Cellular delivery of dinucleotides by conjugation with small molecules: targeting translation initiation for anticancer applications.

Targeting cap-dependent translation initiation is one of the experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5',5'-triphosphates cap analogs are potent antagonists of eukaryotic translation initiation factor 4E (eIF4E) in vitro and could counteract elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic agents remains challenging - they do not easily penetrate into cells and are susceptible to enzymatic cleavage. Here, we tested the potential of several small molecule ligands - folic acid, biotin, glucose, and cholesterol - to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad structure-activity relationship (SAR) study using model fluorescent probes and cap-ligand conjugates showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity towards cancer cells.

Synthesis and in vitro anticancer activity of new gemcitabine-nucleoside analogue dimers containing methyltriazole or ester-methyltriazole linker.

Two series of novel gemcitabine-nucleoside analogue dimers were synthesized using the 'click' chemistry approach. In the first series of dimers (21-30), the nucleoside units were connected with a stable methyltriazole 4N-3'(or 5')C linker whereas in the second series (31-40) with a cleavable ester-methyltriazole 4N-3'(or 5')C linker. Dimers 21-40 were evaluated for their cytotoxic activity in five human cancer cell lines such as cervical (HeLa), nasopharyngeal (KB), lung (A549), brain (U87), liver (HepG2) and normal dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. Compound 29 comprising two gemcitabine (dFdC) units exhibited the highest activity among dimers 21-30. The activity of compound 29 was higher than that of dFdC in all the studied cancer cell lines. A similar order of activity was observed for compounds 25, 28, and 30. The best activity among all the dimers synthesized was displayed by compound 39, comprising two gemcitabine units with a cleavable linker. The activity of compound 39 was 5 to 9 times higher than that of dFdC, depending on the cell line. In addition, marked cytotoxic activity was shown by compounds 31, 36, 38, and 40.

Synthesis and anticancer activity of 3'-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidine analogs and their phosphoramidates.

A series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3'-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidines (20-49) was synthesized by means of phosphorylation of 3'-[4-aryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidines (7-11) with 4-chlorophenyl phosphoroditriazolide (14), followed by a reaction with the appropriate amine. The synthesized compounds 7-11 and 20-49 were evaluated along with four known anticancer compounds for their cytotoxic activity in human cancer cell lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), osteosarcoma (143B) (only selected compounds 20, 24, 28, 32-36, 38, 40, 46) and normal human dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. Among 3'-[4-aryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidines (7-11) the highest activity in all the investigated cancer cells was displayed by 3'-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-3'-deoxythymidine (9) (IC50 in the range of 2.58-3.61 µM) and its activity was higher than that of cytarabine. Among phosphoramidates 20-49 the highest activity was demonstrated by N-n-propyl phosphoramidate of 3'-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-3'-deoxythymidine (35) in all the cancer cells (IC50 in the range of 0.97-1.94 µM). Also N-ethyl phosphoramidate of 3'-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-3'-deoxythymidine (33) exhibited good activity in all the used cell lines (IC50 in the range of 4.79-4.96 µM).

Differences in Antiproliferative Activity Between Salinomycin-AZT Conjugates Obtained via 'Click' and Esterification Reactions.

BACKGROUND: Pharmacophore hybridization by bioconjugation, in which two bioactive moieties are covalently linked, is one of the current strategies in drug discovery for the development of new compounds with improved affinity and efficacy relative to those of the parent molecules. Prompted by the idea that cancer cells may be effectively killed by 3'-azido-3'-deoxythymidine (AZT) and salinomycin (SAL) individually, we synthesized hybrids of these compounds. The development of this type of derivatives, which can easily penetrate the lipid-rich cell membranes and then undergo hydrolysis inside the cancer cells, is an important research area. METHODS: Efficient methods for the synthesis of two new conjugates are presented. The first method is based on the 'click' chemistry and involves the copper(I) catalysed 1,3-dipolar Huisgen cycloaddition reaction. In the second method AZT as well as SAL are connected by the ester bond under mild reaction conditions. The in vitro anti-proliferative activity of both conjugates against several drugsensitive and drug-resistant cancer cell lines as well as toxicity against normal murine embryonic fibroblasts are also determined. RESULTS: Our studies clearly showed that the hybrid obtained via esterification reaction (SAL-OAZT) seems to be attractive in the fight against neoplastic diseases because it helps to overcome a strong drug-resistance of the cancer cell lines examined at low micromolar concentrations. The anticancer activity of this hybrid is also connected with high selectivity indexes (low toxicity) against normal cells.On the other hand, the 'click' conjugate (SAL-AZT) is practically inactive against the drug-resistant cancer cell lines tested and weakly active against the drug-sensitive ones. Also no synergistic effect has been found between SAL and AZT against eight cancer cell lines studied. CONCLUSION: All of our findings support a strategy to decrease the doxorubicin concentration in combination with SAL-O-AZT hybrid in order to reduce the toxicity of this drug, as recently demonstrated for SAL. The advantages of the SAL-O-AZT conjugate over SAL are better RI and SI parameters at similar IC50values.

Synthesis and anticancer activity of some 5-fluoro-2'-deoxyuridine phosphoramidates.

Two series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3'-O-(t-butoxycarbonyl)-5-fluoro-2'-deoxyuridine (3'-BOC-FdU) (9a-9j) and 5-fluoro-2'-deoxyuridine (FdU) (10a-10j) were synthesized by means of phosphorylation of 3'-BOC-FdU (4) with 4-chlorophenyl phosphoroditriazolide (7), followed by a reaction with the appropriate amine. Phosphoramidates 9a-9j were converted to the corresponding 10a-10j by removal of the 3'-t-butoxycarbonyl protecting group (BOC) under acidic conditions. The synthesized phosphoramidates 9a-9j and 10a-10j were evaluated for their cytotoxic activity in five human cancer cell lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), liver (HepG2), osteosarcoma (143B) and normal human dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. Two phosphoramidates 9b and 9j with the N-ethyl and N-(methoxy-(S)-alaninyl) substituents, respectively, displayed remarkable activity in all the investigated cancer cells, and the activity was considerably higher than that of the parent nucleoside 4 and FdU. Among phosphoramidates 10a-10j compound 10c with the N-(2,2,2-trifluoroethyl) substituent showed the highest activity. Phosphoramidate 10c was more active than the FdU in all the cancer cell lines tested.

Synthesis and biological activity of salinomycin conjugates with floxuridine.

As part of our program to develop anticancer agents, we have synthesized new compounds, which are conjugates between well-known anticancer drug, floxuridine and salinomycin which is able to selectivity kill cancer stem cells. The conjugates were obtained in two ways i.e. by copper(I) catalysed click Huisgen cycloaddition reaction performed between 3'-azido-2',3'-dideoxy-5-fluorouridine and salinomycin propargyl amide, and by the ester synthesis starting from salinomycin and floxuridine under mild condition. The compounds obtained were characterized by spectroscopic methods and evaluated for their in vitro cytotoxicity against seven human cancer cell lines as well as antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE). The conjugate obtained by esterification reaction showed a significantly higher antiproliferative activity against the drug-resistant cancer cells and lower toxicity than those of salinomycin and floxuridine towards normal cells, as well as standard anticancer drugs, such as cisplatin and doxorubicin. The conjugate compound revealed also moderate activity against MRSA and MRSE bacterial strains. Very high activity of floxuridine and 5-fluorouracil against MRSA and MRSE has been also observed.

Synthesis of novel 2',3'-didehydro-2',3'-dideoxyinosine phosphoramidate prodrugs and evaluation of their anticancer activity.

An efficient synthesis of 4-chlorophenyl N-alkyl phosphoramidates of 2 ',3 '-didehydro-2 ',3 '-dideoxyinosine employing 4-chlorophenyl phosphoroditetrazolide as a phosphorylating agent is reported. Improved method for the synthesis of 2 ',3 '-didehydro-2 ',3 '-dideoxyinosine starting from inosine is also described. The synthesized phosphoramidates 11-18 were examined for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB), and breast (MCF-7) employing sulforhodamine B assay. The highest activity in all investigated cancer cell lines was displayed by phosphoramidate 13 with N-n-propyl substituent.

Synthesis of 3'-azido-2',3'-dideoxy-5-fluorouridine phosphoramidates and evaluation of their anticancer activity.

A series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3'-azido-2',3'-dideoxy-5-fluorouridine (12-21) were synthesized by means of phosphorylation of 3'-azido-2',3'-dideoxy-5-fluorouridine (4) with 4-chlorophenyl phosphoroditriazolide (10) followed by a reaction with the appropriate amine. The synthesized phosphoramidates (12-21) were evaluated for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7) using the sulforhodamine B (SRB) assay. The highest activity in all the investigated cancer cells was displayed by phosphoramidate 13 with the N-ethyl substituent and its activity was much higher than that of the parent nucleoside. Also phosphoramidate 17 with the N-propargyl substituent exhibited good activity in all the used cell lines.