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, 57 (9), 3874-83

4-Alkyloxyimino Derivatives of uridine-5'-triphosphate: Distal Modification of Potent Agonists as a Strategy for Molecular Probes of P2Y2, P2Y4, and P2Y6 Receptors

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4-Alkyloxyimino Derivatives of uridine-5'-triphosphate: Distal Modification of Potent Agonists as a Strategy for Molecular Probes of P2Y2, P2Y4, and P2Y6 Receptors

P Suresh Jayasekara et al. J Med Chem.

Abstract

Extended N(4)-(3-arylpropyl)oxy derivatives of uridine-5'-triphosphate were synthesized and potently stimulated phospholipase C stimulation in astrocytoma cells expressing G protein-coupled human (h) P2Y receptors (P2YRs) activated by UTP (P2Y2/4R) or UDP (P2Y6R). The potent P2Y4R-selective N(4)-(3-phenylpropyl)oxy agonist was phenyl ring-substituted or replaced with terminal heterocyclic or naphthyl rings with retention of P2YR potency. This broad tolerance for steric bulk in a distal region was not observed for dinucleoside tetraphosphate agonists with both nucleobases substituted. The potent N(4)-(3-(4-methoxyphenyl)-propyl)oxy analogue 19 (EC50: P2Y2R, 47 nM; P2Y4R, 23 nM) was functionalized for chain extension using click tethering of fluorophores as prosthetic groups. The BODIPY 630/650 conjugate 28 (MRS4162) exhibited EC50 values of 70, 66, and 23 nM at the hP2Y2/4/6Rs, respectively, and specifically labeled cells expressing the P2Y6R. Thus, an extended N(4)-(3-arylpropyl)oxy group accessed a structurally permissive region on three Gq-coupled P2YRs, and potency and selectivity were modulated by distal structural changes. This freedom of substitution was utilized to design of a pan-agonist fluorescent probe of a subset of uracil nucleotide-activated hP2YRs.

Figures

Chart 1
Chart 1. Structures of Pyrimidine Nucleotides As Prototypical Agonist Ligands for Studying the P2Y4Ra
Scheme 1
Scheme 1. Synthesis of Various 4-Alkoxyiminopyrimidine Ribonucleoside 5′-Triphosphates
Reagents and conditions: (a) cytidine, pyridine, 110 °C; (b) proton sponge, POCl3, PO(OMe)3, tributylammonium pyrophosphate, DMF; (c) 1 N NaOH, THF, 50 °C; (d) PyBOP, DIEA, but-4-yn-1-amine.
Scheme 2
Scheme 2. Synthesis of 4-Alkoxyiminopyrimidine Dinucleoside Tetraphosphates
Reagents: (a) DIC, MgCl2, DMF.
Scheme 3
Scheme 3. Synthesis of Fluorescent 4-Alkoxyiminopyrimidine Ribonucleoside 5′-Triphosphates 27 and 28 by Click Reaction with Azido-Dyes
Reagents and conditions: (a) azide (e.g., 124, 1 equiv) and alkyne (1.4 equiv) precursors, tBuOH/water, TBTA, sodium ascorbate, cupric sulfate.
Scheme 4
Scheme 4. Synthesis of N-Alkoxyamines: (A) 3-Phenylpropyl and 3-(naphth-2-yl)propyl Intermediates Prepared by the Gabriel Synthesis. (B) N-Alkoxyamine Intermediates Prepared Using N,N′-di-tert-Butoxycarbonylhydroxylamine as Described
Reagents and conditions: (A) (a) diethyl malonate, NaH, THF; (b) NaCl, DMSO; (c) DiBal-H; (d) CBr4, PPh3; (e) Et3N, DMF, 100°C; (f) NH2NH2·H2O; (g) 1 N HCl. Protected 3-(4-carboxymethyloxy-phenyl)propyl (91) and 3-(thien-3-yl)propyl (92). N-alkoxyamine intermediates were prepared by analogous methods (Scheme S1, Supporting information): (a) K2CO3, CH3CN, reflux; (b) CBr4, PPh3; (c) Et3N, DMF, 100 °C; (d) NH2NH2·H2O; (e) 10% Pd/C, H2, MeOH; (f) LiAlH4, THF; (g) Br2, PPh3, 2,6-lutidine. (B) (a) HBr in acetic acid, 50 °C; (b) DBU, CH3CN, 50 °C; (c) 1 N HCl, CH2Cl2.
Figure 1
Figure 1
Comparison of potencies of alkyne 26 and fluorescent analogues 27 and 28 in activation of PLC via the human P2Y2R (A), P2Y4R (B), and P2Y6R (C) in stably transfected 1321N1 human astrocytoma cells.
Figure 2
Figure 2
Fluorescence ligand binding experiments using FCM in stably transfected 1321N1-P2Y6R cells with 28 after preincubation at 37 °C with known P2Y6R agonist or P2Y6R antagonists. Each column shows the brightness of each compound using 28 set at 100% and after correcting the MFI values for autofluorescence. Results are expressed as mean ± SEM (n = 4). Binding of 28 at 20 and 30 min to the P2Y6R was significantly blocked after preincubation with selective antagonist 118, selective agonist 120, and nonselective antagonist 119. No significant difference in MFI was observed for P2Y1R antagonist 117. *, p < 0.05, when compared to cells treated with only 28. Average of MFI after 20 min incubation of 28: 107.0 ± 30.2; after 30 min incubation of 28: 222.8 ± 72.1.
Figure 3
Figure 3
Fluorescent micrographs using a Keyence fluorescent microscope (BZ-9000) of P2Y6R-expressing 1321N1 astrocytoma cells exposed to the fluorescent agonist 28 (1 μM, 30 min incubation at 37 °C). (A) Control cells in the absence of 14. (B) Incubation with 1 μM 14 at 37 °C for 30 min in medium. (C) Incubation with 1 μM 14 at 37 °C for 30 min in cells pretreated with noncompetitive antagonist 118 (10 μM).

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