Design, synthesis, pharmacological characterization of a fluorescent agonist of the P2Y14 receptor.

The P2Y14R is a G(i/o)-coupled receptor of the P2Y family of purinergic receptors that is activated by extracellular UDP and UDP-glucose (UDPG). In an earlier report we described a P2Y14R fluorescent probe, MRS4174, based on the potent and selective antagonist PPTN, a naphthoic acid derivative. Here, we report the design, preparation, and activity of an agonist-based fluorescent probe MRS4183 (11) and a shorter P2Y14R agonist congener, which contain a UDP-glucuronic acid pharmacophore and BODIPY fluorophores conjugated through diaminoalkyl linkers. The design relied on both docking in a P2Y14R homology model and established structure activity relationship (SAR) of nucleotide analogs. 11 retained P2Y14R potency with EC50 value of 0.96 nM (inhibition of adenylyl cyclase), compared to parent UDPG (EC50 47 nM) and served as a tracer for microscopy and flow cytometry, displaying minimal nonspecific binding. Binding saturation analysis gave an apparent binding constant for 11 in whole cells of 21.4±1.1 nM, with a t1/2 of association at 50 nM 11 of 23.9 min. Known P2Y14R agonists and PPTN inhibited cell binding of 11 with the expected rank order of potency. The success in the identification of a new P2Y14R fluorescent agonist with low nonspecific binding illustrates the advantages of rational design based on recently determined GPCR X-ray structures. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands.

N6-Substituted 5'-N-Methylcarbamoyl-4'-selenoadenosines as Potent and Selective A3 Adenosine Receptor Agonists with Unusual Sugar Puckering and Nucleobase Orientation.

Potent and selective A3 adenosine receptor (AR) agonists were identified by the replacement of 4'-oxo- or 4'-thionucleosides with bioisosteric selenium. Unlike previous agonists, 4'-seleno analogues preferred a glycosidic syn conformation and South sugar puckering, as shown in the X-ray crystal structure of 5'-N-methylcarbamoyl derivative 3p. Among the compounds tested, N6-3-iodobenzyl analogue 3d was found to be the most potent A3AR full agonist (Ki = 0.57 nM), which was ≥800- and 1900-fold selective for A1AR and A2AAR, respectively. In the N6-cycloalkyl series, 2-Cl analogues generally exhibited better hA3AR affinity than 2-H analogues, whereas 2-H > 2-Cl in the N6-3-halobenzyl series. N7 isomers 3t and 3u were much weaker in binding than corresponding N9 isomers, but compound 3t lacked A3AR activation, appearing to be a weak antagonist. 2-Cl-N6-3-iodobenzyl analogue 3p inhibited chemoattractant-induced migration of microglia/monocytes without inducing cell death at ≤50 µM. This suggests the potential for the development of 4'-selenonucleoside A3AR agonists as novel antistroke agents.

4-Alkyloxyimino derivatives of uridine-5'-triphosphate: distal modification of potent agonists as a strategy for molecular probes of P2Y2, P2Y4, and P2Y6 receptors.

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.

Exploring a 2-naphthoic acid template for the structure-based design of P2Y14 receptor antagonist molecular probes.

The P2Y14 receptor (P2Y14R), one of eight P2Y G protein-coupled receptors (GPCR), is involved in inflammatory, endocrine, and hypoxic processes and is an attractive pharmaceutical target. The goal of this research is to develop high-affinity P2Y14R fluorescent probes based on the potent and highly selective antagonist 4-(4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl)-2-naphthoic acid (6, PPTN). A model of hP2Y14R based on recent hP2Y12R X-ray structures together with simulated antagonist docking suggested that the piperidine ring is suitable for fluorophore conjugation while preserving affinity. Chain-elongated alkynyl or amino derivatives of 6 for click or amide coupling were synthesized, and their antagonist activities were measured in hP2Y14R-expressing CHO cells. Moreover, a new Alexa Fluor 488 (AF488) containing derivative 30 (MRS4174, Ki = 80 pM) exhibited exceptionally high affinity, as compared to 13 nM for the alkyne precursor 22. A flow cytometry assay employing 30 as a fluorescent probe was used to quantify specific binding to P2Y14R. Known P2Y receptor ligands inhibited binding of 30 with properties consistent with their previously established receptor selectivities and affinities. These results illustrate that potency in this series of 2-naphthoic acid derivatives can be preserved by chain functionalization, leading to highly potent fluorescent molecular probes for P2Y14R. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands. This approach demonstrates the predictive power of GPCR homology modeling and the relevance of newly determined X-ray structures to GPCR medicinal chemistry.