Characterization of Complexes of Nucleoside-5′-Phosphorothioate Analogues with Zinc Ions

On the basis of the high affinity of Zn²⁺ to sulfur and imidazole, we targeted nucleotides such as GDP-β-S, ADP-β-S, and AP₃(β-S)­A, as potential biocompatible Zn²⁺-chelators. The thiophosphate moiety enhanced the stability of the Zn²⁺-nucleotide complex by about 0.7 log units. ATP-α,β-CH₂-γ-S formed the most stable Zn²⁺-complex studied here, log <i>K</i> 6.50, being ∼0.8 and ∼1.1 log units more stable than ATP-γ-S-Zn²⁺ and ATP-Zn²⁺ complexes, and was the major species, 84%, under physiological pH. Guanine nucleotides Zn²⁺ complexes were more stable by 0.3–0.4 log units than the corresponding adenine nucleotide complexes. Likewise, AP₃(β-S)­A-zinc complex was ∼0.5 log units more stable than AP₃A complex. ¹H- and ³¹P NMR monitored Zn²⁺ titration showed that Zn²⁺ coordinates with the purine nucleotide N7-nitrogen atom, the terminal phosphate, and the adjacent phosphate. In conclusion, replacement of a terminal phosphate by a thiophosphate group resulted in decrease of the acidity of the phosphate moiety by approximately one log unit, and increase of stability of Zn²⁺-complexes of the latter analogues by up to 0.7 log units. A terminal phosphorothioate contributed more to the stability of nucleotide-Zn²⁺ complexes than a bridging phosphorothioate

Nucleoside-(5′→P) Methylenebisphosphonodithioate Analogues: Synthesis and Chemical Properties

Nucleoside-(5′→P) methylenebisphosphonodithioate analogues are bioisosteres of natural nucleotides. The potential therapeutic applications of these analogues are limited by their relative instability. With a view toward improving their chemical and metabolic stability as well as their affinity toward zinc ions, we developed a novel nucleotide scaffold, nucleoside-5′-tetrathiobisphosphonate. We synthesized P1-(uridine/adenosine-5′)-methylenebisphosphonodithioate, <b>2</b> and <b>3</b>, and P1,P2-di­(uridine/adenosine-5′)-methylenebisphosphonodithioate, <b>4</b> and <b>5</b>. Using ¹H and ³¹P NMR-monitored Zn²⁺/Mg²⁺ titrations, we found that <b>5</b> coordinated Zn²⁺ by both N7 nitrogen atoms and both dithiophosphonate moieties, whereas <b>3</b> coordinated Zn²⁺ by an N7 nitrogen atom and P_β. Both <b>3</b> and <b>5</b> did not coordinate Mg²⁺ ions. ³¹P NMR-monitored kinetic studies showed that <b>3</b> was more stable at pD 1.5 than <b>5</b>, with <i>t</i>_1/2 of 44 versus 9 h, respectively, and at pD 11 both showed no degradation for at least 2 weeks. However, <b>5</b> was more stable than <b>3</b> under an air-oxidizing atmosphere, with t_1/2 of at least 3 days versus 14 h, respectively. Analogues <b>3</b> and <b>5</b> were highly stable to NPP1,3 and NTPDase1,2,3,8 hydrolysis (0–7%). However, they were found to be poor ectonucleotidase inhibitors. Although <b>3</b> and <b>5</b> did not prove to be effective inhibitors of zinc-containing NPP1/3, which is involved in the pathology of osteoarthritis and diabetes, they may be promising zinc chelators for the treatment of other health disorders involving an excess of zinc ions

Highly Efficient Biocompatible Neuroprotectants with Dual Activity as Antioxidants and P2Y Receptor Agonists

Currently, there is a need for novel, biocompatible, and effective neuroprotectants for the treatment of neurodegenerative diseases and brain injury associated with oxidative damage. Here, we developed nucleotide-based neuroprotectants acting dually as antioxidants and P2Y-R agonists. To improve the potency, selectivity, and metabolic stability of ATP/ADP, we substituted adenine C2-position by Cl and P_α/P_β position by borano group, <b>6</b>–<b>9</b>. Nucleotides <b>6</b>–<b>9</b> inhibited oxidation in cell-free systems (Fe­(II)-H₂O₂), as detected by ESR (IC₅₀ up to 175 μM), and ABTS assay (IC₅₀ up to 40 μM). They also inhibited FeSO₄-induced oxidative stress in PC12 cells (IC₅₀ of 80–200 nM). 2-Cl-ADP­(α-BH₃), <b>7a</b>, was found to be the most potent P2Y₁-R agonist currently known (EC₅₀ 7 nM) and protected primary cortical neurons from FeSO₄ insult (EC₅₀ 170 nM). In addition, it proved to be metabolically stable in human blood serum (<i>t</i>_1/2 7 vs 1.5 h for ADP). Hence, we propose <b>7a</b> as a highly promising neuroprotectant

Highly Potent and Selective Ectonucleotide Pyrophosphatase/Phosphodiesterase I Inhibitors Based on an Adenosine 5′-(α or γ)-Thio-(α,β- or β,γ)-methylenetriphosphate Scaffold

Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure–activity relationship, encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATP-α-thio-β,γ-CH₂ (<b>1</b>), ATP-α-thio-β,γ-CCl₂ (<b>2</b>), ATP-α-CH₂-γ-thio (<b>3</b>), and 8-SH-ATP (<b>4</b>) and established their resistance to hydrolysis by NPP1,3 and NTPDase1,2,3,8 (<5% hydrolysis) (NTPDase = ectonucleoside triphosphate diphosphohydrolase). Analogues <b>1</b>–<b>3</b> at 100 μM inhibited thymidine 5′-monophosphate <i>p</i>-nitrophenyl ester hydrolysis by NPP1 and NPP3 by >90% and 23–43%, respectively, and only slightly affected (0–40%) hydrolysis of ATP by NTPDase1,2,3,8. Analogue <b>3</b> is the most potent NPP1 inhibitor currently known, <i>K</i>_i = 20 nM and IC₅₀ = 0.39 μM. Analogue <b>2a</b> is a selective NPP1 inhibitor with <i>K</i>_i = 685 nM and IC₅₀ = 0.57 μM. Analogues <b>1</b>–<b>3</b> were found mostly to be nonagonists of P2Y₁/P2Y₂/P2Y₁₁ receptors. Docking analogues <b>1</b>–<b>3</b> into the NPP1 model suggested that activity correlates with the number of H-bonds with binding site residues. In conclusion, we propose analogues <b>2a</b> and <b>3</b> as highly promising NPP1 inhibitors