Chemical modifications of a compound-of-interest, such as an enzyme inhibitor, are at the basis of performing robust structure-activity relationships able to address synthetic efforts toward more and more potent ligands. In this context, substitution patterns on phenyl rings, e.g., introduction of electron-donor/withdrawing groups, are widely explored. Generation of derivatives is based on different approaches, including molecular modeling, the emerging artificial intelligence, or the more traditional organic chemistry strategy. The latter usually follows practical guidelines, e.g., the Topliss scheme, or the actual laboratory availability of chemicals. In the last decade, Prof. La Motta’s group has been involved in the development of new anticancer agents targeting Aldehyde Dehydrogenases (ALDHs, EC: 1.2.1.3), enzymes involved in the detoxifying conversion of aldehydes into the safer corresponding carboxylic acids, recognized as Cancer Stem Cells biomarkers and overexpressed in solid tumors. Derivatives libraries containing the imidazo[1,2-a]pyridine scaffold have been proposed as ALDH inhibitors and isoform selectivity has been reached through different phenyl substitution patterns. In particular, a potent and selective ALDH1A3 inhibitor (3q, IC₅₀ = 3.5 µM) endowed with three methoxy substituents in specific phenyl positions was previously reported. Thus, we enlarged the derivatives series of 3q, also synthesizing its corresponding demethylated derivative (1). Unexpectedly, a complete loss of inhibitory activity was observed for 1, still possessing a promising antiproliferative effect on a thyroid primary cell line. In silico analyses on ALDHs were performed to elucidate why such a small chemical modification severely affected the activity. Then, we investigated the actual antiproliferative mechanism of action of 1 by exploring in wet different possibilities, e.g., kinases and carbonic anhydrases, providing partial but interesting information.