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High affinity phenyl-piperidine P2Y 14 R antagonist 1 (PPTN) was modified with piperidine bridging moieties to probe receptor affinity and hydrophobicity. Various 2-azanorbornane, nortropane, isonortropane, isoquinuclidine, and ring-opened cyclopentylamino derivatives preserved human P2Y 14 R affinity (fluorescence binding assay), and their pharmacophoric overlay was compared. Enantiomeric 2-azabicyclo[2.2.1]hept-5-en-3-one precursors assured stereochemically unambiguous, diverse products. Pure ( S , S , S ) 2-azanorbornane enantiomer 15 (MRS4738) displayed higher affinity than 1 (3-fold higher affinity than enantiomer 16 ) and in vivo antihyperallodynic and antiasthmatic activity. Its double prodrug 143 (MRS4815) dramatically reduced lung inflammation in a mouse asthma model. Related lactams 21 - 24 and dicarboxylate 42 displayed intermediate affinity and enhanced aqueous solubility. Isoquinuclidine 34 (IC 50 15.6 nM) and isonortropanol 30 (IC 50 21.3 nM) had lower lipophilicity than 1 . In general, rigidified piperidine derivatives did not lower lipophilicity dramatically, except those rings with multiple polar groups. P2Y 14 R molecular modeling based on a P2Y 12 R structure showed stable and persistent key interactions for compound 15 .

A known zwitterionic, heterocyclic P2Y 14 R antagonist 3a was substituted with diverse groups on the central phenyl and terminal piperidine moieties, following a computational selection process. The most potent analogues contained an uncharged piperidine bioisostere, prescreened in silico, while an aza-scan (central phenyl ring) reduced P2Y 14 R affinity. Piperidine amide 11 , 3-aminopropynyl 19 , and 5-(hydroxymethyl)isoxazol-3-yl) 29 congeners in the triazole series maintained moderate receptor affinity. Adaption of 5-(hydroxymethyl)isoxazol-3-yl gave the most potent naphthalene-containing ( 32 ; MRS4654; IC 50 , 15 nM) and less active phenylamide-containing ( 33 ) scaffolds. Thus, a zwitterion was nonessential for receptor binding, and molecular docking and dynamics probed the hydroxymethylisoxazole interaction with extracellular loops. Also, amidomethyl ester prodrugs were explored to reversibly block the conserved carboxylate group to provide neutral analogues, which were cleavable by liver esterase, and in vivo efficacy demonstrated. We have, in stages, converted zwitterionic antagonists into neutral molecules designed to produce potent P2Y 14 R antagonists for in vivo application.

Dopamine-derived N 6 -substituents, compared to N 6 -(2-phenylethyl), in truncated (N)-methanocarba (bicyclo[3.1.0]hexyl) adenosines favored high A 3 adenosine receptor (AR) affinity/selectivity, e.g., C2-phenylethynyl analogue 15 (MRS7591, K i = 10.9/17.8 nM, at human/mouse A 3 AR). 15 was a partial agonist in vitro (hA 3 AR, cAMP inhibition, 31% E max ; mA 3 AR, [ 35 S]GTP-γ-S binding, 16% E max ) and in vivo and also antagonized hA 3 AR in vitro. Distal H-bonding substitutions of the N 6 -(2-phenylethyl) moiety particularly enhanced mA 3 AR affinity by polar interactions with the extracellular loops, predicted using docking and molecular dynamics simulation with newly constructed mA 3 AR and hA 3 AR homology models. These hybrid models were based on an inactive antagonist-bound hA 1 AR structure for the upper part of TM2 and an agonist-bound hA 2A AR structure for the remaining TM portions. These species-independent A 3 AR-selective nucleosides are low efficacy partial agonists and novel, nuanced modulators of the A 3 AR, a drug target of growing interest.

We investigated the Gi-coupled A 3 adenosine receptor (A 3 AR) activation mechanism by running 7.2 µs of molecular dynamics (MD) simulations. Based on homology to G protein-coupled receptor (GPCR) structures, three constitutively active mutant (CAM) and the wild-type (WT) A 3 ARs in the apo form were modeled. Conformational signatures associated with three different receptor states (inactive R, active R*, and bound to Gi protein mimic) were predicted by analyzing and comparing the CAMs with WT receptor and by considering site-directed mutagenesis data. Detected signatures that were correlated with receptor state included: Persistent salt-bridges involving key charged residues for activation (including a novel, putative ionic lock), rotameric state of conserved W 6.48 , and Na + ions and water molecules present. Active-coupled state signatures similar to the X-ray structures of β 2 adrenergic receptor-Gs protein and A 2A AR-mini-Gs and the recently solved cryo-EM A 1 AR-Gi complexes were found. Our MD analysis suggests that constitutive activation might arise from the D107 3.49 -R108 3.50 ionic lock destabilization in R and the D107 3.49 -R111 3.53 ionic lock stabilization in R* that presumably lowers the energy barrier associated with an R to R* transition. This study provides new opportunities to understand the underlying interactions of different receptor states of other Gi protein-coupled GPCRs.

(N)-Methanocarba ([3.1.0]bicyclohexyl) adenosines and corresponding ribosides were synthesized to identify novel A 1 adenosine receptor (A 1 AR) agonists for CNS or peripheral applications. Human and mouse AR binding was determined to assess the constrained ring system's A 1 AR compatibility. N 6 -Dicyclobutylmethyl ribose agonist (9, MRS7469, >2000-fold selective for A 1 AR) and known truncated N 6 -dicyclopropylmethyl methanocarba 7 (MRS5474) were drug-like. The pure diastereoisomer of known riboside 4 displayed high hA 1 AR selectivity. Methanocarba modification reduced A 1 AR selectivity of N 6 -dicyclopropylmethyl and endo-norbornyladenosines but increased ribavirin selectivity. Most analogues tested (ip) were inactive or weak in inducing mouse hypothermia, despite mA 1 AR full agonism and variable mA 3 AR efficacy, but strong hypothermia by 9 depended on A 1 AR, which reflects CNS activity (determined using A 1 AR or A 3 AR null mice). Conserved hA 1 AR interactions were preserved in modeling of 9 and methanocarba equivalent 24 (∼400-fold A 1 AR-selective). Thus, we identified, and characterized in vivo, ribose and methanocarba nucleosides, including with A 1 AR-enhancing N 6 -dicyclobutylmethyl-adenine and 1,2,4-triazole-3-carboxamide (40, MRS7451) nucleobases.

Recognition of nucleosides at adenosine receptors (ARs) is supported by multiple X-ray structures, but the structure of an adenine complex is unknown. We examined the selectivity of predicted A 1 AR and A 3 AR adenine antagonists that incorporated known agonist affinity-enhancing N 6 and C2 substituents. Adenines with A 1 AR-favoring N 6 -alkyl, cycloalkyl and arylalkyl substitutions combined with an A 3 AR-favoring 2-((5-chlorothiophen-2-yl)ethynyl) group were human (h) A 3 AR-selective, e.g. MRS7497 17 (∼1000-fold over A 1 AR). In addition, binding selectivity over hA 2A AR and hA 2B AR and functional A 3 AR antagonism were demonstrated. 17 was subjected to computational docking and molecular dynamics simulation in a hA 3 AR homology model to predict interactions. The SAR of nucleoside AR agonists was not recapitulated in adenine AR antagonists, and modeling suggested an alternative, inverted binding mode with the key N250 6.55 H-bonding to the adenine N 3 and N 9 , instead of N 6 and N 7 as in adenosine agonists.