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, 51 (15), 4518-28

Selective Nucleoside Triphosphate diphosphohydrolase-2 (NTPDase2) Inhibitors: Nucleotide Mimetics Derived From uridine-5'-carboxamide

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Selective Nucleoside Triphosphate diphosphohydrolase-2 (NTPDase2) Inhibitors: Nucleotide Mimetics Derived From uridine-5'-carboxamide

Andreas Brunschweiger et al. J Med Chem.

Abstract

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases, subtypes 1, 2, 3, 8 of NTPDases) dephosphorylate nucleoside tri- and diphosphates to the corresponding di- and monophosphates. In the present study we synthesized adenine and uracil nucleotide mimetics, in which the phosphate residues were replaced by phosphonic acid esters attached to the nucleoside at the 5'-position by amide linkers. Among the synthesized uridine derivatives, we identified the first potent and selective inhibitors of human NTPDase2. The most potent compound was 19a (PSB-6426), which was a competitive inhibitor of NTPDase2 exhibiting a K i value of 8.2 microM and selectivity versus other NTPDases. It was inactive toward uracil nucleotide-activated P2Y 2, P2Y 4, and P2Y 6 receptor subtypes. Compound 19a was chemically and metabolically highly stable. In contrast to the few known (unselective) NTPDase inhibitors, 19a is an uncharged molecule and may be perorally bioavailable. NTPDase2 inhibitors have potential as novel cardioprotective drugs for the treatment of stroke and for cancer therapy.

Figures

Figure 1
Figure 1
Structures of standard ectonucleotidase inhibitors.
Figure 2
Figure 2
Target structures: adenine and uracil nucleotide mimetics.
Figure 3
Figure 3
Concentration–inhibition curves of selected inhibitors at NTPDase2. The enzyme assays were performed in a final volume of 100 μL using a substrate (ATP) concentration of 400 μM (Km = 70 μM). Product formation was determined by capillary electrophoresis as described in the Experimental Section. Data points represent the mean ± SEM from two separate experiments each performed in triplicate. Determined IC50 values were 42 μM (19a), 101 μM (20c), and 374 μM (20a). Calculated Ki values were 8.2 μM (19a), 29.2 μM (20c), and 71.7 μM (20a).
Figure 4
Figure 4
Lineweaver–Burk plot for NTPDase2 kinetics in the absence and in the presence of two different concentrations of the inhibitor 19a (5 and 10 μM).
Scheme 1
Scheme 1
Synthesis of 2′,3′-Protected Nucleoside-5′-Carboxylic Acidsa a Reagents and conditions: (a) p-methoxybenzaldehyde, ZnCl2, dry THF, room temp, 48 h; (b) TEMPO, bis(acetoxy)iodobenzene, H2O/MeCN = 1:1, room temp, 3 h.
Scheme 2
Scheme 2
Synthesis of ω-Aminoalkylcarboxamidoalkyl- and ω-Aminoalkylcarboxamido-p-benzyl-Substituted Phosphonic Acid Diethyl Estersa a Reagents and conditions: (a) dry THF, N-methylmorpholine, ClCO2CH2CH(CH3)2, –25 °C to room temp, 3 h; (b) 4 N HCl in dry dioxane, room temp, 2h.
Scheme 3
Scheme 3
Synthesis of Nucleoside-5′-Carboxamides and Deprotection of the 2′,3′-Positiona a Reagents and conditions: (a) (i) coupling reagent (PyBOP, HCTU, or HBTU; see Experimental Section), diisopropylethylamine, DMF, room temp, 12 h, (ii) silica gel flash chromatography, CH2Cl2/MeOH = 40:1; (b) (i) 3–5% TFA, CH2Cl2, H2O, 2 h, room temp, (ii) C-18 RP-HPLC, gradient of MeOH/H2O = 25:75 to 100:0.
Scheme 4
Scheme 4
Proposed Metabolization of Compounds 19a and 19c by Rat Liver Homogenate
Scheme 5
Scheme 5
Amide Hydrolysis of Compound 19a

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