SYNTHESIS AND BIOLOGICAL EVALUATION OF NUCLEOSIDES AND NUCLEOTIDES AS ANTITUMOR AGENTS

Nucleotide coenzymes participate in essential enzyme-catalyzed redox reactions and play a fundamental role in cellular metabolic processes such as nicotinamide adenine dinucleotide (NAD) in the case of dehydrogenases. It has been shown that in many living organisms, disturbance of the nucleotide metabolism severely affects cell survival; in fact, altered NAD metabolism has been observed in many cancers. Some NAD analogues have been identified as active metabolites of nucleosides endowed with antitumor and antiviral potency. These dinucleotides are potent inhibitors of inosine 5'-monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme of de novo guanine nucleotides biosynthesis. IMPDH, which catalyzes the NAD-dependent conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP), was shown to be significantly increased in highly proliferative cells. Inhibition of this enzyme results in a decrease in GTP and dGTP biosynthesis, producing inhibition of tumor cell proliferation. It was found that IMPDH exists in two isoforms, type I which is constitutively expressed, and type II which is up-regulated and predominates in neoplastic and fast replicating cells. Thus, the selective inhibition of IMPDH type II may provide improved selectivity against target cells in anticancer chemotherapy. NAD analogues in which the nicotinamide ring is replaced by the thiazole- and selenazole-4-carboxamide moieties (TAD and SAD, respectively), as active metabolites of the antitumor agents tiazofurin and selenazofurin, proved to be potent noncompetitive inhibitors of IMPDH. Nevertheless TAD, SAD and/or other analogues did not show any isoform specificity. Thus, it was conjectured that ligand modification in the dinucleotide adenosine moiety may provide significant isoform specificity. In order to investigate the subdomain of the enzyme that binds the adenosine moiety of TAD, two new dinucleotide analogues in which the ribose ring in the adenosine portion was replaced by 2-C-methyl ribose (T-2'-MeAD) or 3-C-methyl ribose (T-3'-MeAD) were synthesized, and their conformation was investigated in relation to their inhibitory activity against human IMPDH isoforms. Inhibition of recombinant human IMPDH type I and type II isoenzymes by T-2'-MeAD and T-3'-MeAD proved to be noncompetitive with respect to NAD substrate. Binding of T-3'-MeAD was slightly inferior to that of the parent compound TAD, while T-2'-MeAD proved to be a weaker inhibitor. The higher inhibitory activity of T-3'-MeAD, as compared to that of T-2'-MeAD, may be explained by the preference of the first dinucleotide for a conformation of the adenosine moiety [South (2T3)] very close to that of TAD, as determined by molecular modeling techniques of energy minimization, conformational searching, and molecular dynamics simulations. The decrease in activity is more remarkable in the case of the 2'-C-substitution in T-2'-MeAD. It appears that the variation of conformation of the adenosine moiety in T-2'-MeAD and T-3'-MeAD impairs the ability of the 2'- and 3'-hydroxy group of the ribose and/or the purine moiety to bind the NAD site of both IMPDH isoforms. T-2'-MeAD and T-3'-MeAD were also tested for their cytotoxicity against human myelogenous leukemia K562 in culture. T-2'-MeAD and T-3'-MeAD were found to be less cytotoxic than TAD. Interestingly, T-3'-MeAD was found to be active, although to a lesser degree, against K562 cells resistant to tiazofurin. It was found that the activity of T-3'-MeAD was due to 3'-C-methyl-adenosine (3'-Me-Ado), that was formed in culture medium by the hydrolysis of the heterodinucleotide. In fact, 3'-Me-Ado proved to be active against a broad spectrum of tumor cell lines. Another part of the research was addressed to the analogues of mizoribine (MZR), a immunosuppressive agent that behaves as a transition-state analogue inhibitor, able to bind the active site of IMPDH adopting the C3'-endo [North (3T2) conformation] sugar pucker as the xanthosine monophosphate (XMP) in the E-XMP* complex. On the basis of the knowledge that substitution of hydrogen atoms at the 2'-, and 3'-position of the sugar moiety of ribonucleosides with a methyl group induces the stabilization of the conformation into the C3'-endo and C2'-endo forms respectively, two mizoribine analogues containing these modifications (2'-Me-MZR and 3'-Me-MZR, respectively) were synthesized. Surprisingly, mizoribine and its 2'- and 3'-C-methyl derivatives, evaluated for their ability to inhibit the growth of human myelogenous leukemia K562, proved to be inactive. The inactivity of these nucleosides might be due to their inability to be converted into the corresponding 5'-monophosphate derivative in K562 cells. Further experiments are underway to check this hypothesis. On the basis of the activity of T-3'-MeAD against K562 cells resistant to tiazofurin, probably due to 3'-Me-Ado that formed in the culture medium, it was investigated the possibility that this nucleoside could be responsible of the antitumor activity through the inhibition of ribonucleotide reductase (RR). RR catalyzes the conversion of nucleotides to deoxynucleotides in all organisms and thus plays a central role in nucleic acid metabolism. The development of agents that inhibit RR activity is an established strategy in cancer therapy. In this respect, a series of 1'-, 2'-, and 3'-C-methylsubstituted adenosine and 2-chloroadenosine analogues and N6-substituted derivatives were synthesized and evaluated as antitumor agents. From this study 3'-Me-Ado emerged as the most active compound, showing activity against both human leukemia and carcinoma cell lines. Structure-activity relationship studies showed that the structure of 3'-Me-Ado is crucial for the activity. In fact, substitution of a hydrogen atom of the N6-amino group with a small alkyl or cycloalkyl group, the introduction of a chlorine atom in the 2-position of the purine ring or the moving of the methyl group from the 3'-position to other ribose positions brought about a decrease or loss of antitumor activity. The antiproliferative activity of 3'-Me-Ado appears to be related to its ability to deplete both intracellular purine and pyrimidine deoxynucleotides through ribonucleotide reductase inhibition.