4?Alkyloxyimino Derivatives of Uridine-5′-triphosphate: Distal Modification of Potent Agonists as a Strategy for Molecular Probes of P2Y_2, P2Y_4, and P2Y_6 Receptors

摘要

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 (P2Y_(2/4)R) or UDP (P2Y_6R). The potent P2Y_4R-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 (EC_(50): P2Y_2R, 47 nM; P2Y_4R, 23 nM) was functionalized for chain extension using click tethering of fluorophores as prosthetic groups. The BODIPY 630/650 conjugate 28 (MRS4162) exhibited EC_(50) values of 70, 66, and 23 nM at the hP2Y_(2/4/6)Rs, respectively, and specifically labeled cells expressing the P2Y_6R. Thus, an extended N~4-(3- arylpropyl)oxy group accessed a structurally permissive region on three G_q-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.