RESUMEN
Ribavirin and 2'-O-methylcytidine 5'-phosphoramidates derived from L-alanine methyl ester bearing either an O-phenyl or a biodegradable O-[3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl] or O-[3-(acetyloxymethoxy)-2,2-bis(ethoxycarbonyl)propyl] protecting group were prepared. The kinetics of the deprotection of these pro-drugs by porcine liver esterase and by a whole cell extract of human prostate carcinoma was studied by HPLC-ESI-MS/MS. The 3-(acetyloxymethoxy)-2,2-bis(ethoxycarbonyl)propyl and 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl groups were readily removed releasing the l-alanine methyl ester phosphoramidate nucleotide, the deprotection of the 3-(acetyloxymethoxy) derivative being approximately 20 times faster. The chemical stability of the 2'-O-methylcytidine pro-drugs was additionally determined over a pH range from 7.5 to 10.
Asunto(s)
Acetatos/química , Alanina/química , Amidas/química , Antivirales/química , Citidina Monofosfato/química , Esterasas/metabolismo , Malonatos/química , Ácidos Fosfóricos/química , Neoplasias de la Próstata/enzimología , Ribavirina/química , Animales , Antivirales/síntesis química , Citidina Monofosfato/síntesis química , Estabilidad de Enzimas , Esterasas/química , Humanos , Cinética , Hígado/enzimología , Masculino , Estructura Molecular , Porcinos , Factores de TiempoRESUMEN
The applicability of 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl and 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups as biodegradable phosphate protecting groups for nucleoside 5'-monophosphates has been studied in a HEPES buffer at pH 7.5. Enzymatic deacetylation with porcine carboxyesterase triggers the removal of the resulting 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl and 3-hydroxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups by retro-aldol condensation and consecutive half acetal hydrolysis and retro-aldol condensation, respectively. The kinetics of these multistep deprotection reactions have been followed by HPLC, using appropriately protected thymidine 5'-monophosphates as model compounds. The enzymatic deacetylation of the 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl 5'-triester (2) is 25-fold faster than the deacetylation of its 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-protected counterpart 1, and the difference in the deacetylation rates of the resulting diesters, 12b and 12a, is even greater. With 2, conversion to thymidine 5'-monophosphate (5'-TMP) is quantitative, while conversion of 1 to 5'-TMP is accompanied by formation of thymidine. Consistent with the preceding observations, quantitative release of 5'-TMP from 2 has been shown to take place in a whole cell extract of human prostate cancer cells.
