Pyrimidine ribonucleotides with enhanced selectivity as P2Y(6) receptor agonists: novel 4-alkyloxyimino, (S)-methanocarba, and 5'-triphosphate gamma-ester modifications

Abstract

The P2Y(6) receptor is a cytoprotective G-protein-coupled receptor (GPCR) activated by UDP (EC(50) = 0.30 microM). We compared and combined modifications to enhance P2Y(6) receptor agonist selectivity, including ribose ring constraint, 5-iodo and 4-alkyloxyimino modifications, and phosphate modifications such as alpha,beta-methylene and extension of the terminal phosphate group into gamma-esters of UTP analogues. The conformationally constrained (S)-methanocarba-UDP is a full agonist (EC(50) = 0.042 microM). 4-Methoxyimino modification of pyrimidine enhanced P2Y(6), preserved P2Y(2) and P2Y(4), and abolished P2Y(14) receptor potency, in the appropriate nucleotide. N(4)-Benzyloxy-CDP (15, MRS2964) and N(4)-methoxy-Cp(3)U (23, MRS2957) were potent, selective P2Y(6) receptor agonists (EC(50) of 0.026 and 0.012 microM, respectively). A hydrophobic binding region near the nucleobase was explored with receptor modeling and docking. UTP-gamma-aryl and cycloalkyl phosphoesters displayed only intermediate P2Y(6) receptor potency but had enhanced stability in acid and cell membranes. UTP-glucose was inactive, but its (S)-methanocarba analogue and N(4)-methoxycytidine 5'-triphospho-gamma-[1]glucose were active (EC(50) of 2.47 and 0.18 microM, respectively). Thus, the potency, selectivity, and stability of pyrimidine nucleotides as P2Y(6) receptor agonists may be enhanced by modest structural changes.

Chart 1

Structures of prototypical agonist ligands for studying P2Y₆ receptors.

Scheme 1

Synthesis of various pyrimidine ribonucleoside 5′-di and triphosphate derivatives. Reagents and conditions: (a) DCC, methylenebisphosphonate, rt, overnight, 11 % for 11, 48 % for 16; (b) RONH₂, pyridine, 90°C, overnight, 55 – 100 %; (c) POCl₃, Proton Sponge, PO(OMe)₃, 0°C, 2 hr, followed by tributylammonium phosphate, rt, 0.5 hr (for 12 - 15), 14 – 47 %; (d) POCl₃, Proton Sponge, PO(OMe)₃, 0°C, 2hr, followed by tributylammonium pyrophosphate, rt, 0.5 hr, 31 % (for 20).

Scheme 2

Synthesis of various pyrimidine ribonucleoside 5′-triphosphates and dinucleoside triphosphates. Reagents and conditions: (a) DIC, MgCl₂, glucose 1-monophosphate tributylammonium salt, DMF, rt, overnight, 9 - 80 %; (b) DIC, MgCl₂, respective monophosphate tributylammonium salt, e.g., uridine 5′-monophosphate, 35, cyclohexyl monophosphate or phenyl monophosphate, in DMF, rt, overnight, 9–80%. Unless noted, X = H and Y, Z = O.

Scheme 3

Synthesis of uracil ribonucleoside phosphate derivatives constrained with a (S)-methanocarba ring. Reagents and conditions: (a) POCl₃, Proton Sponge, PO(OMe)₃, 0°C, 2 hr; then tributylammonium phosphate, rt, 0.5 hr, 45 %; (b) POCl₃, Proton Sponge, PO(OMe)₃, 0°C, 2 hr; then tributylammonium pyrophosphate, rt, 0.5 hr, 56 %; (c) CDI, DMF, rt, 1 hr, then glucose 1-monophosphate tributylammonium salt, overnight, 23 % for 28, 51 % for 9.

Figure 1

A) Activity of agonists 7, 12, 15, and 23 at the human P2Y₆ receptor as indicated by activation of PLC in stably transfected 1321N1 human astrocytoma cells. The effect of UDP corresponds to 100%. B) Mobilization of intracellular calcium in astrocytoma cells expressing the human P2Y₆ receptor (EC₅₀ values shown in nM) by nucleotide agonists 3 (50), 21 (370), 23 (83), and 24 (630). The EC₅₀ value of 1 was 200 nM.

Figure 1

A) Activity of agonists 7, 12, 15, and 23 at the human P2Y₆ receptor as indicated by activation of PLC in stably transfected 1321N1 human astrocytoma cells. The effect of UDP corresponds to 100%. B) Mobilization of intracellular calcium in astrocytoma cells expressing the human P2Y₆ receptor (EC₅₀ values shown in nM) by nucleotide agonists 3 (50), 21 (370), 23 (83), and 24 (630). The EC₅₀ value of 1 was 200 nM.

Figure 2

Molecular modeling of the human P2Y₆ receptor. A) Details of the nucleotide binding site of the receptor complexed with the agonist 7 as obtained after a fully flexible Monte Carlo conformational search. The ligand is represented as balls and sticks, colored by element. The binding pocket is represented as a van der Waals surface colored according to the electrostatic potential, with positive charges in blue and negative charges in red. For clarity, the residues located in front of the ligand are represented as sticks, colored by element. Labels indicate all of the residues represented as sticks (adjacent) and the key residues represented as van der Waals surfaces (trough arrows). A schematic representation of the receptor-ligand complex is given in the lower left inset. In the tube representations the receptor is colored according to residue positions, with a spectrum of colors that ranges from red (N-terminus) to purple (C-terminus): TM1 is in orange, TM2 in ochre, TM3 in yellow, TM4 in green, TM5 in cyan, TM6 in blue, TM7 in purple. B) Docking of the potent agonist N⁴-methoxy-cytidine-5′-diphosphate 12. A hydrophobic binding pocket for the N⁴-alkyloxy substituent is defined in this docking model. Distances in Å from the methoxy carbon atom to neighboring amino acid residues are shown.

Figure 2

Molecular modeling of the human P2Y₆ receptor. A) Details of the nucleotide binding site of the receptor complexed with the agonist 7 as obtained after a fully flexible Monte Carlo conformational search. The ligand is represented as balls and sticks, colored by element. The binding pocket is represented as a van der Waals surface colored according to the electrostatic potential, with positive charges in blue and negative charges in red. For clarity, the residues located in front of the ligand are represented as sticks, colored by element. Labels indicate all of the residues represented as sticks (adjacent) and the key residues represented as van der Waals surfaces (trough arrows). A schematic representation of the receptor-ligand complex is given in the lower left inset. In the tube representations the receptor is colored according to residue positions, with a spectrum of colors that ranges from red (N-terminus) to purple (C-terminus): TM1 is in orange, TM2 in ochre, TM3 in yellow, TM4 in green, TM5 in cyan, TM6 in blue, TM7 in purple. B) Docking of the potent agonist N⁴-methoxy-cytidine-5′-diphosphate 12. A hydrophobic binding pocket for the N⁴-alkyloxy substituent is defined in this docking model. Distances in Å from the methoxy carbon atom to neighboring amino acid residues are shown.