Bis(styryl)benzene Probes for Targeting Early-Stage Amyloid-β Aggregates in Alzheimer’s Disease

Abstract

With the recent FDA approval of antibody-based therapeutics for Alzheimer’s disease (AD), the need for diagnostic tools capable of detecting the disease at the earliest stages has become increasingly urgent. Although small molecules offer advantages in terms of production, stability, and accessibility, no imaging agent currently enables reliable detection of early-stage aggregates of the amyloid-β (Aβ) peptide, among the earliest biomarkers in AD progression. Herein, we report a series of bis(styryl)benzene (BSB) probes derived from the methoxy-X04 scaffold and a systematic structure–activity investigation examining how targeted molecular modifications, including hydroxyl positioning, (Me)HN or (Me)2N substitution, and incorporation of a 1,4-dimethyl-1,4,7-triazacyclononane (tacn) moiety, modulate Aβ binding, cytotoxicity, and brain uptake. These methoxy-X04 analogues retain Aβ affinity while exhibiting markedly improved cytotoxicity profiles and logD values consistent with in vivo applicability. Amyloid-β binding was evaluated using age-dependent in situ staining of 5xFAD mouse brain sections, supported by in vitro fluorescence-based oligomer and fibril assays and in silico analyses that reveal tunable selectivity toward early versus late Aβ aggregates. In vivo brain uptake studies of BSB5 and Me2tacnBSB5 further support the utility of these probes as modular Aβ-targeting fragments for the development of early-stage AD imaging agents.