Hybridisation potential of 1',3'-Di-O-methylaltropyranoside nucleic acids.

In further study of our series of six-membered ring-containing nucleic acids, different 1',3'-di-O-methyl altropyranoside nucleoside analogs (DMANA) were synthesized comprising all four base moieties, adenine, cytosine, uracil and guanine. Following assembly into oligonucleotides (ONs), their affinity for natural oligonucleotides was evaluated by thermal denaturation of the respective duplexes. Data were compared with results obtained previously for both anhydrohexitol (HNAs) and 3'-O-methylated altrohexitol modified ONs (MANAs). We hereby demonstrate that ONs modified with DMANA monomers, unlike some of our previously described analogues with constrained 6-membered hexitol rings, did not improve thermodynamic stability of dsRNA complexes, most probably in view of an energetic penalty when forced in the required 1C4 pairing conformation. Overall, a single incorporation was more or less tolerated or even positive for the adenine congener, but incorporation of a second modification afforded a slight destabilization (except for A), while a fully modified sequence displayed a thermal stability of -0.3 °C per modification. The selectivity of pairing remained very high, and the new modification upon incorporation into a DNA strand, strongly destabilized the corresponding DNA duplexes. Unfortunately, this new modification does not bring any advantage to be further evaluated for antisense or siRNA applications.

Phospho-carboxylic anhydride of a homologated nucleoside leads to primer degradation in the presence of a polymerase.

Starting from thymidine, through a series of key synthetic transformations (e.g., Wittig reaction, hydroboration, Mitsunobu reaction and TEMPO oxidation) a nucleoside homologue bearing a phospho-carboxylic anhydride group at 6' position was synthesized. The potential of polymerases to catalyze amide bond formation was investigated by using a modified primer with an amino group at 3' position and the synthesized phosphoanhydro compound as substrate. Unfortunately, we did not observe the desired product either by gel electrophoresis or mass spectrometry. In contrast, the instability of the phosphoanhydro compound could lead to pyrophosphate formation and thus, to pyrophosphorolysis of the primer rather than to primer extension.

8-Bromo-9-alkyl adenine derivatives as tools for developing new adenosine A2A and A2B receptors ligands.

Importance of making available selective adenosine receptor antagonists is boosted by recent findings of adenosine involvement in many CNS dysfunctions. In the present work a series of 8-bromo-9-alkyl adenines are prepared and fully characterized in radioligand binding assays or functional cyclase experiments in respect to their interaction with all the four adenosine receptor subtypes. Results show that the presence of the bromine atom in 8-position of 9-substituted adenines promotes in general the interaction with the adenosine receptors, in particular at the A(2A) subtype. The present study also demonstrates that adenine derivatives could be a good starting point to obtain selective adenosine A(2B) receptor antagonists.

Synthesis and stability studies of 2',3',5'-tri-O-acetyl-2-amino(-N6-cyclopentyl)-1-deazaadenosines.

In this article, we report on the synthesis of 2',3',5'-tri-O-acetyl-2-amino-1-deazaadenosine and of 2',3',5'-tri-O-acetyl-2-amino-N(6)-cyclopentyl-1-deazaadenosine, which are very versatile intermediates for the preparation of 2-substituted 1-deazaadenosine derivatives. The two synthesized compounds showed to be quite unstable, with the N(6)-substituted derivatives being less stable than the N(6)-unsubstituted counterpart, according to the calculated HOMO-LUMO energy gap. Stability studies were performed through HPLC-MS analysis.

Evaluation of adenine as scaffold for the development of novel P2X3 receptor antagonists.

Ligands that selectively block P2X3 receptors localized on nociceptive sensory fibres may be useful for the treatment of chronic pain conditions including neuropathic pain, migraine, and inflammatory pain. With the aim at exploring the suitability of adenine moiety as a scaffold for the development of antagonists of this receptor, a series of 9-benzyl-2-aminoadenine derivatives were designed and synthesized. These new compounds were functionally evaluated at rat or human P2X3 receptors expressed in human embryonic kidney (HEK) cells and on native P2X3 receptors from mouse trigeminal ganglion sensory neurons using patch clamp recording under voltage clamp configuration. The new molecules behaved as P2X3 antagonists, as they rapidly and reversibly inhibited (IC50 in the low micromolar range) the membrane currents induced via P2X3 receptor activation by the full agonist α,ß-methyleneATP. Introduction of a small lipophilic methyl substituent at the 6-amino group enhanced the activity, in comparison to the corresponding unsubstituted derivative, resulting in the 9-(5-iodo-2-isopropyl-4-methoxybenzyl)-N(6)-methyl-9H-purine-2,6-diamine (24), which appears to be a good antagonist on recombinant and native P2X3 receptors with IC50 = 1.74 ± 0.21 µM.

Adenine-based acyclic nucleotides as novel P2X3 receptor ligands.

A new series of acyclic nucleotides based on the adenine skeleton and bearing in 9-position a phosphorylated four carbon chain has been synthesized. Various substituents were introduced in 2-position of the adenine core. The new compounds were evaluated on rat P2X3 receptors, using patch clamp recording from HEK transfected cells and the full P2X3 agonist alpha,beta-meATP as reference compound. The results suggest that certain acyclic nucleotides, in particular compounds 28 and 29, are endowed with modest partial agonism on P2X3 receptors. This is an interesting property that can depress the function of P2X3 receptors, whose activation is believed to be involved in a number of chronic pain conditions including neuropathic pain and migraine. In fact, the new acyclic nucleotides are able to persistently block (by desensitization) P2X3 receptor activity after a brief, modest activation, yet leaving the ability of sensory neurons to mediate responses to standard painful stimuli via a lower level of signaling.

New 2,6,9-trisubstituted adenines as adenosine receptor antagonists: a preliminary SAR profile.

A new series of 2,6,9-trisubstituted adenines (5-14) have been prepared and evaluated in radioligand binding studies for their affinity at the human A(1), A(2A) and A(3) adenosine receptors and in adenylyl cyclase experiments for their potency at the human A(2B) subtype. From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N (6) position of 9-propyladenine significantly increased binding affinity at the human A(1) and A(3) adenosine receptors, while the presence of a chlorine atom at the 2 position resulted in a not univocal effect, depending on the receptor subtype and/or on the substituent present in the N (6) position. However, in all cases, the presence in the 2 position of a chlorine atom favoured the interaction with the A(2A) subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A(2B) subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles.