Autor(es): Fidalgo, Lara Gião
Data: 2015
Identificador Persistente: http://hdl.handle.net/10362/18454
Origem: Repositório Institucional da UNL
Assunto(s): Tolvaptan; Phase II metabolites; DNA adducts; 2’-deoxynucleobases; 5’-O-acetyl- tolvaptan; 5’-O-sulfonate-tolvaptan; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química
Tolvaptan is an orally effective nonpeptide arginine vasopressin (AVP) V2-receptor antagonist, used in the treatment of clinically significant euvolemic and hypervolemic hyponatremia. Nonetheless, Tolvaptan was reported to be associated with a high risk of liver injury, prompting the U. S. Food and Drug Administration (FDA) to issue a black box label warning. Whereas the mechanisms of tolvaptan-induced liver injury remain to be elucidated, preliminary results obtained in cytochrome P450 (CYP)-deficient liver cell lines have shown evidence of tolvaptaninduced DNA damage without the need for Phase I metabolism. Taking into consideration that Phase II sulfonation and/or acetylation of tolvaptan’s secondary alcohol are metabolically plausible events, the ultimate goal of this thesis was to study the reactivity of these putative Phase II metabolites towards DNA. Indeed, the covalent adducts thus afforded could explain the tolvaptan-induced DNA damage observed in CYP-deficient liver cell lines. Since the identification of DNA adducts involves the hydrolysis to 2’-deoxynucleobase adducts followed by liquid chromatography coupled with mass spectrometry (LC-ESI-MS) in comparison with adducts standards, the first step of this thesis was the preparation of tolvaptan-DNA adduct standards. Two distinct strategies were followed: 1) biomimetic, involving the reaction of the metabolic plausible sulfate and acetate tolvaptan metabolites with 2’-deoxynucleobases; 2) non-biomimetic, involving a palladium-catalyzed coupling reaction. For the biomimetic approach, the two metabolic plausible Phase II metabolites were first prepared: 1) 5’-O-sulfonate-tolvaptan was prepared in 89 % yield upon tolvaptan sulfonation using sulfur trioxide-pyridine complex; 2) 5’-O-acetyl-tolvaptan was prepared in 80% following tolvaptan acetylation with acetic anhydride and trimethylamine. The reaction of 5’-O-sulfonate-tolvaptan and 5’-O-acetyl-tolvaptan with 2’-deoxynucleobases (and nucleophilic amino acids) were subsequently undergone, with the ultimate goal of preparing adduct standards. However, only the product stemming from the elimination of sulfoxy and acetoxy groups was detected, upon LC-ESI-MS analysis of these reaction mixtures. For the non-biomimetic strategy, the derivative 5’-chloro-tolvaptan was prepared upon reaction of tolvaptan with thionyl chloride, and subsequently coupled with 3′,5′-O-bis(tertbutyldimethylsilyl)-2′-deoxyguanosine, under palladium catalysis. Following deprotection, the LC-ESI-HRMS analysis of the reaction mixture allowed the identification of two covalent adducts, one non-depurinating and one depurinating. The reaction of 5’-O-sulfonate-tolvaptan and 5’-O-acetyl-tolvaptan with DNA were also undergone. However, the LC-ESI-HRMS analysis of the hydrolysates obtained following enzymatic or thermic hydrolysis did not allow the identification of any tolvaptan-DNA adduct. This result suggests that the DNA damage observed in hepatic cell lines does not stem from the formation of DNA adducts with the metabolic plausible Phase II metabolites, 5’-O-sulfonate-tolvaptan and 5’-O-acetyltolvaptan.
