Synthesis, Characterization, and Biological Activity of Purine and Pyrimidine Nucleoside and Nucleotide Analogues

Purine and pyrimidine nucleosides and nucleotides are constituents of fundamental structures of the cells. In fact, they are constituents of nucleic acids and their structure is present in several coenzymes involved in cellular reduction/oxidation processes, like nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD). Furthermore ATP is the source of energy that drive the cellular metabolic reactions. 3'-5'-cyclicadenosinemonophosphate (cAMP) acts as second messenger controlling the activation of metabolic pathways. The biological relevance of these molecules is also due to their extracellular activity performed through the interaction with purinergic receptors. This receptor family is divided in two classes: P1 receptors, activated by adenosine, and P2 receptors, activated by purine and pyrimidine nucleotides. P1 receptors belong to G protein-coupled receptors superfamily and are divided in four subtypes A1, A2A, A2B, and A3 based on their different molecular structure, tissue distribution, and pharmacological profile. P2 receptors are activated by a range of naturally occurring extracellular nucleotides and consist of two families: G protein-coupled or ''metabotropic'' receptors, designated P2Y (P2Y1,2,4,6,11-14), and ligand-gated ion channels or ''ionotropic'' receptors, termed P2X (P2X1-7). The presence of ATP as a normal constituent of the extracellular environment suggests that functional alterations in extracellular levels of ATP and thus P2 receptor function or alteration in receptor number are associated with discrete disease states, and will provide the opportunity to develop novel therapeutic agents that act through P2 receptors. The therapeutic areas, in which P2 receptors are involved, currently of interest are pulmonary (P2Y2/P2Y4, Phase III), thrombosis (P2Y2, Phase III), pain (P2X3, preclinical), and bladder disfunction (P2X3, preclinical). The first part of the present work reports the synthesis and biological evaluation of substituted nucleotides as potential ligands of P2 receptors. Recently, our group published the synthesis of mono-, di- and triphosphate derivatives of 2-alkynyladenosines. These compounds, tested on human platelets, showed to induce or inhibit platelet aggregation, depending on the alkynyl chain present in 2-position, through the interaction with P2Y1 and P2Y12 receptors. With the aims at finding new P2 receptors ligands, and in order to evaluate the influence of alkyl groups in N6 position of adenosine nucleotides, we synthesized adenosine derivatives bearing a methyl or a cyclopentyl group in N6 combined or not with a chlorine atom in 2 position. Moreover, to evaluate the influence of the purine ring nitrogen atoms, 2-chloro-3-deazadenosine and its N6-methyl analogue were synthesized. In all cases mono-, di-, and triphosphate derivatives were prepared and tested on human platelets to assess their ability to modulate platelet aggregation through interaction with P2Y1 and P2Y12 receptors. Functional studies demonstrated that the presence of substituents in N6 position of adenine nucleotides do not favour the interaction with P2 platelet receptor. Anyway, the presence of N6 methyl group or substitution of the purine ring with 3-deazapurine is tolerated leading in some cases to nucleotides able to promote or inhibit platelet aggregation. In general, the diphosphate derivatives acted as promoters while the triphosphate ones inhibited platelet aggregation induced by ADP. Furthermore, on the base that the introduction of alkynyl chains in adenine nucleotides led to new P2 receptor ligands, and taking into account that natural modulatorss of some P2Y receptors are uridine nucleotides, we realized the synthesis of uridine derivatives in which the alkynyl chains were introduced in 5 position. The second part of the work, hence, 5-iodouridine was reacted with different 1-alkynes. The obtained 5-alkynyl uridines, together with 5-iodouridine itself, were phosphorylated to obtain the corresponding mono-, di- and triphosphate derivatives. The newly synthesized triphosphate nucleotides were tested on SH-SY5Y neuroblastoma cells stably transfected with P2Y4 receptor. Overexpression of the receptor in this cell line induces cellular differentiation and then cell death when treated with UTP, so this model has been used to investigate the interaction of our compounds with P2Y4 receptors. Preliminary results showed that the new uridine triphosphate derivatives induces cell death in the above mentioned in vitro model, and in the case of 5- iodoUTP, the effect seems to be due to the interaction with P2Y4 receptors. The third part of the work was carried out at the ''Laboratory of Medicinal Chemistry'' of the Rega Institute of Katholieke Universiteit Leuven (Belgium); in particular, analogues of purines and pyrimidines nucleotides were synthesized and studied as constituents of nucleic acids. Universal nucleobases have attracted attention due to their potential utility in the design of oligonucleotide primers or hybridization probes where the identity of one or more bases in the target sequence are unknown, or when ambiguities still remain due to polymorphic or species-dependent sequence differences. Over the last years, different surrogate bases have been evaluated as universal or degenerate nucleosides, which either cannot associate through hydrogen bonding but provide a polarized stackable heterocycle, like 5-nitroindazole and 4- nitroimidazole, or allow a flexible hydrogen bonding pattern mimicking natural bases, like the azole carboxamide derivatives. On the other hand, duplex stability of nucleic acids can be increased by the modifications of the carbohydrate moiety, like in constrained hexitol nucleic acids. Therefore, it was performed the synthesis of nucleoside analogues bearing 5-nitroindazole, 4-nitroimidazole or 1,2,4-triazole-3-carboxamide as the base moiety linked to 1,5-anhydro-3-deoxy-D-glucitol as the sugar part. Following incorporation into several oligodeoxynucleotide sequences, their base pairing and discriminatory properties have been evaluated. All modifications destabilized the double helix upon a single incorporation; 5-nitroindazole congener was the least destabilizing and showed the lowest spread in Tm values and therefore is behaving almost like a true ambiguous nucleoside analogue.