Sugar-modified nucleosides: synthesis, conformational analysis and biological evaluation

Nucleoside analogues are a pharmacologically diverse family that includes cytotoxic compounds, antiviral agents and immunosuppressive molecules. Considerable progress has been made in the search for novel nucleoside structures with anticancer and/or antiviral activity by modifications in the nucleobase and/or in the sugar moiety and several branched-chain sugar nucleosides have shown potent antitumor activity. These agents behave as antimetabolites that interact with a number of intracellular targets blocking the DNA chain elongation or interfering with biosynthesis of nucleosides and nucleotides, a limiting process in cell proliferation. Many of the enzymes involved in these pathways are highly active in cancer cells, such as ribonucleotide reductase (RNR). RNR is a multisubunit radical-containing enzyme involved in the conversion of ribonucleoside diphosphates to the corresponding 2'-deoxyribonucleotides, which are building blocks for DNA replication and repair. Increased RNR activity has been associated with malignant transformation and tumor cell growth; it is therefore an excellent target for cancer chemotherapy. In recent years, several nucleoside RNR inhibitors have entered clinical trial or application such as arabinosylcytosine (ara-C), gemcitabine, cladribine, fludarabine and clofarabine. A previous structure-activity relationship study reported by us, identified the 3'-C-methyladenosine (3'-Me-Ado) as a mechanism-based RNR inhibitor endowed with a significant antitumor activity against a panel of human leukemia and carcinoma cell lines. To further investigate the structural determinants of 3'-Me-Ado required for the antitumor activity, in the first part of my PhD project, a number of 3'-C-methyl ribonucleosides with different purinic and pyrimidinic nucleobases and the 3'-C-methyl derivatives of the antitumor agents 5-fluorouridine and 9-( β-D-arabinofuranosyl)- adenine were synthesized and evaluated on human tumor cell lines. The biological results showed that the structure of 3'-Me-Ado is crucial for the antitumor activity of this type of ribose-modified nucleosides. A further structure-activity relationship study of synthesized adenine 3'-C-methylribonucleosides containing different substituent at N6-amino group of nucleobase such as a cycloalkyl, aryl or heteroaryl group confirmed that any modification in the structure of 3'-Me-Ado is detrimental for the antitumor activity. The obtained results have been rationalized by docking studies utilizing the X-ray structure of the subunit Rnr1 of the RNR from Saccharomyces cerevisiae complexed with ADP. The ribose recognition domain of adenosine derivatives appears to be of considerable importance not only for cytotoxic activity, but also for agonistic activity at adenosine receptors (ARs). The physiological agonist adenosine modulates a variety of functions through four receptor subtypes classified as A1, A2A, A2B and A3. A1 adenosine receptors (A1 ARs) are expressed in high density in the brain, in adipose tissue and in medium to low levels in many peripheral organ and tissues such as heart, lung and kidney. Thus, A1 agonists have many therapeutic potentials; however the clinical application of A1 full agonists is hampered by several side effects. In this respect, it has been reported that A1 partial agonist may be therapeutically advantageous. Among the adenosine derivatives, some 5'-chloro-5'-deoxy-N6-cycloalkyl(bicycloalkyl)- analogues were found A1 agonists with high affinity and selectivity for rat A1 AR or partial agonists for this AR subtype. In the search for potent and selective A1 AR agonists we have previously investigated a series of 2'-C-methylribofuranosyl analogues of selective A1 AR agonists and 2-chloro- 2'-C-methyl-N6-cyclopentyl-adenosine (2'-Me-CCPA) emerged as a potent and highly selective full agonist at rat, bovine and human A1 vs A2A, A2B and A3 ARs. On the base of these findings, in the second part of my work, a series of 5'-substituted derivatives of potent and selective A1 AR agonists were synthesized and tested for affinity and selectivity at different model of adenosine receptor subtypes. In particular, a series of 5'-carbamoyl or 5'-thionocarbamoyl derivatives of 2'-Me-CPA, 2'-Me-CCPA, N6-[(R)-3-tetrahydrofuranyl]adenosine and 2-chloro analogue were synthesized and evaluated for binding affinity at ARs from bovine, porcine and human species. Moreover, a series of 5'-chloro-5'-deoxy-N6-cycloalkyl(bicycloalkyl)-substituted adenosine and 2'-C-methyladenosine derivatives and 2-chloro analogues were synthesized and evaluated for affinity and efficacy at all cloned human AR subtypes. In this study, two adenosine derivatives displayed the highest affinity in the subnanomolar range and relevant selectivity for hA1 vs the other human receptor subtypes. This result was rationalized by a molecular modeling analysis.