with both CD and competition experiments with the Hoechst 33258 dye suggested a weak affinity of complex 3 for the ctDNA structure. The antitumor activity of 3 was investigated in vivo by tumour xenograft implantation of MCF-7 cells in BALB/c nude mice. Take into account a single dose (i.p) of 5 mg kg-1 day-1, 3 exhibited antitumor activity capable of delaying tumour progression between the 2nd and 7th day of treatment, also tumour regression until the 16th day of treatment. It is important to note that after the 7th day of treatment until the 4th day after treatment, 3 induced 7.8% of body weight loss. These results are promising considering the reduction does not exceed ethical standards. The ability of 3, and its analogues, 1, 2 and 4, to inhibit the kDNA decatenation action mediated by the Topoisomerase IIα enzyme was evaluated using the agarose gel electrophoresis technique. The data showed that only 3 and 4 were able to partially inhibit enzyme function in a concentration ranging at 1-100 μM, whereas 1 was inactive and 2 shows poor activity at 100 μM. In silico calculations suggested that 2, 3 and 4 would be acting as allosteric modulators. Therefore, the results of this thesis are promising and seem to converge to the molecular scaffold [PdII(TSC) PR3] (being, TSC = bidentate thiosemicarbazone and PR3 = tertiary phosphine) as a privileged architecture for the design of new metallodrugs with greater selectivity against tumours cells, as a result of affinity by another therapeutic target than DNA.
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Palavras-chave
Fármacos, Tiossemicarbazonas, Bioinformática, Modelos animais, Inibidores enzimátcos