Rational Design of Sulfonated A₃ Adenosine Receptor-Selective Nucleosides as Pharmacological Tools To Study Chronic Neuropathic Pain

(N)-Methanocarba­(bicyclo­[3.1.0]­hexane)­adenosine derivatives were probed for sites of charged sulfonate substitution, which precludes diffusion across biological membranes, e.g., blood–brain barrier. Molecular modeling predicted that sulfonate groups on C2-phenylethynyl substituents would provide high affinity at both mouse (m) and human (h) A3 adenosine receptors (ARs), while a N6-p-sulfophenylethyl substituent would determine higher hA3AR vs mA3AR affinity. These modeling predictions, based on steric fitting of the binding cavity and crucial interactions with key residues, were confirmed by binding/efficacy studies of synthesized sulfonates. N6-3-Chlorobenzyl-2-(3-sulfophenylethynyl) derivative 7 (MRS5841) bound selectively to h/m A3ARs (Ki(hA3AR) = 1.9 nM) as agonist, while corresponding p-sulfo isomer 6 (MRS5701) displayed mixed A1/A3AR agonism. Both nucleosides administered ip reduced mouse chronic neuropathic pain that was ascribed to either A3AR or A1/A3AR using A3AR genetic deletion. Thus, rational design methods based on A3AR homology models successfully predicted sites for sulfonate incorporation, for delineating adenosine’s CNS vs peripheral actions.本研究针对(N)-甲烷碳双环[3.1.0]己烷腺苷（(N)-Methanocarba(bicyclo[3.1.0]hexane)adenosine）衍生物的带电磺酸盐取代位点开展系统探究，该取代方式可阻断衍生物跨生物膜（如血脑屏障（blood–brain barrier））的扩散。分子建模（molecular modeling）预测，C2-苯乙炔基取代位点上的磺酸盐基团，可对小鼠（mouse, m）及人类（human, h）A3腺苷受体（A3 adenosine receptors, ARs）均展现出高亲和力；而N6-对磺苯乙基取代基则可赋予人类A3腺苷受体（hA3AR）相较于小鼠A3腺苷受体（mA3AR）更高的亲和力。上述建模预测基于结合腔的空间匹配特性与关键残基的关键相互作用，经合成磺酸盐类化合物的结合/药效学实验得到验证。N6-3-氯苄基-2-(3-磺苯乙炔基)衍生物7（MRS5841）作为激动剂可选择性结合h/m A3ARs，其对人类A3腺苷受体的抑制常数Ki=1.9 nM；而对应的对磺基异构体6（MRS5701）则表现出A1/A3AR混合激动活性。两种核苷经腹腔注射（intraperitoneal, ip）给药后，均可缓解小鼠慢性神经病理性疼痛；通过A3腺苷受体（A3AR）基因敲除实验证实，该镇痛作用分别归因于A3AR或A1/A3AR的激活。综上，基于A3腺苷受体同源建模（homology modeling）的理性设计方法可成功预测磺酸盐引入位点，从而用于区分腺苷类化合物的中枢神经系统（central nervous system, CNS）与外周组织活性。