Structure-based design, synthesis by click chemistry and in vivo activity of highly selective A₃ adenosine receptor agonists

Abstract

2-Arylethynyl derivatives of (N)-methanocarba adenosine 5′-uronamides are selective A3AR (adenosine receptor) agonists. Here we substitute a 1,2,3-triazol-1-yl linker in place of the rigid, linear ethynyl group to eliminate its potential metabolic liability. Docking of nucleosides containing possible short linker moieties at the adenine C2 position using a hybrid molecular model of the A3AR (based on the A2AAR agonist-bound structure) correctly predicted that a triazole would maintain the A3AR selectivity, due to its ability to fit a narrow cleft in the receptor. The analogues with various N6 and C2-aryltriazolyl substitution were synthesized and characterized in binding (Ki at hA3AR 0.3 – 12 nM) and in vivo to demonstrate efficacy in controlling chronic neuropathic pain (chronic constriction injury). Among N6-methyl derivatives, a terminal pyrimidin-2-yl group in 9 (MRS7116) increased duration of action (36% pain protection at 3 h) in vivo. N6-Ethyl 5-chlorothien-2-yl analogue 15 (MRS7126) preserved in vivo efficacy (85% protection at 1 h) with short duration. Larger N6 groups, e.g. 17 (MRS7138, >90% protection at 1 and 3 h), greatly enhanced in vivo activity. Thus, we have combined structure-based methods and phenotypic screening to identify nucleoside derivatives having translational potential.

Rigidified A3 Adenosine Receptor Agonists: 1-Deazaadenine Modification Maintains High in Vivo Efficacy

“…c n = 3. delimited by TM6 and EL2 and exposed toward the extracellular environment. Consistent with our previous report, 6 there was a correspondence in A 3 AR affinity between 1-aza alkynes and 1-aza triazoles. We have no structural explanation for the lack of correlation of A 3 AR affinity between 1-aza and 1-deaza variants of the alkynes containing N 6 -Pr or c-Pr substituents.…”

“…A combination with a rigid extension at the C2 position of the adenine in the form of a 2-(5-chlorothiophen-2-yl)ethynyl) or a 2-(4-(5-chlorothiophen-2-yl)-1H-1,2,3-triazol-1-yl) group further enhances A 3 AR selectivity. 5,6 This modification suggested structural plasticity of the second transmembrane helix (TM2) of the A 3 AR to accommodate the highly rigidified analogues.…”

“…The N 6 substituent was modified as small alkyl or cycloalkyl groups because the earlier studies emphasized N 6 -methyl or N 6 -(3-chlorobenzyl), 5,6 i.e., leaving a knowledge gap. The terminal aryl group was 5-chlorothiophen-2-yl, which was previously found to prolong the duration of the protective response in pain models.…”

“…5 Reference alkyne 10 and triazoles 23, 24, 27, and 28 were included for comparison. 5,6 We first optimized affinity in the 1-aza series and then proceeded to introduce the 1-deaza modification. Except for 21, the nucleoside alkyne derivatives only weakly inhibited binding (<50% at 10 μM) at the hA 1 AR and hA 2A AR, and many bound at the hA 3 AR with nearly nanomolar affinity.…”

“…The cLog P values were in a favorable range for drug-like molecules, e.g., 3.49 and 2.71 for N 6 -ethyl analogues 20 and 11, respectively (Supporting Information). 11 The observation that nanomolar A 3 AR affinity could be maintained in the 1-deaza series was explored structurally through molecular modeling using a hybrid homology model 5,6 of the receptor (methodological details have been previously reported). 12 Figure 2 shows the docking pose of compound 20 at the hA 3 AR model.…”