Site-Dependent Modulation of Antitumor Activity and Fluorescence in Thieno[3,2-b]pyridin-5(4H)-ones

We report the design and synthesis of thieno[3,2-<i>b</i>]pyridin-5(4<i>H</i>)-one derivatives exhibiting site-dependent modulation of both antitumor activity and fluorescence, enabled by a regioselective BOP-promoted aza-[3 + 3] cycloaddition. The reaction proceeds between thiophen-3-amines and α,β-unsaturated carboxylic acids, followed by base-induced dehydrogenation. Mechanistic studies reveal that the head-to-tail aza-[3 + 3] annulation involves a <i>C</i>-1,4 conjugate addition, leading to an intramolecular amide coupling. Evaluation of the photophysical properties and antitumor activities demonstrated that the biological and optical behaviours of the thieno[3,2-<i>b</i>]pyridin-5(4<i>H</i>)-one scaffold are dependent on the aryl substitution site. Specifically, 3-aryl derivatives exhibited notable antitumor activity, whereas 2-aryl analogues displayed strong fluorescence, highlighting the potential of this scaffold for dual-function applications. DFT calculations supported the observed divergence in fluorescence by revealing differences in orbital conjugation and HOMO-LUMO gaps. In addition, selected compounds showed low cytotoxicity toward MRC-9 cells, indicating favourable cancer cell selectivity.