Developmentand Preliminary Evaluation of a ¹²⁵I‑Labeled Radioligand([¹²⁵I]iodotrazoline) forIn Vitro Detection of Imidazoline‑2 Binding Site in the Brain

Astrocytes exert multiple functions within the brain, including regulating neuroinflammation and maintaining homeostasis, and the reactive astrocytes are implicated in many neurodegenerative disorders. Imidazoline-2 binding site (I₂BS) has been established as a reliable biomarker for precisely quantifying reactive astrocytes. Here, we reported the development of [¹²⁵I]iodotrazoline ([¹²⁵I]<b>8</b>), a novel I₂BS radioligand with high affinity (<i>K</i>_i = 6.8 nM) and exceptional selectivity over α₂-adrenoceptors (>1400 folds). <i>In vitro</i> autoradiography (ARG) using rat brain sections revealed a heterogeneous distribution of [¹²⁵I]<b>8</b>, with high signals in the medulla, midbrain, pons, and hypothalamus. Pretreatment with unlabeled I₂BS-selective ligands, BU224 and FTIMD, reduced the binding by >30%, indicating high <i>in vitro</i> specificity for I₂BS. <i>Ex vivo</i> ARG results confirmed this distribution pattern in the rat brain. Biodistribution results in mice demonstrated a rapid brain uptake of [¹²⁵I]<b>8</b> (3.35% ID/g at 2 min postinjection) with slow washout. Metabolite analysis exhibited the desirable biostability of [¹²⁵I]<b>8</b> in the rat brain. Altogether, this work provides a new ¹²⁵I-labeled radioligand featuring a novel 2-trans-styryl-imidazoline scaffold, which shows significant specificity binding for I₂BS <i>in vitro</i>, serving as a valuable tool for I₂BS detection and astrocyte-related pathology research.