RESUMEN
The insufficient penetration through the cell membranes is one of the major drawbacks of chemotherapeutics such as 5-fluorouracil (5-FU; 1). To improve the penetration, a useful strategy is the attachment of lipophilic moieties. Thus, we have synthesized a series of nucleolipid derivatives of 5-fluorouridine (5-FUrd; 2a), carrying lipophilic moieties at N(3) and/or at the 2',3'-O position, i.e., 3a, 3b, 4-7, and tested their cytostatic/cytotoxic activities towards three carcinoma cell lines (colon (HT-29), hepatocellular (HepG2), and renal (RENCA)) in comparison with 5-FU (1) and 5-FUrd (2a). After 48â h of incubation, four derivatives, 3a, 3b, 5, and 7, showed inhibitory effects on the survival of HT-29, HepG2, and RENCA cells. Additionally, to differentiate between anticancer and side-effects, we tested the cytotoxicity of the derivatives in human macrophages. Interestingly, the derivatives 4, 5, and 6 did not exhibit any effects on survival of THP-1 macrophages. Furthermore, we investigated the apoptosis induction of compound 1 and 2a, and the above-mentioned derivatives in HT-29 cells. Derivative 5 showed the highest significant (p<0.05; p<0.01) increase of the apoptosis at 80â µM after 2-h or 4-h treatment, as well as after 6-h incubation at 40â µM (p<0.05). Real-time PCR revealed that 40-µM derivative 5 showed a 1.8-fold increase of the pro-apoptotic caspase-3 gene and a twofold significant increase (p<0.01 and p<0.05 vs. control and 1, resp.) of the tumor suppressor TP53 gene, whereas the other compounds did not show any effect. We demonstrated that some 5-FUrd derivatives such as compound 5 are more effective than 5-FU or 5-FUrd concerning a cytotoxic (vs. cytostatic (5-FU, 5-FUrd)) effect on different cancer cell lines, but without cytotoxic side-effects on differentiated macrophages. Thus, compound 5 is suggested as a novel potent cytotoxic multi-anti-cancer drug.
Asunto(s)
Antineoplásicos/química , Uridina/análogos & derivados , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células HT29 , Células Hep G2 , Humanos , ARN Mensajero/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Uridina/síntesis química , Uridina/química , Uridina/farmacologíaRESUMEN
5-Fluorouridine (1a) was converted to its N(3)-farnesylated nucleoterpene derivative 8 by direct alkylation with farnesyl bromide (4). Reaction of the cancerostatic 1a with either acetone, heptan-4-one, nonadecan-10-one, or hentriacontan-16-one afforded the 2',3'-O-ketals 2a-2d. Compound 2b was then first farnesylated (â5) and subsequently phosphitylated to give the phosphoramidite 6. The ketal 2c was directly 5'-phosphitylated without farnesylation of the base to give the phosphoramidite 7. Moreover, the recently prepared cyclic 2',3'-O-ketal 11 was 5'-phosphitylated to yield the phosphoramidite 12. The 2',3'-O-isopropylidene derivative 2a proved to be too labile to be converted to a phosphoramidite. All novel derivatives of 1a were unequivocally characterized by NMR and UV spectroscopy and ESI mass spectrometry, as well as by elemental analyses. The lipophilicity of the phosphoramidite precursors were characterized by both their retention times in RP-18 HPLC and by calculated log P values. The phosphoramidites 6, 7, and 12 were exemplarily used for the preparation of four terminally lipophilized oligodeoxynucleotides carrying a cyanine-3 or a cyanine-5 residue at the 5'-(n-1) position (i.e., 14-17). Their incorporation in an artificial lipid bilayer was studied by single-molecule fluorescence spectroscopy and fluorescence microscopy.
Asunto(s)
Membrana Dobles de Lípidos/química , Lípidos/química , Oligonucleótidos/química , Uridina/análogos & derivados , Lípidos/síntesis química , Conformación Molecular , Uridina/químicaRESUMEN
The 2',3'-O-levulinic acid derivative 2b of the cancerostatic 5-fluorouridine as well as its N(3)-farnesylated nucleolipid 2d were synthesized and coupled to H2 O-soluble chitosanes of different molecular weight and at various pH values (3.5-5.5) leading to 6 and 7. In addition, the coumarine fluorophore ATTO-488 N(9)-butanoate was bound to the biopolymer by a sequential-coupling technique to afford 9 and 10. Moreover, chitosan foils were prepared, to which 2b was coupled. Their degradation by chitosanase (from Streptomyces sp. N174) was studied UV-spectrophotometrically in a Franz diffusion cell.
Asunto(s)
Quitosano/química , Uridina/análogos & derivados , Concentración de Iones de Hidrógeno , Ácidos Levulínicos/química , Prenilación , Espectrofotometría Ultravioleta , Uridina/síntesis química , Uridina/químicaRESUMEN
The cancerostatic 5-fluorouridine (5-FUrd; 1) was sequentially sugar-protected by introduction of a 2',3'-O-heptylidene ketal group (â2), followed by 5'-O-monomethoxytritylation (â3). This fully protected derivative was submitted to Mitsunobu reactions with either phytol ((Z and E)-isomer) or nerol ((Z)-isomer) to yield the nucleoterpenes 4a and 4b. Both were 5'-O-deprotected with 2% Cl2 CHCOOH in CH2 Cl2 to yield compounds 5a and 5b, respectively. These were converted to the 5'-O-cyanoethyl phosphoramidites 6a and 6b, respectively. Moreover, the 2',3'-O-(1-nonyldecylidene) derivative, 7a, of 5-fluorouridine was resynthesized and labelled at C(5') with an Eterneon-480 fluorophor(®) (â7b). The resulting nucleolipid was studied with respect to its incorporation in an artificial bilayer, as well as to its aggregate formation. Additionally, two oligonucleotides carrying terminal phytol-alkylated 5-fluorouridine tags were prepared, one of which was studied concerning its incorporation in an artificial lipid bilayer.
Asunto(s)
Materiales Biocompatibles/química , ADN/metabolismo , Fitol/química , Terpenos/química , Uridina/análogos & derivados , Monoterpenos Acíclicos , Materiales Biocompatibles/metabolismo , ADN/química , Isomerismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Uridina/síntesis química , Uridina/químicaRESUMEN
2'-Deoxyinosine (1) and thymidine (7) were N-alkylated with geranyl and farnesyl moieties. These hydrophobic derivatives, 3a and 3b, and 9a and 9b, respectively, represent the first synthetic biomimetic nucleoterpenes and were subsequently 5'-protected and converted into the corresponding 3'-O-phosphoramidites, 5a and 5b and 11a and 11b, respectively. The latter were used to prepare a series of lipophilized oligonucleotide dodecamers, a part of which were additionally labelled with indocarbocyanine fluorescent dyes (Cy3 or Cy5), 18-23. The insertion of the lipooligonucleotides into, as well as duplex formation at artificial lipid bilayers was studied by single-molecule fluorescence spectroscopy and fluorescence microscopy.
Asunto(s)
Materiales Biomiméticos/química , Inosina/análogos & derivados , Oligonucleótidos/química , Timidina/química , Materiales Biomiméticos/síntesis química , Carbocianinas/química , Inosina/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Microscopía Fluorescente , Espectrometría de FluorescenciaRESUMEN
One of the major drawbacks of chemotherapeutics is their insufficient penetration through cell membranes due to a high hydrophobicity. Thus, we have synthesized a series of selected nucleolipid derivatives of 5-fluorouridine (5-FUrd; 2a), carrying lipophilic moieties at N(3) and/or in the 2',3'-O-position (i.e., 3a-7a and 3c), and tested their cytostatic/cytotoxic activities using HT-29 human colon carcinoma cells, in comparison with, e.g., 5-FU (1) and 5-FUrd (2a). Incorporation and intracellular localization of the substances under test were performed after conjugation with the fluorochrome Atto 425. We showed that all 5'-O-labelled Atto 425 derivatives were incorporated by the human HT-29 cells and accumulated in their cytoplasm. Moreover, after 24-h treatment of HT-29 human colon carcinoma cells, 1 or 2a (10, 20, 40, or 80â µM) revealed a significant (14-23 or 33-45%, resp.) decrease of the viability in comparison with the (negative) control. Interestingly, derivatives 3a and 3c (40 and 80â µM) led to a significant (77-95 or 89-96%, resp.) inhibition of survival of human HT29 cells, i.e., these two substances were ca. 63-72% or ca. 75%, respectively more effective than 5-FU (1; positive control). Furthermore, derivative 5a showed a significant, i.e., 30 and 86%, inhibition of the survival at 40 and 80â µM, respectively in comparison with the (negative) control. Some synthesized 5-FUrd derivatives turned out to be more effective than 5-FU (1) or 5-FUrd (2a).
Asunto(s)
Antineoplásicos/síntesis química , Uridina/análogos & derivados , Antineoplásicos/química , Antineoplásicos/toxicidad , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células HT29 , Humanos , Microscopía Confocal , Uridina/síntesis química , Uridina/química , Uridina/toxicidadRESUMEN
Nucleoside analogs (NSAs) were among the first chemotherapeutic agents and could also be useful for the manipulation of cell fate. To investigate the potential of NSAs for the induction of neuronal differentiation, we developed a novel phenotypic assay based on a human neuron-committed teratocarcinoma cell line (NT2) as a model for neuronal progenitors and constructed a NT2-based reporter cell line that expressed eGFP under the control of a neuron-specific promoter. We tested 38 structurally related NSAs and determined their activity to induce neuronal differentiation by immunocytochemistry of neuronal marker proteins, live cell imaging, fluorometric detection and immunoblot analysis. We identified twelve NSAs, which induced neuronal differentiation to different extents. NSAs with highest activity carried a halogen substituent at their pyrimidine nucleobase and an unmodified or 2'-O-methyl substituted 2-deoxy-ß-D-ribofuranosyl residue as glyconic moiety. Cladribine, a purine nucleoside with similar structural features and in use to treat leukemia and multiple sclerosis, induced also differentiation of adult human neural crest-derived stem cells. Our results suggest that NSAs could be useful for the manipulation of neuronal cell fate in cell replacement therapy or treatment of neurodegenerative disorders. The data on the structure and function relationship will help to design compounds with increased activity and low toxicity.