Search

A study was conducted to examine the imino moiety of N1-deazaadenosine of A9 and A10 bases. Findings revealed that imino moiety of A9 promote a key water-mediated contact that favors transition-state formation.

The requirements for chemical activity using a conservative modification are defined by preparing and incorporating N-deazaadenosine into the full-length ribozyme construct. The results have shown the necessity of the imino moiety as well as the importance of its interaction with the substrate in the precatalytic and transition-state conformations.

The article presents a study which determines the direct Raman measurement of an increased base pKa situated in the active site of small ribozyme during a precatalytic conformation. It examines the crystals of the hairpin ribozyme (HRPZ), a catalytic RNA motif. The study revealed that there is an elevation of pKa at residue A38 of the HPRZ.

New 8-amino-6-aryl-1,2,4-triazolo[4,3- a ]pyrazin-3-ones were designed to obtain dual antioxidant-human A 2A adenosine receptor (hA 2A AR) antagonists. Two sets of compounds were synthesized, the first featuring phenol rings at the 6-position, the second bearing the lipoyl and 4-hydroxy-3,5-di- tert but-benzoyl residues appended by different linkers on the 6-phenyl ring. Several new triazolopyrazines ( 1-21 ) were potent and selective hA 2A AR antagonists ( K i = 0.17-54.5 nM). Compounds 11 , 15 , and 21 , featuring antioxidant moieties, and compound 12 , lacking the antioxidant functionality, reduced oxaliplatin-induced toxicity in microglia cells, the most active being the lipoyl-derivative 15 and the (4-hydroxy-3,5-di- tert -butyl)benzoyl-analogue 21 which were effective in reducing the oxygen free radical level. The lipoyl-derivative 15 was also able to revert oxaliplatin-induced neuropathy in the mouse. In vivo efficacy of 15 makes it a promising neuroprotective agent in oxidative stress-related diseases.

Antagonists of the purinergic P2X3 receptors represent promising drugs for the treatment of inflammation and pain. The ATP derivative 2',3'- O -(2,4,6-trinitrophenyl)-ATP (TNP-ATP) has been described as a potent competitive inhibitor of this receptor. In this work, the design and synthesis of novel TNP-ATP analogues bearing alkyl groups in the 2',3'-position are reported. These compounds were biologically evaluated as P2X3 antagonists using the patch clamp recording technique on mouse trigeminal ganglionic sensory neurons. Some of the compounds showed nanomolar inhibitory potency for the P2X3 receptor. Further modification of these derivatives was made by substitution of the triphosphate chain with different acidic groups. All compounds were additionally tested at five human P2X receptor subtypes stably expressed in 1321N1 astrocytoma cells to evaluate their potency and P2X3 selectivity. Results confirmed the P2X3 antagonist potency for some derivatives., Competing Interests: The authors declare no competing financial interest.

In this work, we describe the identification of the 1,2,4-triazolo[4,3-a]pyrazin-3-one as a new versatile scaffold for the development of adenosine human (h) receptor antagonists. The new chemotype ensued from a molecular simplification approach applied to our previously reported 1,2,4-triazolo[4,3-a]quinoxalin-1-one series. Hence, a set of novel 8-amino-2-aryl-1,2,4-triazolopyrazin-3-one derivatives, featured by different substituents on the 2-phenyl ring (R) and at position 6 (R 6 ), was synthesized with the main purpose of targeting the hA 2A adenosine receptor (AR). Several compounds possessed nanomolar affinity for the hA 2A AR (K i = 2.9-10 nM) and some, very interestingly, also showed high selectivity for the target. One selected potent hA 2A AR antagonist (12, R = H, R 6 = 4-methoxyphenyl) demonstrated some ability to counteract MPP + -induced neurotoxicity in cultured human neuroblastoma SH-SY5Y cells, a widely used in vitro Parkinson's disease model. Docking studies at hAR structures were performed to rationalize the observed affinity data.

The application of frontal affinity chromatography-mass spectrometry (FAC-MS), along with molecular modeling studies, to the screening of potential drug candidates toward the recently deorphanized G-protein-coupled receptor (GPCR) GPR17 is shown. GPR17 is dually activated by uracil nucleotides and cysteinyl-leukotrienes, and is expressed in organs typically undergoing ischemic damage (i.e., brain, heart and kidney), thus representing a new pharmacological target for acute and chronic neurodegeneration. GPR17 was entrapped on an immobilized artificial membrane (IAM), and this stationary phase was used to screen a library of nucleotide derivatives by FAC-MS to select high affinity ligands. The chromatographic results have been validated with a reference functional assay ([(35)S]GTPgammaS binding assay). The receptor nucleotide-binding site was studied by setting up a column where a mutated GPR17 receptor (Arg255Ile) has been immobilized. The chromatographic behavior of the tested nucleotide derivatives together with in silico studies have been used to gain insights into the structure requirement of GPR17 ligands.

A new series of 2-aralkynyl-N(6)-methyl-MECAs 10-13 were synthesized and evaluated in radioligand binding studies and in a new Eu-GTP functional assay that provides a powerful alternative to radioisotope use. The new compounds possess high affinity and selectivity for the AA(3)R with N(6)-methyl-2-phenylethynylMECA (10) showing a subnanomolar affinity and about 100000-fold selectivity vs AA(1)R and AA(2A)R. Furthermore, the new nucleosides showed to be full agonists, the N(6)-methyl-2-(2-pyridinyl)ethynylMECA (13) being the most potent in the series.

Adenine derivatives bearing substituents in the 2-, N(6)-, 7-, 8-, and/or 9-position and a series of deazapurines were synthesized and investigated in [(3)H]adenine binding studies at the adenine receptor in rat brain cortical membrane preparations (rAde1R). Steep structure-activity relationships were observed. Substitution in the 8-position (amino, dimethylamino, piperidinyl, piperazinyl) or in the 9-position (2-morpholinoethyl) with basic residues or introduction of polar substituents at the 6-amino function (hydroxy, amino, acetyl) represented the best modifications. Functional evaluation of selected adenine derivatives in adenylate cyclase assays at 1321N1 astrocytoma cells stably expressing the rAde1R showed that all compounds investigated were agonists or partial agonists. A subset of compounds was additionally investigated in binding studies at human embryonic kidney (HEK293) cells, which also express a high-affinity adenine binding site. Structure-affinity relationships at the human cell line were similar to those at the rAde1R, but not identical. In particular, N(6)-acetyladenine (25, K(i) rat: 2.85 microM; K(i) human: 0.515 microM) and 8-aminoadenine (33, K(i) rat: 6.51 microM; K(i) human: 0.0341 microM) were much more potent at the human as compared to the rat binding site. The new AdeR ligands may serve as lead structures and contribute to the elucidation of the functions of the adenine receptor family.

Catalytic RNA molecules can achieve rate acceleration by shifting base pK(a) values toward neutrality. Prior evidence has suggested that base A38 of the hairpin ribozyme plays an important role in phosphoryl transfer, possibly functioning as a general acid, or by orienting a specific water molecule for proton transfer. To address the role of A38, we used Raman spectroscopy to measure directly the pK(a) of the N1-imino moiety in the context of hairpin ribozyme crystals representative of a "precatalytic" conformation. The results revealed that the pK(a) of A38 is shifted to 5.46 +/- 0.05 relative to 3.68 +/- 0.06 derived from a reference solution of the nucleotide AMP. The elevated pK(a) correlates well with the first titration point of the macroscopic pH-rate profile of the hairpin ribozyme in solution and strongly supports A38 as a general acid catalyst in bond scission. The results confirm that A38 is protonated before the transition state, which would promote phosphorane development. Overall, the results establish a cogent structure-function paradigm that expands our understanding of how RNA structure can enhance nucleobase reactivity to catalyze biological reactions.

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.

The good results obtained as potential antitumor drugs with aza-anthracenediones and aza-anthrapyrazoles, e.g. pixantrone, 1a, and 1b (Chart 1), prompted us to design and synthesize a series of symmetrical bis derivatives, compounds 7-10 (Chart 1). These compounds are dimers of different aza-anthracenedione and aza-anthrapyrazolone monomers connected by the linker found to be the most appropriate among potential bis intercalators synthesized by us. The DNA-binding properties of bis derivatives 7 and 8 have been examined using fluorometric techniques: these target compounds are excellent DNA ligands, with a clear binding site preference for AT-rich duplexes. In vitro cytotoxic activity of all target compounds 7-10 and of reference compound pixantrone toward human cancer adenocarcinoma cell line HT29 is also described. Two selected compounds have been investigated for their capacity of inducing early apoptosis.

A new series of N6-methoxy-2-(ar)alkynyladenosine derivatives has been synthesized and tested at the human recombinant adenosine receptors. Binding studies demonstrated that the new compounds possess high affinity and selectivity for the A3 subtype. Among them, compounds bearing an N-methylcarboxamido substituent in the 4'-position showed the highest A3 affinity and selectivity. In particular, the N6-methoxy-2-p-acetylphenylethynylMECA (40; Ki A3 = 2.5 nM, A3 selectivity versus A1 = 21 500 and A2A = 4200) results in one of the most potent and selective agonists at the human A3 adenosine receptor reported so far. Furthermore, functional assay, performed with selected new compounds, revealed that the presence of an alkylcarboxamido group in the 4'-position seems to be essential to obtain full agonists at the A3 subtype. Finally, results of molecular docking analysis were in agreement with binding and functional data and could explain the high affinity and potency of the new compounds.

Newly synthesized mono-, di-, and triphosphate of 2-alkynyl adenosines showed very different behavior in human platelet P2Y receptor models, according to the different alkynyl chains. In fact, 2-hexynyladenosine di- (5) and triphosphate (7) induced platelet shape change and aggregation and inhibited PGE(1)-induced increase in platelet cyclic AMP. On the contrary, the corresponding 2-phenylethynyladenosine di- (6) and triphosphate (8) did not induce platelet shape change or aggregation, but inhibited platelet aggregation induced by ADP.

A series of N(6)-alkyl-2-alkynyl derivatives of adenosine (Ado) have been synthesized and evaluated for their affinity at human A(1), A(2A), and A(3) receptors and for their potency at A(2B) adenosine receptor subtypes. The corresponding 2-(1-alkynyl) derivatives of 5'-N-ethylcarboxamidoadenosine (NECA) and Ado are used as reference compounds. Binding studies demonstrated that the activities of 2-alkynylAdos were slightly increased for the adenosine A(1) receptor and slightly decreased for both A(3) and A(2B) subtypes compared to those of their corresponding NECA derivatives, whereas the A(2A) receptor affinities of the two series of nucleosides were similar. The presence of a methyl group on N(6) of the 2-alkynyladenosines, inducing an increase in affinity at the human A(3) receptor and a decrease at the other subtypes, resulted in an increase in A(3) selectivity. In particular, 2-phenylethynyl-N(6)-methylAdo (8b) showed an A(3) affinity in the low nanomolar range (K(i)(A(3)) = 3.4 nM), with a A(1)/A(3) and A(2A)/A(3) selectivity of about 500 and 2500, respectively. These findings motivated us to search for the preparation of new selective radioligands for the A(3) subtype; hence, a procedure to introduce a tritiated alkylamino group in these molecules was carried out. As far as the potency at the A(2B) receptor, the type of 2-alkynyl chain and the presence of the ethylcarboxamido group on the sugar seem to be very important; in fact, the (S)-2-phenylhydroxypropynylNECA [(S)-PHPNECA, 1e, EC(50)(A(2B)) = 0.22 microM] proved to be one of the most potent A(2B) agonist reported so far. On the other hand, the (S)-2-phenylhydroxypropynyl-N(6)-ethylAdo (9e, EC(50)(A(2B)) = 0.73 microM) showed a significantly increase of potency at the A(2B) subtype in comparison with the N(6)-methyl, N(6)-isopropyl, and the unsubstituted adenosine derivatives, although it resulted in being less potent than (S)-PHPNECA (1e, EC(50)(A(2B)) = 0.22 microM). These observations suggest that the introduction of an ethyl group in the N(6)-position and an ethylcarboxamido substituent in the 4'-position of (S)-2-phenylhydroxypropynyladenosine could lead to a compound endowed with high potency at the A(2B) receptor.